30761-20002.40 SYNTHETIC CYTOKINE RECEPTORS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application No.63/510,921 filed on June 29, 2023, entitled “SYNTHETIC CYTOKINE RECEPTORS”, U.S. Provisional Patent Application No.63/615,248 filed on December 27, 2023, entitled “SYNTHETIC CYTOKINE RECEPTORS”, and U.S. Provisional Patent Application No.63/555,875 filed on February 20, 2024, entitled “SYNTHETIC CYTOKINE RECEPTORS”, the contents of which are incorporated by reference in their entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING [0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 30761-2000240.XML created June 28, 2024, which is 277,183 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. FIELD [0003] The present disclosure generally relates to synthetic cytokine receptors and engineered cells expressing one or more such synthetic cytokine receptors. BACKGROUND [0004] Therapies that use adoptively transferred genetically engineered T cells have shown substantial anti-tumor activity in patients with hematopoietic malignancies. However, such therapies have limited benefit in patients with solid tumors. One major limitation is the poor in vivo expansion and persistence of adoptively transferred T cells. To circumvent this limitation, patients have been lymphodepleted with chemotherapy and/or radiation prior to being transferred with engineered T cells. However, some patients are too weak to receive toxic regimens such as chemotherapy and radiation. The other major limitation is that the T cells that do successfully expand and persist in vivo become terminally differentiated and dysfunctional. Thus, improved systems and engineered cells are needed to address these problems. Provided embodiments address these needs. SUMMARY [0005] The present disclosure provides synthetic cytokine receptors, engineered cells expressing one or more such synthetic cytokine receptors, and uses thereof.
30761-20002.40 [0006] In some embodiments, provided herein is a synthetic cytokine receptor, comprising an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain. [0007] In some embodiments, provided herein is a polynucleotide encoding a synthetic cytokine receptor, wherein the synthetic cytokine receptor comprises an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain. [0008] In some embodiments, provided herein is a vector comprising a polynucleotide encoding a synthetic cytokine receptor, wherein the synthetic cytokine receptor comprises an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain. [0009] In some embodiments, provided herein is a method of engineering an isolated cell, comprising contacting the cell with a polynucleotide encoding a synthetic cytokine receptor or a vector comprising such a polynucleotide, wherein the synthetic cytokine receptor comprises an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain. [0010] In some embodiments, provided herein is an engineered cell expressing one or more synthetic cytokine receptors, wherein the synthetic cytokine receptor comprises an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain. [0011] In some embodiments, provided herein is a population of cells, comprising at least one engineered cell expressing one or more synthetic cytokine receptors, wherein the synthetic cytokine receptor comprises an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain. [0012] In some embodiments, provided herein is a pharmaceutical composition comprising an engineered cell expressing one or more synthetic cytokine receptors or a population of cells comprising at least one such engineered cell, wherein the synthetic cytokine receptor comprises an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain. [0013] In some embodiments, provided herein is a method of treating a cancer in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. [0014] In some aspects, provided herein is a synthetic cytokine receptor that is a homodimer of identical polypeptide chains each comprising an extracellular domain, a transmembrane domain, and an intracellular domain capable of interleukin 9 receptor (IL-9R) signaling. [0015] In some embodiments, the intracellular domain capable of IL-9R signaling comprises an IL-9R intracellular domain or a variant thereof. In some embodiments, the intracellular domain capable of IL-9R signaling comprises a chimeric JAK/STAT fusion domain. [0016] In some aspects, provided herein is a synthetic cytokine receptor, comprising an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular
30761-20002.40 domain or variant thereof, wherein the synthetic cytokine receptor is a constitutively active cytokine receptor. [0017] In some embodiments, the synthetic cytokine receptor is a multimer. In some embodiments, the synthetic cytokine receptor is a multimer of identical polypeptide chains each comprising the extracellular domain, the transmembrane domain and the IL-9R intracellular domain or variant thereof. In some embodiments, the multimer is a dimer. In some embodiments, the dimer is a homodimer. In some embodiments, each polypeptide chain is constitutively multimerized. [0018] In some embodiments, the synthetic cytokine receptor comprises at least one self- assembly domain. In some embodiments, the at least one self-assembly domain is the extracellular domain and/or the transmembrane domain. [0019] In some embodiments, the synthetic cytokine receptor is multimerized through the transmembrane domain and/or the extracellular domain. In some embodiments, the synthetic cytokine receptor is multimerized through the transmembrane domain and the extracellular domain. [0020] In some aspects, provided herein is a synthetic cytokine receptor that is a homodimer of identical polypeptide chains each comprising an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain or a variant thereof. [0021] In some embodiments, the extracellular domain and/or the transmembrane domain are heterologous to the IL-9R. In some embodiments, the transmembrane domain and extracellular domain are the transmembrane domain and extracellular domain from the same protein. In some embodiments, the transmembrane domain and extracellular domain are the transmembrane domain and extracellular domain from different proteins. [0022] In some embodiments, the transmembrane domain is 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 amino acids in length. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from Glycophorin A (GpA) Carnitine palmitoyltransferase 1 (CPT1), a tumor necrosis factor receptor (TNFR) or Muc24. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from Thrombopoietin receptor. [0023] In some embodiments, the transmembrane domain promotes alpha-helix dimerization. In some embodiments, the transmembrane domain comprises the motif GXXXG (SEQ ID NO: 68). In some embodiments, the transmembrane domain comprises the motif LIxxGVxxGVxxT (SEQ ID NO: 70). [0024] In some embodiments, the transmembrane domain is a transmembrane domain derived from Glycophorin A (GpA) or a variant thereof that comprises one or more mutations (e.g, 1, 2, 3, 4, 5 or 6 mutations) compared to a wild-type GpA transmembrane domain, wherein the variant GpA is sufficient to promote alpha-helix dimerization. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from Glycophorin A (GpA). In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ
30761-20002.40 ID NO: 22 or SEQ ID NO:43. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 43. [0025] In some embodiments, the transmembrane domain comprises GXXXG (SEQ ID NO: 68) and GXXXA (SEQ ID NO: 69) motifs. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from Carnitine palmitoyltransferase 1 (CPT1). In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 45. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 45. [0026] In some embodiments, the synthetic cytokine receptor comprises the motif AXXXA (SEQ ID NO:77) or AXXXS (SEQ ID NO: 78). In some embodiments, the synthetic cytokine receptor comprises the motif ĭPXĭ (SEQ ID NO:75) or ĭTXXAĭ (SEQ ID NO: 76). In some embodiments, the transmembrane domain is derived from a TNFR. In some embodiments, the TNFR is TACI, DR5, p75NTR, Fas, TNFR1, TNFR2 or OX40. [0027] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from DR5. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 46. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 46. [0028] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from TACI. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 44. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 44. [0029] In some embodiments, the transmembrane domain comprises 1 to 6 cysteine residues. In some embodiments, the transmembrane domain promotes disulfide-linked dimerization. In some embodiments, the disulfide-linked dimerization forms 1 to 4 disulfide bridges between polypeptide chains of the synthetic cytokine receptor. [0030] In some embodiments, the transmembrane domain is a variant transmembrane domain that comprises one or more mutations compared to a wild-type transmembrane domain to promote homodimerization of the receptor polypeptide. [0031] In some embodiments, the one or more mutations promote alpha-helix dimerization or disulfide-linked dimerization. In some embodiments, the one or more mutations introduces at least one cysteine into the transmembrane domain. In some embodiments, the one or more mutations introduces a proline into the transmembrane domain. In some embodiments, the one or more mutations introduces a threonine into the transmembrane domain. [0032] In some embodiments, the one or more mutations introduces a trimer peptide of cysteine, proline, and another amino acid other than cysteine or proline into the transmembrane domain. In some embodiments, the one or more mutations introduces a trimer peptide of cysteine, proline, threonine (CPT or TCP) into the transmembrane domain.
