CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/390,114 filed on July 18, 2022, which is incorporated herein by reference in its entirety.

BACKGROUND

[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

[0004] Disclosed herein in include a composition comprising a recombinant polynucleic acid comprising a sequence encoding a cytokine receptor switch polypeptide, the cytokine receptor switch polypeptide comprising: (a) an extracellular domain comprising a cytokine binding domain from a first cytokine receptor, wherein the first cytokine receptor is an IL21 receptor, (b) a transmembrane domain, and (c) 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.

[0005] Further disclosed herein include a composition comprising a cell comprising the composition comprising a recombinant polynucleic acid comprising a sequence encoding a cytokine receptor switch polypeptide, the cytokine receptor switch polypeptide comprising: (a) an extracellular domain comprising a cytokine binding domain from a first cytokine receptor, wherein the first cytokine receptor is an IL21 or IL9 receptor, (b) a transmembrane domain, and (c) 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; wherein the cell expresses the cytokine receptor switch polypeptide. [0006] Further disclosed herein include a composition comprising a recombinant polynucleic acid comprising a sequence encoding a first polypeptide comprising a cytokine receptor switch polypeptide and a recombinant polynucleic acid comprising a sequence encoding a second polypeptide, wherein cell surface expression of the cytokine receptor switch polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the cytokine receptor switch polypeptide that lacks the recombinant polynucleic acid encoding the second polypeptide compared to cell surface expression of the cytokine receptor switch polypeptide on a cell comprising the recombinant polynucleic acid encoding the cytokine receptor switch polypeptide and the recombinant polynucleic acid encoding the second polypeptide.

[0007] In some embodiments, the cytokine receptor switch polypeptide is not expressed extracellularly on a cell comprising a recombinant polynucleic acid encoding the cytokine receptor switch polypeptide that lacks the recombinant polynucleic acid encoding the second polypeptide.

[0008] In some embodiments, the second polypeptide comprises a transmembrane domain. In some cases, the second polypeptide comprises a transmembrane domain that is different than the transmembrane domain of the cytokine receptor switch polypeptide. In some cases, the transmembrane domain of the cytokine receptor switch polypeptide is a Triggering Receptor Expressed on Myeloid Cells 1 (TREM1) transmembrane domain. In some embodiments, the transmembrane domain of the second polypeptide is a DNAX Activating Protein of 12 kDa (DAP 12) transmembrane domain. In some embodiments, the transmembrane domain of the cytokine receptor switch polypeptide is a Triggering Receptor Expressed on Myeloid Cells 1 (TREM1) transmembrane domain and the transmembrane domain of the second polypeptide is a DNAX Activating Protein of 12 kDa (DAP12) transmembrane domain.

[0009] In some embodiments, the transmembrane domain of the cytokine receptor switch polypeptide is from a protein selected from the group consisting of Killer Cell Immunoglobulin Like Receptor Two Ig Domains And Short Cytoplasmic Tail 2 (KIR2DS2), TREM2, Immune receptor expressed by myeloid cells 2 (IREM2), KIR3DS1, KIR2DS5, KIR2DS1, KIR2DS4, KLRD1, KLRC2, Natural Cytotoxicity Triggering Receptor 2 (NCR2), Signal Regulatory Protein Beta 1 (SIRPB1), Paired Immunoglobin Like Type 2 Receptor Beta (PILRB), C-Type Lectin Domain Containing 5A (CLEC5 A), CD300 Molecule Like Family Member B (CD300LB), Sialic Acid Binding Ig Like Lectin 14 (SIGLEC14), SIGLEC15, and SIGLEC16.

[0010] In some embodiments, the cytokine receptor switch polypeptide is retained in the endoplasmic reticulum (ER) of a cell that does not express the second polypeptide. In some cases, expression of the cytokine receptor switch polypeptide on a cell surface of a cell is dependent on expression of the second polypeptide by the cell. In some embodiments, retention of the cytokine receptor switch polypeptide in the endoplasmic reticulum (ER) is higher in a cell comprising a recombinant polynucleic acid encoding the cytokine receptor switch polypeptide that lacks the recombinant polynucleic acid encoding the second polypeptide compared to retention of the cytokine receptor switch polypeptide in a cell comprising the recombinant polynucleic acid encoding the cytokine receptor switch polypeptide and the recombinant polynucleic acid encoding the second polypeptide.

[0011] In some embodiments, the second polypeptide does not comprise an intracellular domain. In some cases, the second polypeptide consists of the second transmembrane domain. In some cases, the second polypeptide does not comprise a full-length intracellular domain. In some cases, the second polypeptide does not comprise an intracellular domain comprising an intracellular signaling domain from a cytokine receptor. In some cases, the second polypeptide does not comprise an intracellular domain comprising an intracellular signaling domain. In some cases, the second polypeptide does not comprise a full-length extracellular domain. In some cases, the second polypeptide comprises an extracellular domain that is at most about 10, 50, 100, 150, 200, 250, or 350 amino acids in length. In some cases, the second polypeptide comprises an extracellular domain that is from about 1-10 amino acids in length, about 1-50 amino acids in length, about 1-100 amino acids in length, about 1-150 amino acids in length, about 1-200 amino acids in length, about 1-250 amino acids in length, about 1-300 amino acids in length, or about 1-350 amino acid in length. In some cases, the second polypeptide is a non-functional receptor or is not capable of signaling to a cell in which it is expressed.

[0012] In some embodiments, the cytokine receptor switch polypeptide and the second polypeptide form a complex. In some cases, the complex is a non-functional receptor complex or is not capable of signaling to a cell in which it is expressed. In some cases, the cytokine receptor switch polypeptide and the second polypeptide form a dimer. In some cases, the cytokine receptor switch polypeptide and the second polypeptide form a trimer. In some cases, the trimer is a trimer containing two molecules of the second polypeptide.

[0013] In some embodiments, the recombinant polynucleic acid comprises a first recombinant polynucleic acid comprising a sequence encoding the cytokine receptor switch polypeptide and a second recombinant polynucleic acid comprising a sequence encoding the second polypeptide.

[0014] In some embodiments, the first recombinant polynucleic acid further comprises a sequence encoding a first gene of interest. In some cases, the sequence encoding the first gene of interest encodes for a first protein of interest, wherein the cytokine receptor switch polypeptide and the first protein of interest are expressed as a single polypeptide molecule. In some cases, the sequence encoding the cytokine receptor switch polypeptide is linked to the sequence encoding the first protein of interest by a sequence encoding a linker. In some cases, the linker comprises a protease cleavage site or a 2A self-cleaving peptide. In some cases, the sequence encoding the first gene of interest encodes for a first protein of interest, wherein the cytokine receptor switch polypeptide and the first protein of interest are expressed as separate polypeptide molecules.

[0015] In some embodiments, the sequence encoding the cytokine receptor switch polypeptide and the sequence encoding the first gene of interest are separated or linked by an internal ribosome entry site (IRES) or have different promoters. In some cases, the second recombinant polynucleic acid further comprises a sequence encoding a second gene of interest. In some cases, the sequence encoding a second 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 cases, the sequence encoding the second polypeptide is linked to the sequence encoding the second protein of interest by a sequence encoding a linker. In some cases, the linker comprises a protease cleavage site or a 2A self-cleaving peptide.

[0016] In some cases, the sequence encoding the second gene of interest encodes for a second protein of interest, wherein the second polypeptide and the second protein of interest are expressed as separate polypeptide molecules. In some cases, the sequence encoding the second polypeptide and the sequence encoding the second gene of interest are separated or linked by an internal ribosome entry site (IRES) or have different promoters. In some cases, the first protein of interest and the second protein of interest are each a chimeric antigen receptor (CAR).

[0017] In some embodiments, the composition comprises a recombinant polynucleic acid comprising a sequence encoding a protease that cleaves the protease cleavage site. In some embodiments, the protease cleavage site is a viral protease cleavage site. In some cases, the viral protease cleavage site is for a viral protease derived from hepatitis C virus (HCV) nonstructural protein 3 (NS3). In some cases, the viral protease cleavage site is for a viral protease that further comprises a cofactor polypeptide derived from HCV nonstructural protein 4A (NS4A). In some cases, the viral protease cleavage site is selected from the group consisting of: an NS4A/4B junction cleavage site, an NS3/NS4A junction cleavage site, an NS4A/NS4B junction cleavage site, an NS4B/NS5A junction cleavage site, an NS5A/NS5B junction cleavage site, and variants thereof cleavable by the viral protease.

[0018] In some embodiments, the protease is fused to a transmembrane domain. In some cases, the protease is conditionally active. [0019] In some cases, the recombinant polynucleic acid comprises a first recombinant polynucleic acid comprising a sequence encoding the cytokine receptor switch polypeptide and a second recombinant polynucleic acid comprising a sequence encoding the protease.

[0020] In some cases, the second recombinant polynucleic acid further comprises a sequence encoding a second gene of interest. In some cases, the sequence encoding the second 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 cases, the sequence encoding the second polypeptide is linked to the sequence encoding the second protein of interest by a sequence encoding a linker.

[0021] In some embodiments, the sequence encoding the second gene of interest encodes for a second protein of interest, wherein the second polypeptide and the second protein of interest are expressed as separate polypeptide molecules. In some cases, the sequence encoding the second polypeptide and the sequence encoding the second gene of interest are separated or linked by an internal ribosome entry site (IRES) or have different promoters. In some cases, the second protein of interest is a chimeric antigen receptor (CAR).

[0022] Further disclosed herein include a composition comprising a recombinant polynucleic acid wherein the recombinant polynucleic acid comprises a first recombinant polynucleic acid comprising a first sequence encoding a cytokine receptor switch polypeptide, a second recombinant polynucleic acid comprising a second sequence encoding a first non-functional portion of a protease and a third recombinant polynucleic acid comprising a third sequence encoding a second non-functional portion of the protease wherein the first non-functional portion of the protease and the second non-functional portion of the protease form a functional protease.

[0023] In some embodiments, the first non-functional portion of the protease is fused to a transmembrane domain. In some cases, the second non-functional portion of the protease is fused to a transmembrane domain. In some cases, the second and/or third recombinant polynucleic acid further comprises a sequence encoding a second gene of interest or a third gene of interest.

[0024] In some cases, the sequence encoding a second gene of interest encodes for a second protein of interest, wherein the first non-functional portion of the protease and the second protein of interest are expressed as a single polypeptide molecule. In some cases, the sequence encoding the first non-functional portion of the protease is linked to the sequence encoding the second protein of interest by a sequence encoding a linker.

[0025] In some embodiments, the sequence encoding the second gene of interest encodes for a second protein of interest, and the third gene of interest encodes for a third protein of interest wherein the first non-functional portion of the protease and the second protein of interest are expressed as separate polypeptide molecules. In some cases, the sequence encoding the first non-functional portion of the protease and the sequence encoding the second gene of interest are separated or linked by an IRES or have different promoters. In some cases, the second and third protein of interest is a chimeric antigen receptor (CAR).

[0026] In some embodiments, the sequence encoding a third gene of interest encodes for a third protein of interest, wherein the second non-functional portion of the protease and the third protein of interest are expressed as a single polypeptide molecule. In some cases, the sequence encoding the second nonfunctional portion of the protease is linked to the sequence encoding the third protein of interest by a sequence encoding a linker.

[0027] In some embodiments, the sequence encoding the third gene of interest encodes for a third protein of interest, wherein the second non-functional portion of the protease and the third protein of interest are expressed as separate polypeptide molecules.

[0028] In some embodiments, the sequence encoding the second non-functional portion of the protease and the sequence encoding the third gene of interest are separated or linked by an IRES or have different promoters.

[0029] 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 cases, the first CAR is an anti- CD22 CAR, the second CAR is an anti-CD20 CAR and the third CAR is an anti-CD19 CAR.

[0030] Further disclosed herein include a composition comprising a cell comprising a recombinant polynucleic acid comprising a sequence encoding a cytokine receptor switch polypeptide, wherein the cell expresses the cytokine receptor switch polypeptide, the cytokine receptor switch polypeptide comprising: (a) an extracellular domain comprising a cytokine binding domain from a first cytokine receptor, (b) a transmembrane domain, and (c) 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some embodiments, the first cytokine receptor is an IL21 receptor. In some embodiments, the cell that expresses the cytokine receptor switch polypeptide expands in the presence of IL2 and a cell that does not express the cytokine receptor switch polypeptide expands in the presence of IL2. In some embodiments, a cell that does not express the cytokine receptor switch 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, the second cytokine receptor is a cytokine receptor that induces STAT5 signaling. In some instances, the intracellular signaling domain that induces STAT5 signaling comprises an intracellular signaling domain from IL2, IL4, IL7, IL13, IL15, thymic stromal lymphopoietin (TSLP), granulocyte-macrophage colony-stimulating factor (GM-CSF), or IL27.