30761-20002.40 [0033] In some embodiments, the transmembrane domain is a variant IL-7R transmembrane domain and the one or more mutations is in the wild-type transmembrane sequence PILLTISILSFFSVALLVILACVLW (SEQ ID NO: 71). In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 6, SEQ ID NO:7 or SEQ ID NO: 21. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:21. [0034] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from Muc24. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 23. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 23. [0035] In some embodiments, the extracellular domain is between about 150 to 260 amino acids in length. [0036] In some embodiments, the extracellular domain is a dimerizing domain. In some embodiments, the dimerizing domain comprises a hinge region. In some embodiments, the extracellular domain promotes disulfide-linked dimerization. In some embodiments, the extracellular domain comprises 1 to 6 cysteine residues. In some embodiments, the disulfide-linked dimerization forms 1 to 4 disulfide bridges between polypeptide chains of the synthetic cytokine receptor. [0037] In some embodiments, the extracellular domain is derived from the extracellular domain of CD34, DAP12, Glycophorin A, CD8, or Muc24. In some embodiments, the extracellular domain comprises an extracellular domain derived from Thrombopoietin receptor. [0038] In some embodiments, the extracellular domain comprises an extracellular domain of CD8 or a truncated portion thereof comprising at least one cysteine residue. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 18 or SEQ ID NO: 19. In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19. [0039] In some embodiments, the extracellular domain comprises an extracellular domain of CD34 or a truncated portion thereof comprising at least one cysteine residue. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 4. In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO: 4. [0040] In some embodiments, the extracellular domain is an extracellular domain of Muc24 or a truncated portion thereof comprising at least one cysteine residue. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 20. In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO: 20. [0041] In some embodiments, the extracellular domain is an extracellular domain of DAP12 or a truncated portion thereof comprising at least one cysteine residue. In some embodiments, the
30761-20002.40 extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 5. In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO: 5. [0042] In some embodiments, the extracellular domain is an extracellular domain of Glycophorin A (GpA) or a truncated portion thereof comprising at least one cysteine residue. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 16 or SEQ ID NO: 17. In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17. [0043] In some embodiments, the IL-9R intracellular domain or variant thereof is about 100 to 260 amino acids in length. In some embodiments, the IL-9R intracellular domain or variant thereof comprises a BOX1 motif and/or a BOX2 motif. In some embodiments, the IL-9R intracellular domain or variant thereof comprises a BOX2 motif. In some embodiments, the IL-9R intracellular domain or variant thereof is 230 amino acids in length. [0044] In some embodiments, the IL-9R intracellular domain is wild-type IL-9R intracellular domain or a variant thereof that comprises one or more mutations compared to the wild-type IL-9R intracellular domain set forth in SEQ ID NO: 8. In some embodiments, the one or more mutations comprises one or more amino acid insertions, deletions, and/or substitutions. In some embodiments, the one or more mutations promote signaling through STAT1, STAT3, and/or STAT5 pathways. In some embodiments, the IL-9R intracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises an amino acid sequence of SEQ ID NO: 8. [0045] In some embodiments, the IL-9R intracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises an amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56. [0046] In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66 or SEQ ID NO: 67. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66 or SEQ ID NO: 67.
30761-20002.40 [0047] In some embodiments, the IL-9R intracellular domain or variant thereof comprises one or more amino acid deletions with reference to wild-type IL-9R intracellular domain (SEQ ID NO: 8). [0048] In some embodiments, the IL-9R intracellular domain or variant thereof is a truncated IL- 9R that lacks a contiguous sequence of amino acids at the C-terminus of wild-type IL-9R intracellular domain. In some embodiments, the truncated IL-9R intracellular domain or variant thereof is truncated by between 62 and 99 contiguous amino acids from the C-terminus of wild-type IL-9R intracellular domain. In some embodiments, the IL-9R intracellular domain or variant thereof is a truncated IL-9R that lacks amino acids 132 to 230 of SEQ ID NO:8 or lacks amino acids 134 to 230 of SEQ ID NO:8. [0049] In some embodiments, the IL-9R intracellular domain or variant thereof comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 54, SEQ ID NO: 103, or SEQ ID NO: 104. In some embodiments, the IL-9R intracellular domain or variant thereof comprises an amino acid sequence of SEQ ID NO: 54, SEQ ID NO: 103, or SEQ ID NO: 104. [0050] In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63. [0051] In some embodiments, the IL-9R intracellular domain or variant thereof comprises one or more amino acid substitutions with reference to wild-type IL-9R intracellular domain (SEQ ID NO: 8). [0052] In some embodiments, the IL-9R intracellular domain or variant thereof comprises a STAT binding motif or a variant thereof. In some embodiments, the STAT binding motif comprises a STAT1, STAT3, and/or STAT5 binding motif. In some embodiments, the STAT binding motif comprises YLPQ (SEQ ID NO: 171). In some embodiments, the STAT binding motif comprises a variant STAT binding motif. In some embodiments, the variant STAT binding motif comprises YRPQ (SEQ ID NO: 172). In some embodiments, the variant STAT binding motif comprises YLPL (SEQ ID NO: 173). In some embodiments, the variant STAT binding motif comprises YLKQ (SEQ ID NO: 174). [0053] In some embodiments, the variant IL-9R intracellular domain or variant thereof comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 107 or SEQ ID NO: 108, or SEQ ID NO: 109. In some embodiments, the variant IL-9R intracellular domain comprises an amino acid sequence of SEQ ID NO: 107 or SEQ ID NO: 108, or SEQ ID NO: 109. In some embodiments, the chimeric JAK/STAT fusion domain comprises a JAK binding domain from a type I cytokine receptor and a STAT binding domain from an IL-9R intracellular domain. [0054] In some embodiments, the IL-9R STAT binding domain comprises amino acid residues 73 to 230 of SEQ ID NO: 8. In some embodiments, the STAT binding domain is 59 to 158 amino
30761-20002.40 acids in length and comprises an IL-9R STAT binding motif. In some embodiments, the IL-9R STAT binding motif comprises YLPQ (SEQ ID NO: 171). In some embodiments, the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks a contiguous sequence of amino acids at the N-terminus of SEQ ID NO: 8. In some embodiments, the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks a contiguous sequence of amino acids at the C- terminus of SEQ ID NO: 8. [0055] In some embodiments, the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks amino acids at positions 1 to 72 and/or 132 to 230 of SEQ ID NO: 8. In some embodiments, the IL-9R STAT binding domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 122 or SEQ ID NO: 123. In some embodiments, the IL-9R STAT binding domain comprises an amino acid sequence of SEQ ID NO: 122 or SEQ ID NO: 123. [0056] In some embodiments, the type I cytokine receptor is selected from the group consisting of interleukin 2 receptor (IL-2R), interleukin 4 receptor (IL-4R), interleukin 7 receptor (IL-7R), interleukin 13 receptor (IL-13R), interleukin 15 receptor (IL-15R), and interleukin 2 receptor (IL- 21R). In some embodiments, the type I cytokine receptor is IL-7R. In some embodiments, the IL-7R JAK binding domain is 65 amino acids in length and comprises a box 1 motif. In some embodiments, the IL-7R JAK binding domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 121. In some embodiments, the IL-7R JAK binding domain comprises an amino acid sequence of SEQ ID NO: 121. [0057] In some embodiments, the chimeric JAK/STAT fusion domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 114 or SEQ ID NO: 116. In some embodiments, the chimeric JAK/STAT fusion domain comprises an amino acid sequence of SEQ ID NO: 114 or SEQ ID NO: 116. [0058] In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 113, SEQ ID NO: 115, or SEQ ID NO: 117. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 113, SEQ ID NO: 115, or SEQ ID NO: 117. [0059] In some embodiments, the synthetic cytokine receptor is a constitutively active cytokine receptor. In some embodiments, the synthetic cytokine receptor elicits signaling through STAT1, STAT3, and/or STAT5 pathways. In some embodiments, signaling through STAT1, STAT3, and/or STAT5 is increased compared to STAT1, STAT3, and/or STAT5 signaling via wild-type IL-9R. In some embodiments, signaling through STAT1, STAT3, and/or STAT5 is sustained for a longer period of time compared to STAT1, STAT3, and/or STAT5 signaling via wild-type IL-9R. In some embodiments, sustained STAT1, STAT3, and/or STAT5 signaling is determined by phosphorylation status of STAT1, STAT3, and/or STAT5.In some aspects, provided herein is a polynucleotide encoding any of the synthetic cytokine receptors provided herein.