[0031] In some embodiments, the second cytokine receptor is IL2 receptor beta. In some embodiments, the second cytokine receptor is IL7 receptor. In some embodiments, the recombinant polynucleic acid comprises a sequence encoding a chimeric antigen receptor (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 cytokine receptor switch polypeptide. In some embodiments, the sequence encoding the CAR is linked to the sequence encoding the cytokine receptor switch peptide by a linker. In some embodiments, the linker comprises a sequence encoding a self-cleaving peptide. In some embodiments, the linker is a P2A cleavable linker. 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 anti-CD22 binding domain. 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, 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, 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, M0V18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD20, CD22, ROR1, and GD2. In some embodiments, the intracellular domain comprises an intracellular signaling domain from CD2. In some embodiments, the intracellular domain comprises an intracellular signaling domain from CD3 , 4-1BB (CD137), CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD38, CD3£, 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 cytokine receptor switch polypeptide further comprises a protein retention tag. In some embodiments, the CAR further comprises a protein retention tag. In some embodiments, a protease cleavage site is disposed between the protein retention tag and the intracellular domain of the cytokine receptor switch polypeptide. In some embodiments, a protease cleavage site is disposed between the protein retention tag and the intracellular domain of the CAR. In some embodiments, a protease cleavage site is disposed between the protein retention tag and the extracellular domain of the cytokine receptor switch polypeptide. In some embodiments, a protease cleavage site is disposed between the protein retention tag and the extracellular domain of the CAR. In some embodiments, the protein retention tag is selected from the group consisting of an endoplasmic reticulum (ER) retention tag, a Golgi apparatus (Golgi) retention tag, a lysosome retention tag, a plasma membrane retention tag, a mitochondria retention tag, a peroxisome retention tag, a cytosolic retention tag, and a nuclear retention tag. In some embodiments, the protein retention tag is an ER retention tag. In some embodiments, the ER retention tag comprises 85% or greater, 90% or greater, or 100% amino acid sequence identity to the amino acid sequence LYKYKSRRSFIDEKKMP (SEQ ID NO: 40). In some embodiments, the ER retention tag comprises 85% or greater, 90% or greater, or 100% amino acid sequence identity to the amino acid sequence KHILFRRRRRGFRQ (SEQ ID NO: 30). In some embodiments, the ER retention tag comprises an amino acid sequence KKMP. In some embodiments, the protein retention tag is a Golgi retention tag. In some embodiments, the Golgi retention tag comprises an amino acid sequence YQRL. In some embodiments, the protein retention tag is a lysosome retention tag. In some embodiments, the lysosome retention tag comprises an amino acid sequence KFERQ. 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 l *10 ^A 5 cells.

[0032] Further disclosed herein is a cell, wherein the cell expresses the cytokine receptor switch polypeptide and a second polypeptide, wherein the second polypeptide comprises a DNAX Activating Protein of 12 kDa (DAP 12) transmembrane domain or a first non-functional portion of a protease. In some cases, the cell expresses the cytokine receptor switch polypeptide, the first non-functional portion of the protease, and a third polypeptide, wherein the third polypeptide comprises a second non-functional portion of the protease. In some cases, the cell expresses the first, second, and third polypeptides. In some cases, the cell expresses a first CAR, a second CAR, and a third CAR when the first, second, and third polypeptides further comprise a first CAR, a second CAR, and a third CAR.

[0033] In some embodiments, the cell is an engineered cell. In some embodiments, the engineered cell is a lymphocyte, a T cell, and/or a B cell.

[0034] In some embodiments, the cell is a population of cells. In some cases, the population of cells comprises at least l *10 ^A 5 cells. In some cases, at least 50%, 60%, 70%, 80%, 90, 95 or more of the cells in the population of cells express the first CAR, the second CAR and the third CAR. In some cases, at least 50%, 60%, 70%, 80%, 90, 95 or more of the cells in the population of cells express the first CAR, the second CAR and/or the third CAR.

[0035] Further disclosed herein includes a pharmaceutical composition comprising any of the compositions disclosed herein, and a pharmaceutically acceptable excipient or carrier.

[0036] Further disclosed herein includes a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein. In some embodiments, the cancer is lymphoma or leukemia.

[0037] Further disclosed herein includes a method of expanding cells, the method comprising culturing the any of the cells disclosed herein 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.

[0038] Further disclosed herein includes a method of making a population of chimeric antigen receptor (CAR)-expressing immune effector cells, comprising (a) contacting any of the cells 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 chimeric antigen receptor (CAR)-expressing immune effector cells.

INCORPORATION BY REFERENCE

[0039] 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

[0040] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0041] FIG. 1 depicts an exemplary structure of the cytokine receptor switch polypeptide.

[0042] FIG. 2 depicts the binding of exemplary cytokine receptor switch polypeptides to cytokines. FIG. 2A shows the binding of IL21 by an exemplary cytokine receptor switch polypeptide with IL2 receptor beta intracellular signaling domain that is expressed on the surface of a cell. FIG. 2B shows the binding of IL21 by an exemplary cytokine receptor switch polypeptide with IL7 receptor intracellular signaling domain that is expressed on the surface of a cell. The extracellular domain of the cytokine receptor switch polypeptide binds to IL21 with the assistance of the common gamma chain (IL2RG). The binding activates the downstream STAT5 signaling to promote proliferation and/or enhance cytotoxicity of the cell.

[0043] FIG. 3 illustrates that IL21 can selectively expand the transduced cells expressing an exemplary cytokine receptor switch polypeptide IL21R/IL2RB (i.e., IL21 receptor as the extracellular domain and IL2 receptor beta as the intracellular domain). Specifically, FIG. 3A shows that both IL2 and IL21 could promote proliferation of the transduced cells. FIG. 3B shows that only IL2 but not IL21 could promote proliferation of the non -transduced cells. FIG. 3C is a bar graph showing the percentage of IL21 receptor (IL21R)-positive cells in each of the cell cultures.

[0044] FIG. 4 depicts the growth and selection of cells expressing an IL21/IL2RB cytokine receptor switch polypeptide with a protease cleavage site. FIG. 4A depicts an exemplary IL21/IL2RB cytokine receptor switch polypeptide construct with a protease cleavage site (Pl) and an exemplary protease construct (P2). FIG. 4B depicts the results of a cell count experiment of T cells transduced with either Pl alone or Pl and P2, and grown in the presence of IL21. FIG. 4C depicts the results of an enrichment experiment, demonstrating the % of Pl and P2 double positive T cells grown in the presence of IL21. FIG.4D depicts flow cytometry plots of Pl or Pl and P2 transduced T cells grown in the presence of IL2 or IL21.

[0045] FIG. 5 depicts an exemplary construct for the creation of a trispecific CAR T cell. FIG. 5A depicts exemplary polypeptide constructs (P1-P3) for the creation of a trispecific CAR T cell with a IL21/IL2RB cytokine receptor switch polypeptide. FIG. 5B depicts the results of an enrichment experiment, demonstrating the percentage (%) of P1-P3 triple positive cells grown in the absence of cytokine (54.77%), IL2 (47.86%, or IL21 (86.62%). FIG. 5C depicts the results of a CAR T cell functional assay, demonstrating increased killing with trispecific CAR T cells comprising a IL21/IL2RB cytokine receptor switch polypeptide.

[0046] FIG. 6 depicts an exemplary IL21/TREM1/IL2RB cytokine receptor switch polypeptide. FIG. 6A depicts an IL21/IL2RB/TREM1 cytokine receptor switch polypeptide in complex with a truncated DAP12 domain. FIG. 6B depicts an exemplary IL21/TREM1/IL2RB cytokine receptor switch polypeptide construct (Pl) and an exemplary truncated DAP12 construct (P2). FIG. 6C depicts flow cytometry plots of Pl or Pl and P2 transduced T cells grown in the presence of IL2 or IL21.

[0047] FIG. 7 depicts an exemplary IL9/TREM1/IL2RB cytokine receptor switch polypeptide. FIG. 7A depicts an exemplary IL9/TREM1/IL2RB cytokine receptor switch polypeptide in complex with a DAP12 domain. FIG. 7B depicts an exemplary IL9/TREM1/IL2RB cytokine receptor switch polypeptide construct (Pl) and an exemplary truncated DAP12 construct (P2). FIG. 7C depicts the results of a cell count experiment of T cell counts following 14 days of primary T cell activation. Cell counts are shown for T cells transduced with Pl alone or Pl and P2 in the presence of IL-9 or IL-2. FIG. 7D depicts the % of double positive (Pl + P2) T cells in the presence of IL-9 or IL-2.

[0048] FIG. 8A depicts an exemplary polynucleotide encoding a cytokine receptor switch polypeptide and a chimeric antigen receptor separated by a sequence encoding a P2A cleavage site. FIG. 8B depicts an exemplary cytokine receptor switch polypeptide. FIG. 8C depicts an exemplary cytokine receptor switch polypeptide.

[0049] FIG. 9A depicts an exemplary first polynucleotide molecule encoding a cytokine receptor switch polypeptide and a first chimeric antigen receptor separated by a sequence encoding a P2A cleavage site and an exemplary second polynucleotide molecule encoding a second polypeptide containing a transmembrane domain and a second chimeric antigen receptor separated by a sequence encoding a P2A cleavage site. FIG. 9B depicts an exemplary cytokine receptor switch polypeptide expressed at the cellsurface when the second polypeptide is expressed in the same cell. FIG. 9C depicts an exemplary cytokine receptor switch polypeptide expressed at the cell-surface when the second polypeptide is expressed in the same cell (which forms a multimer with the cytokine receptor switch polypeptide).

[0050] FIG. 10A depicts an exemplary first polynucleotide molecule encoding a first polypeptide containing a cytokine receptor switch polypeptide and a first chimeric antigen receptor separated by a sequence encoding a P2A cleavage site, an exemplary second polynucleotide molecule encoding a second polypeptide containing a transmembrane domain and a second chimeric antigen receptor separated by a sequence encoding a P2A cleavage site, and an exemplary third polynucleotide molecule encoding a third polypeptide containing a transmembrane domain and a third chimeric antigen receptor separated by a sequence encoding a P2A cleavage site. FIG. 10B depicts an exemplary cytokine receptor switch polypeptide expressed at the cell-surface when the second polypeptide and the third polypeptide of FIG. 10A are expressed in the same cell (which form a multimer with the cytokine receptor switch polypeptide). FIG. IOC depicts an exemplary cytokine receptor switch polypeptide expressed at the cell-surface when the second polypeptide of FIG. 10A is expressed in the same cell.

[0051] FIG. 11A depicts an exemplary first polynucleotide molecule encoding a first polypeptide containing cytokine receptor switch polypeptide with a protease cleavage site and a retention tag and a first chimeric antigen receptor separated by a sequence encoding a P2A cleavage site, and an exemplary second polynucleotide molecule encoding second polypeptide containing a protease and a second chimeric antigen receptor separated by a sequence encoding a P2A cleavage site. FIG. 11B depicts an exemplary cytokine receptor switch polypeptide expressed at the cell-surface when the protease is active and expressed in the same cell. As depicted, the protease cleaves the cytokine receptor switch polypeptide at the protease cleavage site in the ER or Golgi, releasing the retention tag and promoting cell-surface expression of the cytokine receptor switch polypeptide. FIG. 11C depicts an exemplary cytokine receptor switch polypeptide expressed at the cell-surface when the protease is active and expressed in the same cell. As depicted, the protease cleaves the cytokine receptor switch polypeptide at the protease cleavage site in the ER or Golgi, releasing the retention tag and promoting cell-surface expression of the cytokine receptor switch polypeptide.

[0052] FIG. 12A depicts an exemplary first polynucleotide molecule encoding a cytokine receptor switch polypeptide with a protease cleavage site and a retention tag and a first chimeric antigen receptor separated by a sequence encoding a P2A cleavage site, an exemplary second polynucleotide molecule encoding a C-terminal portion of a protease and a second chimeric antigen receptor separated by a sequence encoding a P2A cleavage site, and an exemplary third polynucleotide molecule encoding a N-terminal portion of the protease and a third chimeric antigen receptor separated by a sequence encoding a P2A cleavage site. FIG. 12B depicts an exemplary cytokine receptor switch polypeptide expressed at the cell-surface when the C-terminal portion of the protease and the N-terminal portion of the protease are expressed in the same cell and form a functional protease. As depicted, the C-terminal portion and N-terminal portion of the protease come together to form a functional protease that cleaves the cytokine receptor switch polypeptide at the protease cleavage site in the ER or Golgi, releasing the retention tag and promoting cell-surface expression of the cytokine receptor switch polypeptide. FIG. 12C depicts an exemplary cytokine receptor switch polypeptide expressed at the cell-surface when the C-terminal portion of the protease and the N- terminal portion of the protease are expressed in the same cell and form a functional protease. As depicted, the C-terminal portion and N-terminal portion of the protease come together to form a functional protease that cleaves the cytokine receptor switch polypeptide at the protease cleavage site in the ER or Golgi, releasing the retention tag and promoting cell-surface expression of the cytokine receptor switch polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

[0053] 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 cytokine receptor switch polypeptide comprising 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. Definitions

[0054] 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.” [0055] 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.

[0056] 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.

[0057] ‘ ‘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.

[0058] ‘ ‘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.

[0059] 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 nonlimiting 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.

[0060] 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.

[0061] The term “binding domain”, as used herein, refers to a protein, or polypeptide sequence, which specifically binds to a target.

[0062] 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 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 immunoglobulin 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 immunoglobulin binding functionality of its target.

[0063] 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.

[0064] 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 linker and/or a spacer.

[0065] As used herein, “specifically binds” means that the binding domain preferentially binds the corresponding target over other proteins. In some instances, “specifically binds” means that the binding domains have a higher affinity for the target than for other proteins. In some instances, the binding domain is a cytokine binding domain and the target is the corresponding cytokine.

[0066] As used herein, a “therapeutically effective amount” of an agent is an amount 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.

[0067] As used herein, the term “treat,” “treating” or “treatment” of any disease or disorder refers, in one instance, to ameliorating the disease or disorder (z. 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. [0068] 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.

Cytokine Receptor Switch Polypeptides

[0069] In some aspects, the present disclosure describes a cytokine 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.