30761-20002.40 [0060] In some aspects, provided herein is a vector, comprising any one of the polynucleotides provided herein. [0061] In some aspects, provided herein is a method of engineering an isolated cell, comprising contacting the cell with any one of the polynucleotides or any one of the vectors provided herein. In some aspects, provided herein is an engineered cell expressing any one of the synthetic cytokine receptors provided herein. [0062] In some aspects, provided herein is a synthetic cytokine receptor that is a homodimer of identical polypeptide chains each comprising an extracellular domain, a transmembrane domain, and an intracellular domain capable of interleukin 9 receptor (IL-9R) signaling. In some embodiments, the intracellular domain capable of IL-9R signaling comprises an IL-9R intracellular domain or a variant thereof. In some embodiments, the intracellular domain capable of IL-9R signaling comprises a chimeric JAK/STAT fusion domain. [0063] In some aspects, provided herein is an engineered cell expressing a synthetic cytokine receptor, wherein the synthetic cytokine receptor comprises an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain or a variant thereof, wherein the synthetic cytokine receptor is a constitutively active cytokine receptor. [0064] In some embodiments, the synthetic cytokine receptor is a multimer. In some embodiments, the synthetic cytokine receptor is a multimer of identical polypeptide chains each comprising the extracellular domain, the transmembrane domain and the IL-9R intracellular domain or a variant thereof. In some embodiments, the multimer is a dimer. In some embodiments, the dimer is a homodimer. In some embodiments, each polypeptide chain is constitutively multimerized. [0065] In some embodiments, the synthetic cytokine receptor comprises at least one self- assembly domain. In some embodiments, the at least one self-assembly domain is the extracellular domain and/or the transmembrane domain. In some embodiments, the synthetic cytokine receptor is multimerized through the transmembrane domain and/or the extracellular domain. [0066] In some embodiments, the synthetic cytokine receptor is multimerized through the transmembrane domain and the extracellular domain. [0067] In some aspects, provided herein is an engineered cell expressing a synthetic cytokine receptor that is a homodimer of identical polypeptide chains each comprising an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain or variant thereof. In some embodiments, the extracellular domain and/or the transmembrane domain are heterologous to the IL-9R. In some embodiments, the transmembrane domain and extracellular domain are the transmembrane domain and extracellular domain from the same protein. In some embodiments, the transmembrane domain and extracellular domain are the transmembrane domain and extracellular domain from different proteins. [0068] In some embodiments, the transmembrane domain is 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 amino acids in length. In some embodiments, the transmembrane domain comprises
30761-20002.40 a transmembrane domain derived from Glycophorin A (GpA) Carnitine palmitoyltransferase 1 (CPT1), a tumor necrosis factor receptor (TNFR) or Muc24. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from Thrombopoietin receptor. [0069] In some embodiments, the transmembrane domain promotes alpha-helix dimerization. In some embodiments, the transmembrane domain comprises the motif GXXXG (SEQ ID NO: 68). In some embodiments, the transmembrane domain comprises the motif LIxxGVxxGVxxT (SEQ ID NO: 70). [0070] In some embodiments, the transmembrane domain is a transmembrane domain derived from Glycophorin A (GpA) or a variant thereof that comprises one or more mutations (e.g, 1, 2, 3, 4, 5 or 6 mutations) compared to a wild-type GpA transmembrane domain, wherein the variant GpA is sufficient to promote alpha-helix dimerization. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from Glycophorin A (GpA). [0071] In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 22 or SEQ ID NO:43. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 43. [0072] In some embodiments, the transmembrane domain comprises GXXXG (SEQ ID NO: 68) and GXXXA (SEQ ID NO: 69) motifs. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from Carnitine palmitoyltransferase 1 (CPT1). In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 45. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 45. [0073] In some embodiments, the synthetic cytokine receptor comprises the motif AXXXA (SEQ ID NO:77) or AXXXS (SEQ ID NO: 78). In some embodiments, the synthetic cytokine receptor comprises the motif ĭPXĭ (SEQ ID NO:75) or ĭTXXAĭ (SEQ ID NO: 76). In some embodiments, the transmembrane domain is derived from a TNFR. In some embodiments, the TNFR is TACI, DR5, p75NTR, Fas, TNFR1, TNFR2 or OX40. [0074] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from DR5. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 46. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 46. [0075] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from TACI. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 44. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 44. [0076] In some embodiments, the transmembrane domain comprises 1 to 6 cysteine residues. In some embodiments, the transmembrane domain promotes disulfide-linked dimerization. In some