[0070] The extracellular domain of the cytokine receptor switch polypeptide functions to receive the cytokine signal outside of a cell by specifically binding to the cytokine. In some instances, the extracellular domain comprises a cytokine binding domain from a first cytokine receptor. In some instances, the first cytokine receptor is an interleukin 21 (IL21) receptor. In some embodiments, the first cytokine receptor is an interleukin 9 (IL9) receptor. The cytokine binding domain from the first cytokine receptor can specifically bind to the first cytokine. In some instances, the cytokine binding domain from the first cytokine receptor specifically binds to IL21. In some instances, the cytokine binding domain from the first cytokine receptor comprises an amino acid sequence of SEQ ID NO: 7. In some instances, the cytokine binding domain from the first cytokine receptor consists of an amino acid sequence of SEQ ID NO: 7. In some instances, the cytokine binding domain from the first cytokine receptor comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 7. In some instances, the cytokine binding domain from the first cytokine receptor comprises an amino acid sequence with at least 96% identity to SEQ ID NO: 7. In some instances, the cytokine binding domain from the first cytokine receptor comprises an amino acid sequence with at least 97% identity to SEQ ID NO: 7. In some instances, the cytokine binding domain from the first cytokine receptor comprises an amino acid sequence with at least 98% identity to SEQ ID NO: 7. In some instances, the cytokine binding domain from the first cytokine receptor comprises an amino acid sequence with at least 99% identity to SEQ ID NO: 7. In some instances, the cytokine binding domain from the first cytokine receptor comprises an amino acid sequence with at least 99.5% identity to SEQ ID NO: 7. In some instances, the cytokine binding domain from the first cytokine receptor comprises an amino acid sequence with at least 99.9% identity to SEQ ID NO: 7. In some instances, the cytokine binding domain from the first cytokine receptor consists of an amino acid sequence with at least 95% identity to SEQ ID NO: 7. In some instances, the cytokine binding domain from the first cytokine receptor consists of an amino acid sequence with at least 96% identity to SEQ ID NO: 7. In some instances, the cytokine binding domain from the first cytokine receptor consists of an amino acid sequence with at least 97% identity to SEQ ID NO: 7. In some instances, the cytokine binding domain from the first cytokine receptor consists of an amino acid sequence with at least 98% identity to SEQ ID NO: 7. In some instances, the cytokine binding domain from the first cytokine receptor consists of an amino acid sequence with at least 99% identity to SEQ ID NO: 7. In some instances, the cytokine binding domain from the first cytokine receptor consists of an amino acid sequence with at least 99.5% identity to SEQ ID NO: 7. In some instances, the cytokine binding domain from the first cytokine receptor consists of an amino acid sequence with at least 99.9% identity to SEQ ID NO: 7.

[0071] The extracellular domain of the cytokine receptor switch polypeptide is operably connected to the transmembrane domain. In some instances, the extracellular domain is connected to the transmembrane domain by a spacer. The transmembrane domain of the cytokine receptor switch polypeptide serves to transduce the cytokine signal received by the extracellular domain to the intracellular domain. 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 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 cytokine receptor switch polypeptide. A glycine-serine doublet provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain. In some instances, the transmembrane domain is an IL21 receptor transmembrane domain. In some instances, the transmembrane domain is an IL7 receptor transmembrane domain. In some instances, the transmembrane domain is an IL2 receptor beta transmembrane domain. In some instances, the transmembrane domain is a TREM1 transmembrane domain. In some instances, the transmembrane domain is a DAP12 transmembrane domain.

[0072] In some instances, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 8. In some instances, the transmembrane domain comprises an amino acid sequence with least 90% identity to SEQ ID NO: 8. In some instances, the transmembrane domain comprises an amino acid sequence with least 95% identity to SEQ ID NO: 8. In some instances, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 8. In some instances, the transmembrane domain consists of an amino acid sequence with least 90% identity to SEQ ID NO: 8. In some instances, the transmembrane domain consists of an amino acid sequence with least 95% identity to SEQ ID NO: 8. In some instances, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 9. In some instances, the transmembrane domain comprises an amino acid sequence with least 90% identity to SEQ ID NO: 9. In some instances, the transmembrane domain comprises an amino acid sequence with least 95% identity to SEQ ID NO: 9. In some instances, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 9. In some instances, the transmembrane domain consists of an amino acid sequence with least 90% identity to SEQ ID NO: 9. In some instances, the transmembrane domain consists of an amino acid sequence with least 95% identity to SEQ ID NO: 9. In some instances, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 10. In some instances, the transmembrane domain comprises an amino acid sequence with least 90% identity to SEQ ID NO: 10. In some instances, the transmembrane domain comprises an amino acid sequence with least 95% identity to SEQ ID NO: 10. In some instances, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 10. In some instances, the transmembrane domain consists of an amino acid sequence with least 90% identity to SEQ ID NO: 10. In some instances, the transmembrane domain consists of an amino acid sequence with least 95% identity to SEQ ID NO: 10. In some instances, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 11. In some instances, the transmembrane domain comprises an amino acid sequence with least 90% identity to SEQ ID NO: 11. In some instances, the transmembrane domain comprises an amino acid sequence with least 95% identity to SEQ ID NO: 11. In some instances, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 11. In some instances, the transmembrane domain consists of an amino acid sequence with least 90% identity to SEQ ID NO: 11. In some instances, the transmembrane domain consists of an amino acid sequence with least 95% identity to SEQ ID NO: 11. In some instances, the transmembrane domain comprises an amino acid sequence with least 90% identity to SEQ ID NO: 51. In some instances, the transmembrane domain comprises an amino acid sequence with least 95% identity to SEQ ID NO: 51. In some instances, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 51. In some instances, the transmembrane domain consists of an amino acid sequence with least 90% identity to SEQ ID NO: 51. In some instances, the transmembrane domain consists of an amino acid sequence with least 95% identity to SEQ ID NO: 51. In some instances, the transmembrane domain comprises an amino acid sequence with least 90% identity to SEQ ID NO: 52. In some instances, the transmembrane domain comprises an amino acid sequence with least 95% identity to SEQ ID NO: 52. In some instances, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 52. In some instances, the transmembrane domain consists of an amino acid sequence with least 90% identity to SEQ ID NO: 52. In some instances, the transmembrane domain consists of an amino acid sequence with least 95% identity to SEQ ID NO: 52.

[0073] The transmembrane domain of the cytokine receptor switch polypeptide is operably connected to the intracellular domain. The intracellular domain serves to transduce the received cytokine signal to activate the downstream signaling cascade. 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 instances, the first cytokine receptor is an IL21 receptor and the second cytokine receptor is a cytokine receptor other than an IL21 receptor. In some instances, the intracellular signaling domain from the second cytokine receptor induces a downstream signaling that enhances cytotoxicity, promotes stem memory T cells (T _S cm) formation, and/or promotes central memory T cells (Tcm) formation. In some instances, the intracellular signaling domain from the second cytokine receptor induces STAT5 signaling. In some instances, the second cytokine receptor is IL2 receptor beta. In some instances, the second cytokine receptor is IL7 receptor. In some instances, the second cytokine receptor is IL9 receptor.

[0074] In some instances, the intracellular signaling domain that induces STAT5 signaling comprises an intracellular signaling domain from IL2, IL4, IL7, IL13, IL15, thymic stromal lymphopoietin (TSLP), granulocyte-macrophage colony-stimulating factor (GM-CSF), or IL27.

[0075] In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence of SEQ ID NO: 12. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence of SEQ ID NO: 12. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 12. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 96% identity to SEQ ID NO: 12. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 97% identity to SEQ ID NO: 12. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 98% identity to SEQ ID NO: 12. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 99% identity to SEQ ID NO: 12. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 99.5% identity to SEQ ID NO: 12. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 99.9% identity to SEQ ID NO: 12. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 95% identity to SEQ ID NO: 12. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 96% identity to SEQ ID NO: 12. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 97% identity to SEQ ID NO: 12. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 98% identity to SEQ ID NO: 12. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 99% identity to SEQ ID NO: 12. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 99.5% identity to SEQ ID NO: 12. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 99.9% identity to SEQ ID NO: 12.

[0076] In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence of SEQ ID NO: 13. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence of SEQ ID NO: 13. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 13. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 96% identity to SEQ ID NO:

13. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 97% identity to SEQ ID NO: 13. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 98% identity to SEQ ID NO: 13. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 99% identity to SEQ ID NO: 13. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 99.5% identity to SEQ ID NO: 13. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 99.9% identity to SEQ ID NO: 13. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 95% identity to SEQ ID NO: 13. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 96% identity to SEQ ID NO: 13. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 97% identity to SEQ ID NO: 13. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 98% identity to SEQ ID NO: 13. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 99% identity to SEQ ID NO: 13. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 99.5% identity to SEQ ID NO: 13. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 99.9% identity to SEQ ID NO: 13.

[0077] In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence of SEQ ID NO: 14. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence of SEQ ID NO: 14. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 14. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 96% identity to SEQ ID NO: 14. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 97% identity to SEQ ID NO: 14. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 98% identity to SEQ ID NO: 14. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 99% identity to SEQ ID NO: 14. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 99.5% identity to SEQ ID NO: 14. In some instances, the intracellular signaling domain from the second cytokine receptor comprises an amino acid sequence with at least 99.9% identity to SEQ ID NO: 14. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 95% identity to SEQ ID NO: 14. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 96% identity to SEQ ID NO: 14. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 97% identity to SEQ ID NO: 14. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 98% identity to SEQ ID NO: 14. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 99% identity to SEQ ID NO: 14. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 99.5% identity to SEQ ID NO: 13. In some instances, the intracellular signaling domain from the second cytokine receptor consists of an amino acid sequence with at least 99.9% identity to SEQ ID NO: 14.

[0078] In some instances, the cytokine receptor switch polypeptide further comprises a protein localization tag. The protein localization tag can be operably linked to the intracellular domain of the cytokine receptor switch polypeptide. The protein localization tag can be operably linked to the extracellular domain of the cytokine receptor switch polypeptide. The term “protein localization tag,” as used herein, refers to a specific amino acid sequence (i.e., “a signal peptide”) that can be recognized by a corresponding signal receptor and thus guide the signal peptide and the protein connected to the signal peptide to the target site, usually a subcellular compartment such as cell membrane, nucleus, lysosomes, endoplasmic reticulum (ER), ribosomes, Golgi apparatus, and mitochondria. Such signal peptides include but not limited to nuclear localization signal and ER localization signal. 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 cytokine receptor switch polypeptide further comprises a protein retention tag, e.g. , an ER retention tag, a Golgi apparatus (Golgi) retention tag, a lysosome retention tag, a plasma membrane retention tag, a mitochondria retention tag, a peroxisome retention tag, a cytosolic retention tag, or a nuclear retention tag. In some instances, the protein retention tag is an ER retention tag. In some instances, the ER retention tag comprises an amino acid sequence of SEQ ID NO: 23. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 23. In some instances, the ER retention tag comprises an amino acid sequence of SEQ ID NO: 24. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 24. In some instances, the ER retention tag comprises an amino acid sequence of SEQ ID NO: 25. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 25. In some instances, the ER retention tag comprises an amino acid sequence of SEQ ID NO: 26. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 26. In some instances, the ER retention tag comprises an amino acid sequence of SEQ ID NO: 27. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 27. In some instances, the ER retention tag comprises an amino acid sequence of SEQ ID NO: 28. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 28. In some instances, the ER retention tag comprises an amino acid sequence of SEQ ID NO: 29. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 29. In some instances, the ER retention tag comprises an amino acid sequence of SEQ ID NO: 30. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 30. In some instances, the ER retention 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 SEQ ID NO: 23. In some instances, the ER retention 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 to SEQ ID NO: 23. In some instances, the ER retention 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 SEQ ID NO: 24. In some instances, the ER retention 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 to SEQ ID NO: 24. In some instances, the ER retention 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 SEQ ID NO: 25. In some instances, the ER retention 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 to SEQ ID NO: 25. In some instances, the ER retention 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 SEQ ID NO: 25. In some instances, the ER retention 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 to SEQ ID NO: 25. In some instances, the ER retention 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 SEQ ID NO: 26. In some instances, the ER retention 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 to SEQ ID NO: 26. In some instances, the ER retention 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 SEQ ID NO: 27. In some instances, the ER retention 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 to SEQ ID NO: 27. In some instances, the ER retention 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 SEQ ID NO: 28. In some instances, the ER retention 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 to SEQ ID NO: 28. In some instances, the ER retention 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 SEQ ID NO: 29. In some instances, the ER retention 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 to SEQ ID NO: 29. In some instances, the ER retention 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 SEQ ID NO: 30. In some instances, the ER retention 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 to SEQ ID NO: 30.

[0079] In some instances, the ER retention tag comprising an amino acid sequence KHILFRRRRRGFRQ (SEQ ID NO:30). In some instances, the ER retention 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 SEQ ID NO: 30. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 30. In some instances, the ER retention 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 to SEQ ID NO: 30.

[0080] In some instances, the ER retention tag comprising an amino acid sequence LYKYKSRRSFIDEKKMP (SEQ ID NO: 40). In some instances, the ER retention 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 SEQ ID NO: 40. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 40. In some instances, the ER retention 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 to SEQ ID NO: 40. In some instances, the ER retention tag comprises the amino acid sequence KKMP (SEQ ID NO: 41). In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 41. [0081] In some instances, the protein retention tag is a Golgi retention tag. In some instances, the Golgi retention tag comprises the amino acid sequence YQRL (SEQ ID NO: 38). In some instances, the Golgi retention tag consists of the amino acid sequence YQRL (SEQ ID NO: 38). In some instances, the protein retention tag is a lysosome retention tag. In some instances, the lysosome retention tag comprises the amino acid sequence KFERQ (SEQ ID NO: 39). In some instances, the lysosome retention tag consists of the amino acid sequence KFERQ (SEQ ID NO: 39).

[0082] In some instances, a protease cleavage site is disposed between the protein retention tag and the cytokine receptor switch peptide. In some instances, the protease cleavage site is disposed between the protein retention tag and the intracellular domain of the cytokine receptor switch polypeptide. In some instances, the protease cleavage site is disposed between the protein retention tag and the extracellular domain of the cytokine 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 instances, 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 and/or cleaved by the protease.

[0083] 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 subtilisin-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 instances, the protease cleavage site is TEV protease cleavage site.

[0084] In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL21 receptor, and an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL21 receptor, and an intracellular domain comprising an intracellular signaling domain from IL7 receptor. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL21 receptor, and an intracellular domain comprising an intracellular signaling domain from IL9 receptor. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL7 receptor, and an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL7 receptor, and an intracellular domain comprising an intracellular signaling domain from IL7 receptor. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL7 receptor, and an intracellular domain comprising an intracellular signaling domain from IL9 receptor. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL2 receptor beta, a transmembrane domain from IL2 receptor beta, and an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL2 receptor beta, and an intracellular domain comprising an intracellular signaling domain from IL7 receptor. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL2 receptor beta, and an intracellular domain comprising an intracellular signaling domain from IL9 receptor. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from GDP Glucuronosyltransferase Family 2 Member B17 (UGT2B17), and an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from UGT2B17, and an intracellular domain comprising an intracellular signaling domain from IL7 receptor. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from UGT2B17, and an intracellular domain comprising an intracellular signaling domain from IL9 receptor.

[0085] In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL21 receptor, an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and an ER retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL21 receptor, an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and a Golgi retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL21 receptor, an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and a lysosome retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL21 receptor, an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and an ER retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL21 receptor, an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and a Golgi retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL21 receptor, an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and a lysosome retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL21 receptor, an intracellular domain comprising an intracellular signaling domain from IL7 receptor, and an ER retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL21 receptor, an intracellular domain comprising an intracellular signaling domain from IL7 receptor, and a Golgi retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL21 receptor, an intracellular domain comprising an intracellular signaling domain from IL7 receptor, and a lysosome retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL21 receptor, an intracellular domain comprising an intracellular signaling domain from IL9 receptor, and an ER retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL21 receptor, an intracellular domain comprising an intracellular signaling domain from IL9 receptor, and a Golgi retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL21 receptor, an intracellular domain comprising an intracellular signaling domain from IL9 receptor, and a lysosome retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL7 receptor, an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and an ER retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL7 receptor, an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and a Golgi retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL7 receptor, an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and a lysosome retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL7 receptor, an intracellular domain comprising an intracellular signaling domain from IL7 receptor, and an ER retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL7 receptor, an intracellular domain comprising an intracellular signaling domain from IL7 receptor, and a Golgi retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL7 receptor, an intracellular domain comprising an intracellular signaling domain from IL7 receptor, and a lysosome retention tag.

[0086] In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL7 receptor, an intracellular domain comprising an intracellular signaling domain from IL9 receptor, and an ER retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL7 receptor, an intracellular domain comprising an intracellular signaling domain from IL9 receptor, and a Golgi retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL7 receptor, an intracellular domain comprising an intracellular signaling domain from IL9 receptor, and a lysosome retention tag.

[0087] In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL2 receptor beta, a transmembrane domain from IL2 receptor beta, an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and an ER retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL2 receptor beta, a transmembrane domain from IL2 receptor beta, an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and a Golgi retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL2 receptor beta, a transmembrane domain from IL2 receptor beta, an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and a lysosome retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL2 receptor beta, an intracellular domain comprising an intracellular signaling domain from IL7 receptor, and an ER retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL2 receptor beta, an intracellular domain comprising an intracellular signaling domain from IL7 receptor, and a Golgi retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL2 receptor beta, an intracellular domain comprising an intracellular signaling domain from IL7 receptor, and a lysosome retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL2 receptor beta, an intracellular domain comprising an intracellular signaling domain from IL9 receptor, and an ER retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL2 receptor beta, an intracellular domain comprising an intracellular signaling domain from IL7 receptor, and a Golgi retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from IL2 receptor beta, an intracellular domain comprising an intracellular signaling domain from IL7 receptor, and a lysosome retention tag.

[0088] In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from TREM 1, an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and an ER retention tag. [0089] In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL9 receptor, a transmembrane domain from TREM 1 , an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and an ER retention tag.

[0090] In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from UDP Glucuronosyltransferase Family 2 Member Bl 7 (UGT2B17), an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and an ER retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from UGT2B 17, an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and a Golgi retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from UGT2B 17, an intracellular domain comprising an intracellular signaling domain from IL2 receptor beta, and a lysosome retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from UGT2B 17, an intracellular domain comprising an intracellular signaling domain from IL7 receptor, and an ER retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from UGT2B 17, an intracellular domain comprising an intracellular signaling domain from IL7 receptor, and a Golgi retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from UGT2B 17, an intracellular domain comprising an intracellular signaling domain from IL7 receptor, and a lysosome retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from UGT2B 17, an intracellular domain comprising an intracellular signaling domain from IL9 receptor, and an ER retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from UGT2B 17, an intracellular domain comprising an intracellular signaling domain from IL9 receptor, and a Golgi retention tag. In some instances, the cytokine receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from IL21 receptor, a transmembrane domain from UGT2B 17, an intracellular domain comprising an intracellular signaling domain from IL9 receptor, and a lysosome retention tag. [0091] In some instances, the cytokine receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 2. In some instances, the cytokine receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 3. In some instances, the cytokine receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 4. In some instances, the cytokine receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 5. In some instances, the cytokine receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 6. In some instances, the cytokine 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% identity to SEQ ID NO: 2. In some instances, the cytokine 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% identity to SEQ ID NO: 3. In some instances, the cytokine 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% identity to SEQ ID NO: 4. In some instances, the cytokine 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% identity to SEQ ID NO: 5. In some instances, the cytokine 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% identity to SEQ ID NO: 6.

[0092] In some instances, the cytokine receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 15. In some instances, the cytokine receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 16. In some instances, the cytokine receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 17. In some instances, the cytokine receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 18. In some instances, the cytokine receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 19. In some instances, the cytokine receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 20. In some instances, the cytokine receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 21. In some instances, the cytokine receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 22. In some instances, the cytokine 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% identity to SEQ ID NO: 15. In some instances, the cytokine 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% identity to SEQ ID NO: 16. In some instances, the cytokine 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% identity to SEQ ID NO: 17. In some instances, the cytokine 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% identity to SEQ ID NO: 18. In some instances, the cytokine 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% identity to SEQ ID NO: 19. In some instances, the cytokine 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% identity to SEQ ID NO: 20. In some instances, the cytokine 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% identity to SEQ ID NO: 21. In some instances, the cytokine 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% identity to SEQ ID NO: 22.

[0093] In some embodiments, the cytokine receptor switch polypeptide comprises an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In some embodiments, the cytokine receptor switch polypeptide consists of an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In some instances, the cytokine receptor switch polypeptide comprises an amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22. In some instances, the cytokine receptor switch polypeptide consists of an amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 50. In some instances, the cytokine receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to a sequence in Table 1.

Polynucleotides [0094] In some aspects, the present disclosure describes polynucleic acid sequences encoding any of the cytokine receptor switch polypeptides disclosed herein.

[0095] In some instances, the recombinant polynucleic acid further comprises a sequence encoding a chimeric antigen receptor (CAR). 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 instances, 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 cytokine receptor switch peptide. The sequence encoding the CAR can be linked to the sequence encoding the cytokine receptor switch peptide by a linker comprising or consisting of a sequence encoding a selfcleaving peptide. In some instances, the linker is a P2A cleavable linker.

[0096] 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 instances, the antigen is an antigen expressed by a tumor cell. In some instances, the antigen binding domain comprises an scFv, a nanobody, a ligand, or a receptor.

[0097] 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. Bamhashemi 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).

[0098] In some instances, the antigen binding domain specifically binds to CD19. In some instances, 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 (SEQ ID NO: 53), SRLHSGV (SEQ ID NO: 54) and GNTLPYTFG (SEQ ID NO: 55), 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

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSG VPSRFSGS GSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT. 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 (SEQ ID NO: 56), VIWGSETTYYNSALKS (SEQ ID NO: 57) and YAMDYWG (SEQ ID NO: 58), 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

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALK SRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 59).

[0099] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSG VPSRFSGS GSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVK LQES GPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL TIIKD NSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 33).

[0100] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALK SRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGG SG GGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHT SRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 60). [0101] In some instances, the antigen binding domain comprises an amino acid sequence of SEQ ID NO:

32. In some instances, the antigen binding domain comprises an amino acid sequence with at least 85% identity to SEQ ID NO: 32. In some instances, the antigen binding domain comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 32. In some instances, the antigen binding domain comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 32. In some instances, the antigen binding domain comprises an amino acid sequence with at least 96% identity to SEQ ID NO: 32. In some instances, the antigen binding domain comprises an amino acid sequence with at least 97% identity to SEQ ID NO: 32. In some instances, the antigen binding domain comprises an amino acid sequence with at least 98% identity to SEQ ID NO: 32. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99% identity to SEQ ID NO: 32. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99.5% identity to SEQ ID NO:

32. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99.9% identity to SEQ ID NO: 32. In some instances, the antigen binding domain consists of an amino acid sequence of SEQ ID NO: 32. In some instances, the antigen binding domain consists of an amino acid sequence with at least 85% identity to SEQ ID NO: 32. In some instances, the antigen binding domain consists of an amino acid sequence with at least 90% identity to SEQ ID NO: 32. In some instances, the antigen binding domain consists of an amino acid sequence with at least 95% identity to SEQ ID NO: 32. In some instances, the antigen binding domain consists of an amino acid sequence with at least 96% identity to SEQ ID NO: 32. In some instances, the antigen binding domain consists of an amino acid sequence with at least 97% identity to SEQ ID NO: 32. In some instances, the antigen binding domain consists of an amino acid sequence with at least 98% identity to SEQ ID NO: 32. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99% identity to SEQ ID NO: 32. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99.5% identity to SEQ ID NO: 32. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity to SEQ ID NO: 32.

[0102] In some instances, the antigen binding domain comprises an amino acid sequence of SEQ ID NO:

33. In some instances, the antigen binding domain comprises an amino acid sequence with at least 85% identity to SEQ ID NO: 33. In some instances, the antigen binding domain comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 33. In some instances, the antigen binding domain comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 33. In some instances, the antigen binding domain comprises an amino acid sequence with at least 96% identity to SEQ ID NO: 33. In some instances, the antigen binding domain comprises an amino acid sequence with at least 97% identity to SEQ ID NO: 33. In some instances, the antigen binding domain comprises an amino acid sequence with at least 98% identity to SEQ ID NO: 33. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99% identity to SEQ ID NO: 33. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99.5% identity to SEQ ID NO: 33. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99.9% identity to SEQ ID NO: 33. In some instances, the antigen binding domain consists of an amino acid sequence of SEQ ID NO: 33. In some instances, the antigen binding domain consists of an amino acid sequence with at least 85% identity to SEQ ID NO: 33. In some instances, the antigen binding domain consists of an amino acid sequence with at least 90% identity to SEQ ID NO: 33. In some instances, the antigen binding domain consists of an amino acid sequence with at least 95% identity to SEQ ID NO: 33. In some instances, the antigen binding domain consists of an amino acid sequence with at least 96% identity to SEQ ID NO: 33. In some instances, the antigen binding domain consists of an amino acid sequence with at least 97% identity to SEQ ID NO: 33. In some instances, the antigen binding domain consists of an amino acid sequence with at least 98% identity to SEQ ID NO: 33. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99% identity to SEQ ID NO: 33. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99.5% identity to SEQ ID NO: 33. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity to SEQ ID NO: 33.

[0103] In some instances, the antigen binding domain specifically binds to CD22. In some instances, the antigen binding domain is an anti-CD22 binding domain. In some embodiments, the antigen binding domain is a mouse anti -human CD22 binding domain. In some embodiments, the antigen binding domain is a chimeric mouse anti-human CD22 binding domain. In some embodiments, the antigen binding domain is a humanized anti-human CD22 binding domain. In some embodiments, the antigen binding domain is a fully human anti-human CD22 binding domain. In some embodiments, the antigen binding domain is the m971 antibody.

[0104] 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 (SEQ ID NO: 61). AAS and QOSYSfPQT (SEQ ID NO: 62). 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

DIOMTOSPSSLSASVGDRVTITCRASOTIWSYLNWYOORPGKAPNLLIYAASSLOSG VPSRFSGR GSGTDFTLTISSLOAEDFATYYCOQSYSIPOTFGOGTKLEI (SEQ ID NO: 63). [0105] 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 (SEQ ID NO: 64). TYYRSKWYN (SEQ ID NO: 65) and AREVTGDLEDAFDI (SEQ ID NO: 66). 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 OVOLOOSGPGLVKPSOTLSLTCAISGDSVSSNSAAWNWIROSPSRGLEWLGRTYYRSKWY NDY AVSVKSRITINPDTSKNOFSLOLNSVTPEDTAVYYCAREVTGDLEDAFDIWGOGTMVTVS S (SEQ ID NO: 67).

[0106] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to

OVOLOOSGPGLVKPSOTLSLTCAISGDSVSSNSAAWNWIROSPSRGLEWLGRTYYRS KWYNDY AVSVKSRITINPDTSKNOFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGOGTMVTVS SGG GGSDIOMTOSPSSLSASVGDRVTITCRASOTIWSYLNWYOORPGKAPNLLIYAASSLOSG VPSRF SGRGSGTDFTLTISSLOAEDFATYYCOQSYSIPOTFGOGTKLEIK (SEQ ID NO: 34).

[0107] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to

DIOMTOSPSSLSASVGDRVTITCRASOTIWSYLNWYOORPGKAPNLLIYAASSLOSG VPSRFSGR GSGTDFTLTISSLOAEDFATYYCOOSYSIPOTFGOGTKLEIKGGGGSOVOLOOSGPGLVK PSOTL SLTCAIS GDSVSSNSAA WNWIROSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNOF SLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGOGTMVTVSS. In some embodiments, the antigen binding domain comprises an scFv with at least about 85, 90, 95, 97, 98, or 99% sequence identity to OVOLOOSGPGLVKPSOTLSLTCAISGDSVSSNSAAWNWIROSPSRGLEWLGRTYYRSKWY NDY AVSVKSRITINPDTSKNOFSLOLNSVTPEDTAVYYCAREVTGDLEDAFDIWGOGTMVTVS SGG GGSDIOMTOSPSSLSASVGDRVTITCRASOTIWSYLNWYOORPGKAPNLLIYAASSLOSG VPSRF SGRGSGTDFTLTISSLOAEDFATYYCOOSYSIPOTFGOGTKLEIK (SEQ ID NO: 34). In some embodiments, the antigen binding domain comprises an scFv with 100% sequence identity to OVOLOOSGPGLVKPSOTLSLTCAISGDSVSSNSAAWNWIROSPSRGLEWLGRTYYRSKWY NDY AVSVKSRITINPDTSKNOFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGOGTMVTVS SGG GGSDIOMTOSPSSLSASVGDRVTITCRASOTIWSYLNWYOORPGKAPNLLIYAASSLOSG VPSRF SGRGSGTDFTLTISSLOAEDFATYYCOQSYSIPOTFGOGTKLEIK (SEQ ID NO: 34).