30761-20002.40 embodiments, the disulfide-linked dimerization forms 1 to 4 disulfide bridges between polypeptide chains of the synthetic cytokine receptor. [0077] In some embodiments, the transmembrane domain is a variant transmembrane domain that comprises one or more mutations compared to a wild-type transmembrane domain to promote homodimerization of the receptor polypeptide. In some embodiments, the one or more mutations promote alpha-helix dimerization or disulfide-linked dimerization. [0078] In some embodiments, the one or more mutations introduces at least one cysteine into the transmembrane domain. In some embodiments, the one or more mutations introduces a proline into the transmembrane domain. In some embodiments, the one or more mutations introduces a threonine into the transmembrane domain. In some embodiments, the one or more mutations introduces a trimer peptide of cysteine, proline, and another amino acid other than cysteine or proline into the transmembrane domain. In some embodiments, the one or more mutations introduces a trimer peptide of cysteine, proline, threonine (CPT or TCP) into the transmembrane domain. [0079] In some embodiments, the transmembrane domain is a variant IL-7R transmembrane domain and the one or more mutations is in the wild-type transmembrane sequence PILLTISILSFFSVALLVILACVLW (SEQ ID NO: 71). [0080] In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 6, SEQ ID NO:7 or SEQ ID NO: 21. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:21. [0081] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from Muc24. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 23. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 23. [0082] In some embodiments, the extracellular domain is between about 150 to 260 amino acids in length. In some embodiments, the extracellular domain is a dimerizing domain. In some embodiments, the dimerizing domain comprises a hinge region. In some embodiments, the extracellular domain promotes disulfide-linked dimerization. In some embodiments, the extracellular domain comprises 1 to 6 cysteine residues. In some embodiments, the disulfide-linked dimerization forms 1 to 4 disulfide bridges between polypeptide chains of the synthetic cytokine receptor. [0083] In some embodiments, the extracellular domain is derived from the extracellular domain of CD34, DAP12, Glycophorin A, CD8, or Muc24. In some embodiments, the extracellular domain comprises an extracellular domain derived from Thrombopoietin receptor. [0084] In some embodiments, the extracellular domain comprises an extracellular domain of CD8 or a truncated portion thereof comprising at least one cysteine residue. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ
30761-20002.40 ID NO: 18 or SEQ ID NO: 19. In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19. [0085] In some embodiments, the extracellular domain comprises an extracellular domain of CD34 or a truncated portion thereof comprising at least one cysteine residue. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 4. In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO: 4. [0086] In some embodiments, the extracellular domain is an extracellular domain of Muc24 or a truncated portion thereof comprising at least one cysteine residue. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 20. In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the extracellular domain is an extracellular domain of DAP12 or a truncated portion thereof comprising at least one cysteine residue. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 5. In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO: 5. [0087] In some embodiments, the extracellular domain is an extracellular domain of Glycophorin A (GpA) or a truncated portion thereof comprising at least one cysteine residue. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 16 or SEQ ID NO: 17. In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17. [0088] In some embodiments, the IL-9R intracellular domain or variant thereof is about 100 to 260 amino acids in length. In some embodiments, the IL-9R intracellular domain or variant thereof comprises a BOX1 motif and/or a BOX2 motif. In some embodiments, the IL-9R intracellular domain or variant thereof comprises a BOX2 motif. In some embodiments, the IL-9R intracellular domain or variant thereof is 230 amino acids in length. [0089] In some embodiments, the IL-9R intracellular domain is wild-type IL-9R intracellular domain or a variant thereof that comprises one or more mutations compared to the wild-type IL-9R intracellular domain set forth in SEQ ID NO: 8. [0090] In some embodiments, the one or more mutations comprises one or more amino acid insertions, deletions, and/or substitutions. In some embodiments, the one or more mutations promote signaling through STAT1, STAT3, and/or STAT5 pathways. [0091] In some embodiments, the IL-9R intracellular domain or variant thereof comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain or variant thereof comprises an amino acid sequence of SEQ ID NO: 8. [0092] In some embodiments, the IL-9R intracellular domain or variant thereof comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID
30761-20002.40 NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain or variant thereof comprises an amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56. [0093] In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66 or SEQ ID NO: 67. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66 or SEQ ID NO: 67. [0094] In some embodiments, the IL-9R intracellular domain or variant thereof comprises one or more amino acid deletions with reference to wild-type IL-9R intracellular domain (SEQ ID NO: 8). In some embodiments, the IL-9R intracellular domain or variant thereof is a truncated IL-9R that lacks a contiguous sequence of amino acids at the C-terminus of wild-type IL-9R intracellular domain. In some embodiments, the truncated IL-9R intracellular domain or variant thereof is truncated by between 62 and 99 contiguous amino acids from the C-terminus of wild-type IL-9R intracellular domain. [0095] In some embodiments, the IL-9R intracellular domain or variant thereof is a truncated IL- 9R that lacks amino acids 132 to 230 of SEQ ID NO:8 or lacks amino acids 134 to 230 of SEQ ID NO:8. [0096] In some embodiments, the IL-9R intracellular domain or variant thereof comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 54, SEQ ID NO: 103, or SEQ ID NO: 104. In some embodiments, the IL-9R intracellular domain or variant thereof comprises an amino acid sequence of SEQ ID NO: 54, SEQ ID NO: 103, or SEQ ID NO: 104. [0097] In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63. [0098] In some embodiments, the IL-9R intracellular domain or variant thereof comprises one or more amino acid substitutions with reference to wild-type IL-9R intracellular domain (SEQ ID NO: 8).