[0108] In some instances, the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 34. In some instances, the antigen binding domain comprises an amino acid sequence with at least 85% identity to SEQ ID NO: 34. In some instances, the antigen binding domain comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 34. In some instances, the antigen binding domain comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 34. In some instances, the antigen binding domain comprises an amino acid sequence with at least 96% identity to SEQ ID NO: 34. In some instances, the antigen binding domain comprises an amino acid sequence with at least 97% identity to SEQ ID NO: 34. In some instances, the antigen binding domain comprises an amino acid sequence with at least 98% identity to SEQ ID NO: 34. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99% identity to SEQ ID NO: 34. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99.5% identity to SEQ ID NO: 34. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99.9% identity to SEQ ID NO: 34. In some instances, the antigen binding domain consists of an amino acid sequence of SEQ ID NO: 34. In some instances, the antigen binding domain consists of an amino acid sequence with at least 85% identity to SEQ ID NO: 34. In some instances, the antigen binding domain consists of an amino acid sequence with at least 90% identity to SEQ ID NO: 34. In some instances, the antigen binding domain consists of an amino acid sequence with at least 95% identity to SEQ ID NO: 34. In some instances, the antigen binding domain consists of an amino acid sequence with at least 96% identity to SEQ ID NO: 34. In some instances, the antigen binding domain consists of an amino acid sequence with at least 97% identity to SEQ ID NO: 34. In some instances, the antigen binding domain consists of an amino acid sequence with at least 98% identity to SEQ ID NO: 34. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99% identity to SEQ ID NO: 34. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99.5% identity to SEQ ID NO: 34. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity to SEQ ID NO: 34.

[0109] In some instances, 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, 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, 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, M0V18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD 19, CD20, CD22, R0R1, and GD2, or a mixture thereof.

[0110] 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 (SEQ ID NO: 68). 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 (SEQ ID NO: 69).

[OHl] 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.

[0112] 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.

[0113] 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 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.

[0114] 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.

[0115] Alternatively, the spacer domain can be a synthetic polypeptide spacer, such as a spacer having a random sequence, a (gly-gly-ser)n (“GGS _n ”) sequence, or a variation thereof such as (SGG) _n , (GGGS) _n , (SGGG) _n , (GSGGG) _n , and the like, where n is an integer ranging 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.

[0116] The extracellular domain of the CAR is operably connected to the transmembrane domain. In some instances, 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, CD8 hinge/transmembrane domain (CD8H), 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.

[0117] 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, CD35, CD3E, CD3 , CD35, CD22, CD79a, CD79, and CD665. Such a CAR may further include a spacer domain between the antigen-binding 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 (SEQ ID NO: 70). In some embodiments, the CAR comprises a transmembrane domain from CD8. In some embodiments, the CAR comprises a transmembrane domain with the sequence IWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 71).

[0118] 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, CD3 , CD4, CD5, CD8a, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and/or PD-1.

[0119] The transmembrane domain may include, for example without limitation, all or part of the transmembrane domain of the CD3 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 CD3^ 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 CD3 , 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 CD3 , CD28, CD2, CD4, 0X40, 4-1BB (CD137), FcERIy, ICOS (CD278), ILRB (CD122), IL-2RG (CD132), or CD40 transmembrane domain.

[0120] 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.

[0121] 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 instances, the intracellular domain comprises an intracellular signaling domain from 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), DAP10, LAT, KD2C, SLP76, TRIM, and/or ZAP70.

[0122] 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 (SEQ ID NO: 72).

[0123] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3 . In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN EL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 73). In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 74). [0124] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3 and an intracellular signaling domain from 4- IBB (CD 137).

[0125] 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 (SEQ ID NO: 75). In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence PATSQHPPPPPGHRSQAPSHRPPPPGHRVQH (SEQ ID NO: 76).

[0126] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3 epsilon. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence RPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 77). In some embodiments, the CAR comprises an intracellular domain comprising a truncated CD3 epsilon intracellular domain.

[0127] 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. [0128] 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, CD33, CD3s, CD3 , CD35, CD22, CD79a, CD79b, and CD665. In certain embodiments, a CAR includes a CD3 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.

[0129] 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), DAP10, 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 CD 134, and a CD3^ 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 CD3^ 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 CD3^ 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 CD3^ 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 CD3 intracellular signaling domain, a second intracellular signaling domain or fragment thereof that is a 4- IBB intracellular signaling domain, and a third intracellular signaling domain or fragment thereof that is a CD3 epsilon intracellular signaling domain.

[0130] CARs of the disclosure may comprise a CD3 , 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- IBB), CD278 (ICOS), DAP 10, 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, 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. 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 (CD150), CD58, ikaros, CD53, integnn a4, CD82, integnn a4bl, CD90, integnn 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, integnn a4, CD82, integral a4bl, CD90, integnn a4b7, CD96, LAG-3, CD160, LMIR, CRTAM, TCL1A, DAP12; TIM-1, Dectin-1, TIM-4, TSLP, EphB6, TSLP-R, and/or HLA-DR domains.

[0131] In some instances, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD28H hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD8H hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD28H hinge/transmembrane domain, and an intracellular domain comprising a 4- IBB (CD 137) intracellular signaling domain. In some instances, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD8H hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the CAR comprises an extracellular domain comprising an anti- CD22 binding domain, a CD28H hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD8H hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD28H hinge/transmembrane domain, and an intracellular domain comprising a 4- IBB (CD 137) intracellular signaling domain. In some instances, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD8H hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain.

[0132] In some instances, the CAR comprises an amino acid sequence of SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37. In some instances, the CAR comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 35. In some instances, the CAR comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 36. In some instances, the CAR comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 37. In some instances, the CAR consists of an amino acid sequence of SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37. In some instances, the CAR consists of an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 35. In some instances, the CAR consists of an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 36. In some instances, the CAR consists of an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to SEQ ID NO: 37.

[0133] In some instances, the CAR further comprises a protein retention tag. The protein retention tag can be operably linked to the intracellular domain of the CAR. The protein retention tag can be operably linked to the extracellular domain of the CAR. The protein retention tag can be an ER retention tag, a Golgi apparatus (Golgi) retention tag, a lysosome retention tag, a plasma membrane retention tag, a mitochondria retention tag, a peroxisome retention tag, a cytosolic retention tag, or a nuclear retention tag. In some instances, the protein retention tag is an ER retention tag. In some instances, the ER retention tag comprises an amino acid sequence of SEQ ID NO: 23. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 23. In some instances, the ER retention tag comprises an amino acid sequence of SEQ ID NO: 24. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 24. In some instances, the ER retention tag comprises an amino acid sequence of SEQ ID NO: 25. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 25. In some instances, the ER retention tag comprises an amino acid sequence of SEQ ID NO: 26. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 26. In some instances, the ER retention tag comprises an amino acid sequence of SEQ ID NO: 27. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 27. In some instances, the ER retention tag comprises an amino acid sequence of SEQ ID NO: 28. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 28. In some instances, the ER retention tag comprises an amino acid sequence of SEQ ID NO: 29. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 29. In some instances, the ER retention tag comprises an amino acid sequence of SEQ ID NO: 30. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 30. In some instances, the ER retention 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 SEQ ID NO: 23. In some instances, the ER retention 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 to SEQ ID NO: 23. In some instances, the ER retention 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 SEQ ID NO: 24. In some instances, the ER retention 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 to SEQ ID NO: 24. In some instances, the ER retention 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 SEQ ID NO: 25. In some instances, the ER retention 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 to SEQ ID NO:

25. In some instances, the ER retention 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 SEQ ID NO: 25. In some instances, the ER retention 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 to SEQ ID NO: 25. In some instances, the ER retention 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 SEQ ID NO: 26. In some instances, the ER retention 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 to SEQ ID NO:

26. In some instances, the ER retention 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 SEQ ID NO: 27. In some instances, the ER retention 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 to SEQ ID NO: 27. In some instances, the ER retention 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 SEQ ID NO: 28. In some instances, the ER retention 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 to SEQ ID NO: 28. In some instances, the ER retention 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 SEQ ID NO: 29. In some instances, the ER retention 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 to SEQ ID NO: 29. In some instances, the ER retention 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 SEQ ID NO: 30. In some instances, the ER retention 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 to SEQ ID NO: 30. [0134] In some instances, the ER retention tag comprising an amino acid sequence LYKYKSRRSFIDEKKMP (SEQ ID NO: 40). In some instances, the ER retention 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 SEQ ID NO: 40. In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 40. In some instances, the ER retention 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 to SEQ ID NO: 40. In some instances, the ER retention tag comprises the amino acid sequence KKMP (SEQ ID NO: 41). In some instances, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 41.

[0135] In some instances, the protein retention tag is a Golgi retention tag. In some instances, the Golgi retention tag comprises the amino acid sequence YQRL (SEQ ID NO: 38). In some instances, the Golgi retention tag consists of the amino acid sequence YQRL (SEQ ID NO: 38). In some instances, the protein retention tag is a lysosome retention tag. In some instances, the lysosome retention tag comprises the amino acid sequence KFERQ (SEQ ID NO: 39). In some instances, the lysosome retention tag consists of the amino acid sequence KFERQ (SEQ ID NO: 39).

[0136] In some instances, a protease cleavage site is disposed between the protein retention tag and the CAR. In some instances, the protease cleavage site is disposed between the protein retention tag and the intracellular domain of the CAR. In some instances, the protease cleavage site is disposed between the protein retention 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 instances, 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.

[0137] 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 subtilisin-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 instances, the protease cleavage site is TEV protease cleavage site. [0138] The sequence encoding the cytokine receptor switch polypeptide disclosed herein and the sequence encoding the CAR disclosed herein can be a single polynucleotide sequence. In some instances, the single polynucleotide sequence comprises a self-cleaving site (e.g., 2A peptides) separating the sequence encoding the cytokine receptor switch polypeptide and the sequence encoding the CAR. In some instances, the sequence encoding the cytokine receptor switch polypeptide and the sequence encoding the CAR are separated sequences. The sequence encoding the cytokine receptor switch polypeptide or the sequence encoding the CAR can be inserted to a vector for expression in a cell. In some instances, the sequence encoding the cytokine receptor switch polypeptide and the sequence encoding the CAR are inserted into the same vector. In some instances, the sequence encoding the cytokine receptor switch peptide and the sequence encoding the CAR are inserted into different vectors. In some instances, the vector comprising the sequence encoding the cytokine receptor switch polypeptide is different from the vector comprising the sequence encoding the CAR. In some instances, the vector comprising the sequence encoding the cytokine receptor switch polypeptide and the vector comprising the sequence encoding the CAR are the same.

[0139] In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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-CD19 binding domain, a CD28H or CD8H hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain.

[0140] In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. [0141] In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain.

[0142] In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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-CD19 binding domain, a CD28H or CD8H hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain.

[0143] In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain.

[0144] In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain.

[0145] In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain.

[0146] In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain.

[0147] In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain.

[0148] In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain.

[0149] In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain.

[0150] In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises (a) the sequence encoding the cytokine receptor switch peptide 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 hinge/transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain.

[0151] In some instances, the recombinant polynucleic acid comprises a first recombinant polynucleic acid molecule comprising a sequence encoding a first polypeptide comprising a cytokine receptor switch polypeptide, and a second recombinant polynucleic acid molecule comprising a sequence encoding a second polypeptide comprising a transmembrane domain that forms a dimer or a trimer with the cytokine receptor switch polypeptide. In some instances, cell-surface expression of the cytokine receptor switch polypeptide occurs when the second polypeptide is expressed in the same cell. In some instances, the extracellular domain of the cytokine receptor switch polypeptide is from IL21R or IL9R. In some instances, the transmembrane domain of the cytokine receptor switch polypeptide is from TREM1. In some instances, the intracellular domain of the cytokine receptor switch polypeptide comprises an intracellular signaling domain from IL2RB, IL7R, or IL9R. In some instances, the transmembrane domain of the second polypeptide is from DAP 12. In some instances, the first recombinant polynucleic acid molecule comprises a first CAR sequence encoding a first CAR and the second recombinant polynucleic acid molecule comprises a second CAR sequence encoding a second CAR. In some instances, the first CAR sequence encoding a first CAR is linked to the sequence encoding the cytokine receptor switch polypeptide by a sequence encoding a cleavable linker, such as a self-cleaving peptide, e.g., a P2A sequence. In some instances, the second CAR sequence encoding a second CAR is linked to the sequence encoding the second polypeptide by a sequence encoding a cleavable linker, such as a self-cleaving peptide, e.g., a P2A sequence.

[0152] In some instances, the recombinant polynucleic acid comprises a first recombinant polynucleic acid molecule comprising a sequence encoding a first polypeptide comprising a cytokine receptor switch polypeptide comprising a protease cleavage site and a retention tag, and a second recombinant polynucleic acid molecule comprising a sequence encoding a second polypeptide comprising a protease. In some instances, the protease cleavage site is disposed between the terminus of the intracellular domain of the cytokine receptor switch polypeptide and the retention tag. In some instances, the protease is fused to a transmembrane domain. In some instances, the protease is not fused to a transmembrane domain. In some instances, cell-surface expression of the cytokine receptor switch polypeptide occurs when the protease is expressed and active in the same cell. In some instances, the extracellular domain of the cytokine receptor switch polypeptide comprises an extracellular domain from IL21R or IL9R. In some instances, the intracellular domain of the cytokine receptor switch polypeptide comprises an intracellular signaling domain from IL2RB, IL7R, or IL9R. In some instances, the first recombinant polynucleic acid molecule comprises a first CAR sequence encoding a first CAR and the second recombinant polynucleic acid molecule comprises a second CAR sequence encoding a second CAR. In some instances, the first CAR sequence encoding a first CAR is linked to the sequence encoding the cytokine receptor switch polypeptide by a sequence encoding a cleavable linker, such as a self-cleaving peptide, e.g., a P2A sequence. In some instances, the second CAR sequence encoding a second CAR is linked to the sequence encoding the protease by a sequence encoding a cleavable linker, such as a self-cleaving peptide, e.g., a P2A sequence. [0153] In some instances, the recombinant polynucleic acid comprises a first recombinant polynucleic acid molecule comprising a sequence encoding a first polypeptide comprising a cytokine receptor switch polypeptide, a second recombinant polynucleic acid molecule comprising a sequence encoding a second polypeptide, and a third recombinant polynucleic acid molecule comprising a sequence encoding a third polypeptide. In some instances, cell-surface expression of the cytokine receptor switch polypeptide occurs when the second polypeptide and the third polypeptide are expressed in the same cell. In some instances, the extracellular domain of the cytokine receptor switch polypeptide comprises an extracellular domain from IL21R or IL9R. In some instances, the transmembrane domain of the cytokine receptor switch polypeptide is from TREM1. In some instances, the intracellular domain of the cytokine receptor switch polypeptide comprises an intracellular signaling domain from IL2RB, IL7R, or IL9R. In some instances, the second polypeptide comprises a transmembrane domain. In some instances, the transmembrane domain of the second polypeptide is from DAP12. In some instances, the third polypeptide comprises a transmembrane domain. In some instances, the transmembrane domain of the third polypeptide is from DAP12. In some instances, the first recombinant polynucleic acid molecule comprises a first CAR sequence encoding a first CAR, the second recombinant polynucleic acid molecule comprises a second CAR sequence encoding a second CAR and the third recombinant polynucleic acid molecule comprises a third CAR sequence encoding a third CAR. In some instances, the first CAR sequence encoding a first CAR is linked to the sequence encoding the cytokine receptor switch polypeptide by a sequence encoding a cleavable linker, such as a self-cleaving peptide, e.g., a P2A sequence. In some instances, the second CAR sequence encoding a second CAR is linked to the sequence encoding the second polypeptide by a sequence encoding a cleavable linker, such as a self-cleaving peptide, e.g., a P2A sequence. In some instances, the third CAR sequence encoding a third CAR is linked to the sequence encoding the third polypeptide by a sequence encoding a cleavable linker, such as a self-cleaving peptide, e.g., a P2A sequence.

[0154] In some instances, the recombinant polynucleic acid comprises a first recombinant polynucleic acid molecule comprising a sequence encoding a first polypeptide comprising a cytokine receptor switch polypeptide comprising a protease cleavage site and a retention tag, a second recombinant polynucleic acid molecule comprising a sequence encoding a second polypeptide comprising a C-terminal portion of a protease, and a third recombinant polynucleic acid molecule comprising a sequence encoding a third polypeptide comprising an N-terminal portion of the protease. In some instances, the C-terminal portion of a protease is fused to a transmembrane domain. In some instances, the protease cleavage site is disposed between the terminus of the intracellular domain of the cytokine receptor switch polypeptide and the retention tag. In some instances, the C-terminal portion of a protease is not fused to a transmembrane domain. In some instances, the N-terminal portion of a protease is fused to a transmembrane domain. In some instances, the N-terminal portion of a protease is not fused to a transmembrane domain. In some instances, cell-surface expression of the cytokine receptor switch polypeptide occurs when the C-terminal portion of the protease and the N-terminal portion of the protease are expressed in the same cell and form a functional protease. In some instances, the extracellular domain of the cytokine receptor switch polypeptide comprises an extracellular domain from IL21R or IL9R. In some instances, the intracellular domain of the cytokine receptor switch polypeptide comprises an intracellular signaling domain from IL2RB, IL7R, or IL9R. In some instances, the first recombinant polynucleic acid molecule comprises a first CAR sequence encoding a first CAR, the second recombinant polynucleic acid molecule comprises a second CAR sequence encoding a second CAR, and the third recombinant polynucleic acid molecule comprises a third CAR sequence encoding a third CAR. In some instances, the first CAR sequence encoding a first CAR is linked to the sequence encoding the cytokine receptor switch polypeptide by a sequence encoding a cleavable linker, such as a self-cleaving peptide, e.g., a P2A sequence. In some instances, the second CAR sequence encoding a second CAR is linked to the sequence encoding the C- terminal portion of the protease by a sequence encoding a cleavable linker, such as a self-cleaving peptide, e.g., a P2A sequence. In some instances, the third CAR sequence encoding a third CAR is linked to the sequence encoding the N-terminal portion of the protease by a sequence encoding a cleavable linker, such as a self-cleaving peptide, e.g., a P2A sequence.

[0155] In some embodiments, the first polypeptide comprises a cytokine receptor switch polypeptide with a protease cleavage site and the second polypeptide comprises a protease. In some embodiments, the protease cleavage site comprises a tobacco etch virus (TEV) protease cleavage site and the protease comprises a TEV protease.

[0156] In some embodiments, the first polypeptide comprises a cytokine receptor switch polypeptide with a protease cleavage site, the second polypeptide comprises an N-terminal portion of a protease or a first non-functional portion of a protease, and the third polypeptide comprises the C-terminal portion of the protease or a second non-functional portion of a protease. In some embodiments, the protease cleavage site comprises a TEV protease cleavage site, the N-terminal portion of the protease comprises the N- terminal portion of a TEV protease, and the C-terminal portion of the protease comprises the C-terminal portion of a TEV protease.

[0157] In some embodiments, the protease cleavage site is a viral protease cleavage site. In some cases, the viral protease cleavage site is for a viral protease derived from hepatitis C virus (HCV) nonstructural protein 3 (NS3). In some cases, the viral protease cleavage site is for a viral protease that further comprises a cofactor polypeptide derived from HCV nonstructural protein 4A (NS4A).

[0158] In some embodiments, the viral protease cleavage site is selected from the group consisting of: an NS4A/4B junction cleavage site, an NS3/NS4A junction cleavage site, an NS4A/NS4B junction cleavage site, an NS4B/NS5 A junction cleavage site, an NS5A/NS5B junction cleavage site, and variants thereof cleavable by the viral protease. In some embodiments, the protease is conditionally active.

[0159] In some embodiments, cell surface expression of the cytokine receptor switch polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the cytokine receptor switch polypeptide that lacks the recombinant polynucleic acid encoding the second polypeptide compared to cell surface expression of the cytokine receptor switch polypeptide on a cell comprising the recombinant polynucleic acid encoding the cytokine receptor switch polypeptide and the recombinant polynucleic acid encoding the second polypeptide (see, e.g., FIG. 4A-4D, 6A-6C, 7A-7D, 9A-9C, 10A-10C and 11A-11C).

[0160] In some embodiments, a cytokine receptor switch polypeptide is not expressed extracellularly on a cell comprising a recombinant polynucleic acid encoding the cytokine receptor switch polypeptide that lacks the recombinant polynucleic acid encoding the second polypeptide, or a cell that lacks the recombinant polynucleic acid encoding the second polypeptide and the third polypeptide (see, e.g., FIG. 5A-5C, 6A-6C, 7A-7D, 9A-9C, lOA-lOCand 12A-12C)

[0161] In some embodiments, the second polypeptide comprises a transmembrane domain. In some embodiments, the transmembrane domain of the second polypeptide is different than the transmembrane domain of the cytokine receptor switch polypeptide. In some embodiments, the first polypeptide comprises a first transmembrane domain and the second polypeptide comprises a second transmembrane domain. In some embodiments, the transmembrane domain of the cytokine receptor switch polypeptide is a TREM1 transmembrane domain, and the transmembrane domain of the second polypeptide is a DAP12 transmembrane domain. In some embodiments, the cell surface expression of the cytokine receptor switch polypeptide with a TREM1 transmembrane domain is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% when the second polypeptide comprising the DAP12 transmembrane domain is expressed in a cell.

[0162] In some embodiments, the transmembrane domain of the cytokine receptor switch polypeptide comprises a Killer Cell Immunoglobulin Like Receptor Two Ig Domains And Short Cytoplasmic Tail 2 (KIR2DS2), TREM2, immune receptor expressed by myeloid cells 2 (IREM2), KIR3DS1, KIR2DS5, KIR2DS1, KIR2DS4, KLRD1, KLRC2, Natural Cytotoxicity Triggering Receptor 2 (NCR2), Signal Regulatory Protein Beta 1 (SIRPB1), Paired Immunoglobin Like Type 2 Receptor Beta (PILRB), C-Type Lectin Domain Containing 5A (CLEC5A), CD300 Molecule Like Family Member B (CD300LB), Sialic Acid Binding Ig Like Lectin 14 (SIGLEC14), SIGLEC15, or SIGLEC16 transmembrane domain.

[0163] In some embodiments, the first polypeptide comprising the cytokine receptor switch polypeptide further comprises an ER retention tag. In some embodiments, the cytokine receptor switch polypeptide is retained in the endoplasmic reticulum (ER) of a cell that does not express the second polypeptide.

[0164] In some embodiments, the second polypeptide does not comprise an intracellular domain or does not comprise a full-length intracellular domain. In some embodiments, the second polypeptide consists of a transmembrane domain. In some embodiments, the second polypeptide does not comprise an intracellular domain comprising an intracellular signaling domain from a cytokine receptor.

[0165] In some embodiments, the second polypeptide does not comprise an intracellular domain comprising an intracellular signaling domain.

[0166] In some embodiments, the second polypeptide does not comprise a full-length extracellular domain. In some embodiments, the second polypeptide comprises an extracellular domain that is at most about 200, 250, 300, 350, 400, or 450 amino acids in length. In some cases, the second polypeptide is a non-functional receptor or is not capable of signaling to a cell in which it is expressed.

[0167] In some embodiments, the cytokine receptor switch polypeptide and the second polypeptide form a complex. In some cases, the complex is a non-functional receptor complex or is not capable of signaling to a cell in which it is expressed. In some cases, the cytokine receptor switch polypeptide and the second polypeptide form a dimer or a trimer. In some cases, the trimer is a trimer containing two copies of the second polypeptide.

[0168] In some embodiments, the first, second, and/or third recombinant polynucleic acids further comprise a sequence encoding a gene of interest. In some embodiments, the first recombinant polynucleic acid further encodes a first gene of interest, the second recombinant polynucleic acid further encodes a second gene of interest, and the third recombinant polynucleic acid further encodes a third gene of interest. In some cases, the first, second and/or third gene(s) of interest encodes a first, second, and/or third protein(s) of interest. In some cases, the first, second, or third protein(s) of interest comprises a CAR. In some embodiments, each of the first, second, or third CARs target different antigens. In some embodiments, each of the first, second, or third CARS target the same antigen with different antigen binding elements.

[0169] In some cases, the recombinant polynucleic acid encoding the first gene of interest encodes for a first protein of interest, wherein the cytokine receptor switch polypeptide and the first protein of interest are expressed as a single polypeptide molecule. In some cases, the recombinant polynucleic acid sequence encoding the cytokine receptor switch polypeptide is linked to the sequence encoding the first protein of interest by a sequence encoding a linker. In some cases, the recombinant polynucleic acid sequence encoding the first gene of interest encodes for a first protein of interest, wherein the cytokine receptor switch polypeptide and the first protein of interest are expressed as separate polypeptide molecules. In some cases, the recombinant polynucleic acid sequence encoding the cytokine receptor switch polypeptide and the sequence encoding the first gene of interest are separated or linked by an internal ribosome entry site (IRES) or have different promoters.

[0170] In some embodiments, the recombinant polynucleic acid sequence encoding a second 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 cases, the recombinant polynucleic acid sequence encoding the second polypeptide is linked to the sequence encoding the second protein of interest by a sequence encoding a linker. In some cases, the recombinant polynucleic acid sequence encoding the second gene of interest encodes for a second protein of interest, wherein the second polypeptide and the second protein of interest are expressed as separate polypeptide molecules. In some cases, the recombinant polynucleic acid sequence encoding the first polypeptide and the sequence encoding the first gene of interest are separated or linked by an IRES or have different promoters. In some cases, the recombinant polynucleic acid sequence encoding the second polypeptide and the sequence encoding the second gene of interest are separated or linked by an IRES or have different promoters. In some cases, the recombinant polynucleic acid sequence encoding the third polypeptide and the sequence encoding the third gene of interest are separated or linked by an IRES or have different promoters.

[0171] In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 2 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 to SEQ ID NO: 35. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 3 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 to SEQ ID NO: 35. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 4 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 to SEQ ID NO: 35. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 5 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 to SEQ ID NO: 35. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 6 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 to SEQ ID NO: 35. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 15 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 to SEQ ID NO: 35. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 16 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 to SEQ ID NO: 35. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 17 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 to SEQ ID NO: 35. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 18 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 to SEQ ID NO: 35. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 19 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 to SEQ ID NO: 35. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 20 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 to SEQ ID NO: 35. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 21 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 to SEQ ID NO: 35. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 22 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 to SEQ ID NO: 35. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 2 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 to SEQ ID NO: 36. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 3 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 to SEQ ID NO: 36. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 4 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 to SEQ ID NO: 36. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 5 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 to SEQ ID NO: 36. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 6 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 to SEQ ID NO: 36. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 15 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 to SEQ ID NO: 36. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 16 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 to SEQ ID NO: 36. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 17 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 to SEQ ID NO: 36. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 18 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 to SEQ ID NO: 36. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 19 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 to SEQ ID NO: 36. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 20 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 to SEQ ID NO: 36. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 21 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 to SEQ ID NO: 36. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 22 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 to SEQ ID NO: 36. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 2 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 to SEQ ID NO: 37. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 3 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 to SEQ ID NO: 37. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 4 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 to SEQ ID NO: 37. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 5 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 to SEQ ID NO: 37. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 6 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 to SEQ ID NO: 37. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 15 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 to SEQ ID NO: 37. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 16 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 to SEQ ID NO: 37. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 17 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 to SEQ ID NO: 37. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 18 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 to SEQ ID NO: 37. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 19 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 to SEQ ID NO: 37. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 20 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 to SEQ ID NO: 37. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 21 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 to SEQ ID NO: 37. In some instances, the recombinant polynucleic acid comprises the sequence encoding the cytokine 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 to SEQ ID NO: 22 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 to SEQ ID NO: 37.