30761-20002.40 [0099] In some embodiments, the IL-9R intracellular domain or variant thereof comprises a STAT binding motif or a variant thereof. In some embodiments, the STAT binding motif comprises a STAT1, STAT3, and/or STAT5 binding motif. In some embodiments, the STAT binding motif comprises YLPQ (SEQ ID NO: 171). In some embodiments, the STAT binding motif comprises a variant STAT binding motif. In some embodiments, the variant STAT binding motif comprises YRPQ (SEQ ID NO: 172). In some embodiments, the variant STAT binding motif comprises YLPL (SEQ ID NO: 173). In some embodiments, the variant STAT binding motif comprises YLKQ (SEQ ID NO: 174). In some embodiments, the variant IL-9R intracellular domain or variant thereof comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 107 or SEQ ID NO: 108, or SEQ ID NO: 109. [0100] In some embodiments, the variant IL-9R intracellular domain comprises an amino acid sequence of SEQ ID NO: 107 or SEQ ID NO: 108, or SEQ ID NO: 109. [0101] In some embodiments, the chimeric JAK/STAT fusion domain comprises a JAK binding domain from a type I cytokine receptor and a STAT binding domain from an IL-9R intracellular domain. [0102] In some embodiments, the IL-9R STAT binding domain comprises amino acid residues 73 to 230 of SEQ ID NO: 8. [0103] In some embodiments, the STAT binding domain is 59 to 158 amino acids in length and comprises an IL-9R STAT binding motif. In some embodiments, the IL-9R STAT binding motif comprises YLPQ (SEQ ID NO: 171). In some embodiments, the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks a contiguous sequence of amino acids at the N- terminus of SEQ ID NO: 8. In some embodiments, the IL-9R STAT binding domain is a truncated IL- 9R STAT binding domain that lacks a contiguous sequence of amino acids at the C-terminus of SEQ ID NO: 8. [0104] In some embodiments, the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks amino acids at positions 1 to 72 and/or 132 to 230 of SEQ ID NO: 8. In some embodiments, the IL-9R STAT binding domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 122 or SEQ ID NO: 123. In some embodiments, the IL-9R STAT binding domain comprises an amino acid sequence of SEQ ID NO: 122 or SEQ ID NO: 123. [0105] In some embodiments, the type I cytokine receptor is selected from the group consisting of interleukin 2 receptor (IL-2R), interleukin 4 receptor (IL-4R), interleukin 7 receptor (IL-7R), interleukin 13 receptor (IL-13R), interleukin 15 receptor (IL-15R), and interleukin 2 receptor (IL- 21R). In some embodiments, the type I cytokine receptor is IL-7R. [0106] In some embodiments, the IL-7R JAK binding domain is 65 amino acids in length and comprises a box 1 motif. In some embodiments, the IL-7R JAK binding domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 121. In some embodiments, the IL- 7R JAK binding domain comprises an amino acid sequence of SEQ ID NO: 121. In some
30761-20002.40 embodiments, the chimeric JAK/STAT fusion domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 114 or SEQ ID NO: 116. In some embodiments, the chimeric JAK/STAT fusion domain comprises an amino acid sequence of SEQ ID NO: 114 or SEQ ID NO: 116. [0107] In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 113, SEQ ID NO: 115, or SEQ ID NO: 117. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 113, SEQ ID NO: 115, or SEQ ID NO: 117. [0108] In some embodiments, the synthetic cytokine receptor is a constitutively active cytokine receptor. [0109] In some embodiments, the synthetic cytokine receptor elicits signaling through STAT1, STAT3, and/or STAT5 pathways. In some embodiments, signaling through STAT1, STAT3, and/or STAT5 is increased compared to STAT1, STAT3, and/or STAT5 signaling via wild-type IL-9R. In some embodiments, signaling through STAT1, STAT3, and/or STAT5 is sustained for a longer period of time compared to STAT1, STAT3, and/or STAT5 signaling via wild-type IL-9R. In some embodiments, sustained STAT1, STAT3, and/or STAT5 signaling is determined by phosphorylation status of STAT1, STAT3, and/or STAT5. [0110] In some embodiments, the engineered cell further expresses at least one different type of engineered receptor. In some embodiments, the at least one different type of engineered receptor is a chimeric antigen receptor. In some embodiments, the extracellular domain of the chimeric antigen receptor binds to an antigen expressed on a cancer cell. In some embodiments, the extracellular domain of the chimeric antigen receptor binds to an idiotype of an antibody. In some embodiments, the antibody is against an antigen expressed on a cancer cell. In some embodiments, the cancer cell is a blood cancer cell or a solid tumor cancer cell. [0111] In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the engineered cell is an immune effector cell. In some embodiments, the cell is a T cell or a Natural Killer (NK) cell. In some embodiments, the cell is a T cell and the T cell is a CD4+ T cell or a CD8+ T cell. In some embodiments, the immune effector cell is a cytotoxic T cell. In some embodiments, the immune effector cell is a natural killer cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a human cell. In some aspects, provided herein is a population of cells, comprising at least one of any of the engineered cells provided herein. In some embodiments, the at least one engineered cell comprises engineered CD4+ T cells and engineered CD8+ T cells. [0112] In some aspects, provided herein is a pharmaceutical composition comprising any of the engineered cells provided herein or any of the populations of cells provided herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutical acceptable carrier.