[0172] 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 phospho rami dite linkages, 2 ’-modified ribose or deoxyribose, morpholino phosphorami dites, 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 doublestranded 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.

[0173] In some instances, the recombinant polynucleic acid comprises a sequence encoding the cytokine receptor switch polypeptide, wherein the cytokine 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 instances, the recombinant polynucleic acid comprises a sequence encoding the cytokine receptor switch polypeptide, wherein the cytokine 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 instances, the recombinant polynucleic acid further comprises a sequence encoding a protein retention tag.

[0174] In some instances, 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 instances, the recombinant nucleic acid is further defined as an expression cassette or a vector. In some instances, the vector is an adenovirus vector, an adeno-associated virus vector, or a retroviral vector. In some embodiments, the retroviral vector is a lentiviral vector.

[0175] 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.”

[0176] Also provided herein are vectors, plasmids or viruses containing one or more of the nucleic acid sequences encoding any of the cytokine receptor switch peptides 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).

[0177] Accordingly, in some instances, the cytokine receptor switch peptides 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.

[0178] 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., (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.

[0179] Vectors suitable for use include the pMSXND expression vector for use in mammalian cells. In some instances, nucleic acid inserts, which encode the cytokine receptor switch peptides 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, e.g. , Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

[0180] In some instances, 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). [0181] 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, e.g., Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

[0182] In some instances, 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 instances, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some instances, 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 instances, the nucleic acid molecule is present in the recombinant host cell as a mini circle expression vector for stable or transient expression.

[0183] 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.

[0184] 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. Compositions

[0185] In other aspects, the present disclosure describes compositions comprising any of the cytokine receptor switch polypeptides disclosed herein. In another aspect, the present disclosure provides compositions comprising any of the recombinant polynucleic acids disclosed herein, either alone or in combination. In some instances, the composition comprises a cytokine receptor switch polypeptide disclosed herein and a CAR disclosed herein. In some instances, the composition comprises a recombinant polynucleic acid encoding a cytokine receptor switch polypeptide disclosed herein and a CAR disclosed herein. In some instances, the composition comprises a cytokine switch receptor polypeptide disclosed herein, a second polypeptide disclosed herein, and a third polypeptide disclosed herein. In some instances, the composition comprises a cytokine receptor switch polypeptide disclosed herein, a second polypeptide disclosed herein, and a third polypeptide disclosed herein, wherein the cytokine receptor switch polypeptide further comprises a first CAR, the second polypeptide further comprises a second CAR, and the third polypide further comprises a third CAR. In some instances, the composition comprises a first recombinant polynucleic acid encoding the cytokine receptor switch polypeptide and the first CAR, a second recombinant polynucleic acid encoding the second polypeptide and the second CAR, and a third recombinant polynucleic acid encoding the third CAR.

Cells

[0186] In some aspects, the present disclosure describes cells comprising the cytokine receptor switch polypeptides disclosed herein, cells comprising the recombinant polynucleic acids encoding the cytokine receptor switch polypeptides disclosed herein, cells comprising the compositions comprising the cytokine receptor switch polypeptides disclosed herein, and cells comprising the compositions comprising the recombinant polynucleic acids encoding the cytokine receptor switch polypeptides disclosed herein. In some instances, the cell expresses the cytokine receptor switch polypeptides disclosed herein. In some instances, the cytokine 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 retention tag (e.g., ER retention tag, Golgi retention 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, and the like) - 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. [0187] In some instances, 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 instances, the host cell is an immune cell, a stem cell, a mammalian cell, a primate cell, or a human cell. In some instances, the host cell is autologous or allogeneic. In some instances, the host cell is a T cell. In some instances, the T cell is a CD8-positive T cell, a CD4-positive T cell, a regulatory T cell, a cytotoxic T cell, or a tumor infdtrating lymphocyte.

[0188] Host cells can be transduced with a nucleic acid encoding the cytokine receptor switch polypeptide and/or a CAR, with or without the protein retention tag. In some instances, a host cell can be transduced with a nucleic acid encoding a cytokine receptor switch polypeptide. In some instances, a host cell can be transduced with a bicistronic nucleic acid encoding a cytokine receptor switch polypeptide and a CAR. In some instances, a host cell can be transduced with a nucleic acid encoding a cytokine receptor switch polypeptide and an additional nucleic acid encoding a CAR. In some instances, a host cell can be transduced with a bicistronic nucleic acid encoding a cytokine receptor switch polypeptide with a protein retention tag and a CAR. In some instances, a host cell can be transduced with a bicistronic nucleic acid encoding a cytokine receptor switch polypeptide and a CAR with a protein retention tag. In some instances, a host cell can be transduced with a bicistronic nucleic acid encoding a cytokine receptor switch polypeptide with a protein retention tag and a CAR with another protein retention tag. In some instances, 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.

[0189] In some instances, the recombinant cell is an animal cell. In some instances, the animal cell is a mammalian cell. In some instances, the animal cell is a mouse cell. In some instances, the animal cell is a human cell. In some instances, 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 instances, the immune system cell is selected from the group consisting of B cells, T cells, monocytes, dendritic cells, and epithelial cells. In some instances, the immune system cell is a T lymphocyte. In some instances, the T cell is a CD8-positive T cell, a CD4-positive T cell, a regulatory T cell, a cytotoxic T cell, or a tumor infiltrating lymphocyte. The immune cell can also be a precursor cell, i.e., a cell that is capable of differentiating into an immune cell.

[0190] 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 instances, 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.

[0191] 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 instances, the cells are T cells obtained from a mammal. In some instances, the T cells obtained from a mammal are CD8-positive T cells, CD4-positive T cells, regulatory T cells (Tregs), cytotoxic T cells (CTLs), or tumor infiltrating lymphocytes (TILs). In some instances, the mammal is a primate. In some instances, the primate is a human.

[0192] 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 instances, 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.

[0193] In some instances, an isolated or purified population of T cells is used. In some instances, TCTL and TH lymphocytes are purified from PBMCs. In some instances, 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.

[0194] 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 instances, a specific subpopulation of T cells, expressing one or more of the markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD 122, CD 127, CD197; or CD38 or CD62L, CD127, CD197, and CD38, is further isolated by positive or negative selection techniques. In some instances, 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 instances, 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.

[0195] Further disclosed herein are compositions comprising a cell comprising a recombinant polynucleic acid comprising a sequence encoding a cytokine receptor switch polypeptide, wherein the cell expresses the cytokine receptor switch polypeptide, the cytokine 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell

-n- 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, 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 cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, 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 cytokine receptor switch polypeptide in the presence of the cytokine.

[0196] In some instances, the cell expands at least 2-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 3 -fold greater by day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 4-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 5 -fold greater by day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 10-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 20-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 30-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 40-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 50-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 100-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 200-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 300-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 400-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 500-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine.

[0197] In some instances, the cell expands at least 2-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 10-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 50-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 100-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 200-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 500-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 1000-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 2000-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 3000-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 4000-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the cell expands at least 5000-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the cytokine receptor switch polypeptide in the presence of the cytokine.

[0198] In some instances, the cell is a mammalian cell. In some instances, the mammalian cell is a primate cell or a human cell. In some instances the mammalian cell is a human cell. In some instances, the human cell is a blood cell. The cell can be a human cell. The cell can be a blood cell. In some instances, the blood cell is a lymphocyte. In some instances, the lymphocyte is a T cell. In some instances, the T cell obtained from a mammal is a CD8-positive T cell, a CD4-positive T cell, a regulatory T cell (Treg), a cytotoxic T cells (CTL), or a tumor infiltrating lymphocyte (TIL). In some instances, the cell is a population of cells. In some instances, the population of cells is a population of blood cells. The blood cells can be lymphocytes. The lymphocytes can be T cells. In some instances, the T cells are CD8- positive T cells, CD4-positive T cells, regulatory T cells (Tregs), cytotoxic T cells (CTLs), or tumor infiltrating lymphocytes (TILs). In some instances, the population of cells is a homogeneous mixture of cells of the same cell type. In some instances, the population of cells is a heterogeneous mixture of cells of different cell types. In some instances, the population of cells comprises at least about 1 * 10 ⁵ cells. In some instances, the population of cells comprises at least about l *10 ⁶ cells. In some instances, the population of cells comprises at least about l * 10 ⁷ cells. In some instances, the population of cells comprises at least about I xlO ⁸ cells. In some instances, the population of cells comprises at least about I xlO ⁹ cells. In some instances, the population of cells comprises from about I xlO ⁵ cells to about I x lO ⁹ cells. In some instances, the population of cells comprises from about I x lO ⁵ cells to about I x lO ⁸ cells. In some instances, the population of cells comprises from about I x lO ⁵ cells to about I xlO ⁷ cells. In some instances, the population of cells comprises from about I xlO ⁵ cells to about I xlO ⁶ cells.

[0199] In some instances, addition of IL2 can stimulate and expand both the cell expressing the cytokine receptor switch polypeptide and the cell not expressing the cytokine receptor switch polypeptide. Specifically, the cell that expresses the cytokine receptor switch polypeptide expands in the presence of IL2 and a cell that does not express the cytokine receptor switch polypeptide expands in the presence of IL2.

[0200] In some instances, addition of the cytokine that specifically binds to the extracellular domain of the cytokine receptor switch polypeptide can stimulate and expand only the cell expressing the cytokine receptor switch polypeptide but not the cell not expressing the cytokine receptor switch polypeptide. Specifically, the cell that does not express the cytokine receptor switch polypeptide does not expand in the presence of the cytokine that binds to the extracellular domain of the cytokine receptor switch polypeptide. In some instances, the cytokine is IL21.

Pharmaceutical Compositions

[0201] In some aspects, the present disclosure describes a pharmaceutical composition comprising the composition comprising the cytokine 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 l *10 ⁵ to 5* 10 ¹⁰ of the cells of the present disclosure. [0202] 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.

[0203] 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.

[0204] 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.

[0205] 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).

[0206] 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 7.0 to 8.0, 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. [0207] 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.

[0208] In some embodiments, 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 polyoxy ethylensorbitan 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 polyethylene-polypropylene 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.

[0209] In some instances, 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

[0210] 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 virus, 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 instances, 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, anticancer agents, or anti-cancer therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery. “Chemotherapy” and “anticancer agent” are used interchangeably herein. Various classes of anti-cancer agents can be used. Nonlimiting 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.

[0211] In some instances, 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 instances, the disease, disorder, or condition is cancer. In some instances, the cancer is lymphoma or leukemia. In some instances, 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).

[0212] 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.

[0213] 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, 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, intra-abdominal 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.

[0214] 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 [0215] 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 cytokine receptor switch polypeptide, thereby expanding the cell. In some instances, the cell is expanded at least 2-fold. In some instances, the cell is expanded at least 3 -fold. In some instances, the cell is expanded at least 4-fold. In some instances, the cell is expanded at least 5-fold. In some instances, the cell is expanded at least 10-fold. In some instances, the cell is expanded at least 20-fold. In some instances, the cell is expanded at least 30-fold. In some instances, the cell is expanded at least 40-fold. In some instances, the cell is expanded at least 50-fold. In some instances, the cell is expanded at least 100-fold. In some instances, the cell is expanded at least 200-fold. In some instances, the cell is expanded at least 300-fold. In some instances, the cell is expanded at least 400-fold. In some instances, the cell is expanded at least 500-fold. In some instances, the cell is expanded at least 1000-fold.

[0216] In some instances, the cell is expanded a least 2-fold by day 10 after addition of the cytokine that binds to the extracellular domain of the cytokine receptor switch polypeptide. In some instances, the cell is expanded at least 3 -fold by day 10 after addition of the cytokine. In some instances, the cell is expanded at least 4-fold by day 10 after addition of the cytokine. In some instances, the cell is expanded at least 5- fold by day 10 after addition of the cytokine. In some instances, the cell is expanded at least 10-fold by day 10 after addition of the cytokine. In some instances, the cell is expanded at least 20-fold by day 10 after addition of the cytokine. In some instances, the cell is expanded at least 30-fold by day 10 after addition of the cytokine. In some instances, the cell is expanded at least 40-fold by day 10 after addition of the cytokine. In some instances, the cell is expanded at least 50-fold by day 10 after addition of the cytokine. In some instances, the cell is expanded at least 100-fold by day 10 after addition of the cytokine. In some instances, the cell is expanded at least 200-fold by day 10 after addition of the cytokine. In some instances, the cell is expanded at least 300-fold by day 10 after addition of the cytokine. In some instances, the cell is expanded at least 400-fold by day 10 after addition of the cytokine. In some instances, the cell is expanded at least 500-fold by day 10 after addition of the cytokine. In some instances, the cell is expanded at least 1000-fold by day 10 after addition of the cytokine.

[0217] In some instances, the cell is expanded at least 2-fold by day 14 after addition of the cytokine. In some instances, the cell is expanded at least 3 -fold by day 14 after addition of the cytokine. In some instances, the cell is expanded at least 4-fold by day 14 after addition of the cytokine. In some instances, the cell is expanded at least 5 -fold by day 14 after addition of the cytokine. In some instances, the cell is expanded at least 10-fold by day 14 after addition of the cytokine. In some instances, the cell is expanded at least 50-fold by day 14 after addition of the cytokine. In some instances, the cell is expanded at least 100-fold by day 14 after addition of the cytokine. In some instances, the cell is expanded at least 500-fold by day 14 after addition of the cytokine. In some instances, the cell is expanded at least 1000-fold by day 14 after addition of the cytokine. In some instances, the cell is expanded at least 1500-fold by day 14 after addition of the cytokine. In some instances, the cell is expanded at least 2000-fold by day 14 after addition of the cytokine.

Methods for Preparing CAR-Expressing Immune Effector Cells

[0218] Further disclosed herein are methods for making a population of CAR-expressing immune effector cells, comprising contacting any one of the cells or compositions 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.

[0219] In some instances, the first cytokine receptor is an IL21 receptor. In some instances, the cytokine is IL21. In some instances, the first cytokine receptor is an IL21 receptor and the cytokine is IL21. IL21 can be added to make a population of the CAR-expressing immune effector cells expressing the cytokine receptor switch polypeptide. In some instances, the CAR-expressing immune effector cells, after culturing with the cytokine, for example IL21, are expanded at least 2-fold. In some instances, the CAR-expressing immune effector cells are expanded at least 3 -fold. In some instances, the CAR-expressing immune effector cells are expanded at least 4-fold. In some instances, the CAR-expressing immune effector cells are expanded at least 5 -fold. In some instances, the CAR-expressing immune effector cells are expanded at least 10-fold. In some instances, the CAR-expressing immune effector cells are expanded at least 20- fold. In some instances, the CAR-expressing immune effector cells are expanded at least 30-fold. In some instances, the CAR-expressing immune effector cells are expanded at least 40-fold. In some instances, the CAR-expressing immune effector cells are expanded at least 50-fold. In some instances, the CAR-expressing immune effector cells are expanded at least 100-fold. In some instances, the CAR- expressing immune effector cells are expanded at least 200-fold. In some instances, the CAR-expressing immune effector cells are expanded at least 300-fold. In some instances, the CAR-expressing immune effector cells are expanded at least 400-fold. In some instances, the CAR-expressing immune effector cells are expanded at least 500-fold. In some instances, the CAR-expressing immune effector cells are expanded at least 1000-fold.

[0220] In some instances, the CAR-expressing immune effector cells are expanded at least 2-fold by day 10 after addition of the cytokine that binds to the extracellular domain of the cytokine receptor switch polypeptide, for example, IL21. In some instances, the CAR-expressing immune effector cells are expanded at least 3 -fold by day 10 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 4-fold by day 10 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 5 -fold by day 10 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 10- fold by day 10 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 20-fold by day 10 after addition of the cytokine. In some instances, the CAR- expressing immune effector cells are expanded at least 30-fold by day 10 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 40-fold by day 10 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 50-fold by day 10 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 100-fold by day 10 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 200-fold by day 10 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 300-fold by day 10 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 400-fold by day 10 after addition of the cytokine. In some instances, the CAR- expressing immune effector cells are expanded at least 500-fold by day 10 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 1000-fold by day 10 after addition of the cytokine.

[0221] In some instances, the CAR-expressing immune effector cells are expanded at least 2-fold by day 14 after addition of the cytokine (e.g., IL21). In some instances, the CAR-expressing immune effector cells are expanded at least 3 -fold by day 14 after addition of the cytokine. In some instances, the CAR- expressing immune effector cells are expanded at least 4-fold by day 14 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 5-fold by day 14 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 10-fold by day 14 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 50-fold by day 14 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 100-fold by day 14 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 500-fold by day 14 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 1000-fold by day 14 after addition of the cytokine. In some instances, the CAR- expressing immune effector cells are expanded at least 1500-fold by day 14 after addition of the cytokine. In some instances, the CAR-expressing immune effector cells are expanded at least 2000-fold by day 14 after addition of the cytokine.

[0222] In some instances, the CAR-expressing immune effector cells expand at least 2-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 3 -fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 4-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 5 -fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 10-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 15-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 20-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 30-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 40-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 50-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 100-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 200-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 300-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 400-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 500-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 700-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 1000-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine.

[0223] In some instances, the CAR-expressing immune effector cells expand at least 2-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR- expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 3-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 4-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 5-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR- expressing immune effector cells expand at least 10-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain (e.g. , IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 20-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain (e.g. , IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 30-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 40-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 50-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 100-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 200-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 300-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain (e.g. , IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 400-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 500-fold greater by day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine.

[0224] In some instances, the CAR-expressing immune effector cells expand at least 2-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR- expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 10-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 50-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 100-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 200-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain (e.g. , IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 500-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 1000-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 2000-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 3000-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that doe not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 4000-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine. In some instances, the CAR-expressing immune effector cells expand at least 5000-fold greater by day 14 after addition of the cytokine that binds to the extracellular domain (e.g. , IL21) compared to CAR-expressing immune effector cells that do not express the cytokine receptor switch polypeptide in the presence of the cytokine.

[0225] In some instances, the immune effector cell is a mammalian cell. In some instances, the mammalian cell is a human cell. In some instances, the immune effector cell is a lymphocyte. In some instances, the lymphocyte is a T cell. In some instances, the T cell is a CD8-positive T cell, a CD4-positive T cell, a regulatory T cell (Treg), a cytotoxic T cells (CTL), or a tumor infiltrating lymphocyte (TIL). In some instances, the cell is a population of cells. In some instances, the population of cells is a homogeneous mixture of cells of the same cell type. In some instances, the population of cells is a heterogeneous mixture of cells of different cell types. In some instances, the population of cells comprises at least about 1 *10 ⁵ cells. In some instances, the population of cells comprises at least about 1 *10 ⁶ cells. In some instances, the population of cells comprises at least about I xlO ⁷ cells. In some instances, the population of cells comprises at least about I x lO ⁸ cells. In some instances, the population of cells comprises at least about I xlO ⁹ cells. In some instances, the population of cells comprises from about I xlO ⁵ cells to about I x lO ⁹ cells. In some instances, the population of cells comprises from about I xlO ⁵ cells to about I x lO ⁸ cells. In some instances, the population of cells comprises from about I xlO ⁵ cells to about I xlO ⁷ cells. In some instances, the population of cells comprises from about I xlO ⁵ cells to about I xlO ⁶ cells.

[0226] 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 instances, 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.

[0227] In some instances, 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, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combinations thereof. For example, the immune effector cells can comprise T lymphocytes. In some embodiments, the T lymphocytes is a CD8-positive T cell, a CD4-positive T cell, a regulatory T cell (Treg), a cytotoxic T cells (CTL), or a tumor infdtrating lymphocyte (TIL).

EXAMPLES

[0228] 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.

[0229] 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 cytokine receptor switch peptide constructs

[0230] Exemplary cytokine receptor switch peptide and components constructs are listed in Table 1.

Table 1: Exemplary Cytokine Receptor Switch Peptide Constructs

[0231] “R” refers to receptor, for example, “IL21R” refers to IL21 receptor; “IL2RB” refers to IL2 receptor beta. “Ecto” refers to extracellular domain. “TM” refers to transmembrane domain. “Endo” refers to intracellular domain.

Example 2 - Exemplary cytokine receptor switch peptide with ER retention tag and/or CAR constructs

[0232] Exemplary cytokine receptor switch peptide with ER retention tag and/or CAR constructs are listed in Table 2.

Table 2: Exemplary Cytokine Receptor Switch Peptide with ER Retention Tag Constructs

[0233] “R” refers to receptor. “TM” refers to transmembrane domain. “Cyto” refers to intracellular domain.

[0234] Exemplary ER retention tag constructs are listed in Table 3.

Table 3: Exemplary ER Retention Tag Constructs

[0235] In some instances, the protease cleavage site is a protease cleavage site from the Tobacco Etch Virus (TEV). An exemplary TEV site sequence is ENLYFQS (SEQ ID NO.: 31).

Example 3 - Exemplary CAR constructs

[0236] Exemplary antigen binding domains for the CAR are listed in Table 4.

Table 4: Exemplary Antigen Binding Domain for CAR

[0237] Exemplary CAR constructs are listed in Table 5.

Table 5: Exemplary CAR Constructs

Example 4 - Selective Expansion of Transduced Cells

Synthesis of cytokine receptor switch peptides

[0238] Codon optimized genes encoding the cytokine receptor switch peptides were synthesized and cloned into retrovirus vector MSGV1. Viral supernatant was prepared by transfecting HEK293gp with MSGV1 using lipofectamine. After 2 days, supernatant containing virus was harvested, fdtered and centrifuged for 2 hours at 3000g onto retronectin coated 24 well plates.

T cell transduction

[0239] T cells were activated on day 0 using Transact (Miltenyi 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. 3A-3C, IL2 promoted expansion of both transduced and non-transduced cells; however, IL21 could promote the expansion of only the transduced cells but not the non-transduced cells. [0240] 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 5 - Selective Expansion of a Cytokine Receptor Switch Polypeptide with a Protease Cleavage Site

[0241] T cells expressing two components/proteins (GFP and BFP) were creating by transducing the cells with the constructs (Pl and P2) shown in FIG 4 A. Pl contained green fluorescent protein (GFP) connected to the IL21/2RB fusion protein containing a TEV cleavage site and ER retention tag using a P2A linker. Eight different Pl plasmids were prepared, each containing a different ER retention tag. P2 contained blue fluorescent protein (BFP) connected to TEV protease using a P2A linker. T cells transduced with Pl and/or P2 were grown in the presence of IL-21 and cell counts were monitored over time as shown in FIG. 4B. High cell counts in Pl only transduced cells only suggest lack of ER retention of the IL21/2RB fusion protein. ERtag2, ERtag4, ERtag5, ERtag6 and ERtag8 all show a differential in cell growth comparing T cells transduced with Pl only compared to co-transduced with Pl and P2, with ERtag6 and ERtag8 having the lowest Pl only background. The % of Pl and P2 double positive T cells grown in IL21 was measured as shown in FIG. 4C. Enrichment of double positive T cells grown in IL-21 over time suggests specific cell surface expression of IL21/2RB fusion protein in double positive cells. Flow cytometry plots of primary T cells transduced with Pl(ERtag8) only or Pl(ERtag8) + P2 were generated as shown in FIG. 4D. T cells grown with either IL-2 to expand all populations or IL-21 to expand only populations expressing the IL21/2RB fusion protein. Only in the condition with Pl(ERtag8)+P2 using IL-21 to grow out the cells is there selection of the double positive population. In some embodiments, the GFP and BFP can be replaced with a protein or gene of interest, such as a CAR.

Example 6 - Creation and Function of a Trispecific CAR T cell

[0242] Three plasmids were prepared to make a trispecific CAR T cell as shown in FIG. 5A. The first plasmid, Pl, contained a CD20 CAR connected to an IL21/2RB cytokine receptor switch polypeptide with TEV cleavage site and ERtag8 using a P2A linker. The second plasmid, P2, contained a CD22 CAR containing the m971 binder connected to the N-terminal domain of TEV protease using a P2A linker. The third plasmid, P3, contained a CD 19 CAR containing the FMC63 binder connected to the C-terminal domain of TEV protease by a P2A linker. T cells were transduced with P1-P3 to create the trispecific CAR T cell.

[0243] Flow cytometry was performed and plots of primary T cells co-transduced with all three plasmids, P1+P2+P3, and grown without cytokine, with IL-2 or with IL-21 were created as shown in FIG. 5B. Using IL-21 to expand the cells selected for cells that contained all three CARs (86.62%) whereas IL-2 expanded all CAR populations equally. The condition without cytokine added did not result in significant cell expansion.

[0244] CAR T cell function was assessed in a serial killing assay in which Nalm6 target cells expressing luciferase were added to wells containing CAR T cells every 2-3 days and killing was monitored using a luciferin substrate as shown in FIG. 5C. IL-21 was added into the serial killing assay when new target cells were added. Trispecific CAR T cells containing the IL21/2RB cytokine receptor switch polypeptide maintained good cell killing over 3 weeks while all other controls, including monospecific, bispecific and trispecific CAR T cells not expressing the IL21/2RB cytokine receptor switch polypeptide lost serial killing ability by 3 weeks, FIG. 5C.

Example 7 - Selective Expansion of a Cytokine Receptor Switch Polypeptide with a TREM 1 Transmembrane Domain

-I l l- [0245] A cytokine receptor switch polypeptide was created that contained the extracellular domain of the IL-21 receptor (IL21R), the transmembrane domain of TREM1 and the intracellular signaling domain of IL-2 receptor beta (IL2RB) as shown in FIG. 6A. The TREM1 transmembrane domain prevents the fusion protein from getting to the cell surface. When truncated DAP12, in which the intracellular domain has been removed, is co-expressed with the IL-21R/TREM1TM/IL2RB cytokine receptor switch polypeptide, this allows the fusion protein to traffic to the cell surface.

[0246] Retrovirus plasmid Pl was prepared with green fluorescent protein (GFP) connected to the IL21R/TREM1TM/IL2RB cytokine receptor switch polypeptide using a P2A linker FIG. 6B. Retrovirus plasmid P2 was prepared with blue fluorescent protein (BFP) connected to truncated DAP12 using a P2A linker FIG. 6B. In some embodiments, the GFP and BFP can be replaced with a protein or gene of interest, such as a CAR.

[0247] Flow cytometry was performed and flow data was obtained FIG. 6C. Primary T cells cotransduced with Pl and P2 were grown in IL-2 to expand all cell populations or IL-21 to expand only cells expressing the IL21R/TREM1TM/IL2RB cytokine receptor switch polypeptide on the cell surface.

[0248] A cytokine receptor switch polypeptide was created that contained the extracellular domain of the IL-9 receptor (IL9R), the transmembrane domain of TREM1 and the intracellular signaling domain of IL- 2 receptor beta (IL2RB) as shown in FIG. 7A. The TREM1 transmembrane domain prevents the cytokine receptor switch polypeptide from getting to the cell surface. When truncated DAP12, in which the intracellular domain has been removed, is co-expressed with the IL-9R/TREM1TM/IL2RB fusion protein, this allows the fusion protein to traffic to the cell surface.

[0249] Retrovirus plasmid Pl was prepared with green fluorescent protein (GFP) connected to the IL9R/TREM1TM/IL2RB fusion protein using aP2A linker FIG. 7B. Retrovirus plasmid P2 contains blue fluorescent protein (BFP) connected to truncated DAP12 using a P2A linker FIG. 7B.

[0250] Primary T cells were transduced with Pl and P2 and cell counts were obtained 14 days after primary T cell activation. T cells transduced with P1+P2 and grown in IL-9 showed growth that was dosedependent on IL-9 concentration. 100 ng/ml IL-9 resulted in cell proliferation similar to that observed with cells grown in IL-2 as shown in FIG. 7C.

[0251] The % of cells that are double positive for GFP and BFP (Pl and P2) was higher for T cells grown with IL-9 than those grown with IL-2 thereby showing enrichment of cells transduced with both vectors FIG. 7D

[0252] While preferred embodiments of the present invention 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 invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.