30761-20002.40 In some embodiments, the pharmaceutical composition further comprises a cryoprotectant. In some embodiments, the pharmaceutical composition is for use in treating a cancer in a subject. [0113] In some aspects, provided herein is a method of treating a disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions provided herein. In some embodiments, the disease or condition is a cancer. [0114] In some embodiments, the engineered cell of the pharmaceutical composition expresses an engineered antigen receptor that binds to an antigen expressed on a cell of the cancer. In some embodiments, the engineered antigen receptor is a chimeric antigen receptor. In some embodiments, the engineered antigen receptor is a T cell receptor. [0115] In some aspects, provided herein is a method of improving function of an immune cell, comprising introducing to the immune cell any one of the polynucleotides or vectors provided herein, wherein improving immune cell function comprises increased STAT1, STAT3, and/or STAT5 signaling compared to an immune cell expressing wild-type IL-9R. [0116] In some aspects, provided herein is a method of improving cytotoxicity of an immune cell, comprising introducing to the immune cell any one of the polynucleotides or vectors provided herein, wherein improving immune cell cytotoxicity comprises increased target cell killing compared to an immune cell expressing wild-type IL-9R. [0117] In some aspects, provided herein is a method of improving viability of an immune cell, comprising introducing to the immune cell any one of the polynucleotides or vectors provided herein, wherein improving viability of the immune cell comprises decreased immune cell death compared to an immune cell expressing wild-type IL-9R. [0118] In some aspects, provided herein is a method of improving cytokine secretion by an immune cell, comprising introducing to the immune cell any one of the polynucleotides or vectors provided herein, wherein improving cytokine secretion comprises increased secretion of interferon compared to an immune cell expressing wild-type IL-9R. [0119] In some embodiments, the interferon comprises IFNȖ. In some embodiments, the immune cell further comprises an engineered antigen receptor. In some embodiments, the one different type of engineered receptor is a chimeric antigen receptor (CAR). In some embodiments, the extracellular domain of the CAR binds to an antigen expressed on a cancer cell. In some embodiments, the cancer cell is a blood cancer cell or a solid tumor cancer cell. [0120] In some embodiments, the immune cell is a lymphocyte. In some embodiments, the immune cell is an effector cell. In some embodiments, the immune cell is a T cell or NK cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a T cell and the T cell is a CD4+ T cell or a CD8+ T cell. In some embodiments, the immune cell is a cytotoxic T cell. In some embodiments, the immune cell is a NK cell. In some embodiments, the immune cell is a primary cell.
30761-20002.40 [0121] In some embodiments, provided herein is a method of improving function of an immune cell, comprising introducing to the immune cell any of the polynucleotides or vectors provided herein, thereby improving function of the immune cell. [0122] In some embodiments, improving immune cell function comprises one or more of increased STAT signaling, increased cytotoxicity, increased proliferation, increased viability, increased cytokine secretion, and increased cytotoxic protein secretion compared to a reference cell. [0123] In some embodiments, the reference cell comprises a non-engineered immune cell or an engineered immune cell. In some embodiments, the engineered immune cell expresses an engineered antigen receptor. In some embodiments, the engineered antigen receptor is a wild-type IL-9R, a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [0124] In some embodiments, increased STAT signaling comprises increased STAT1, STAT3 and/or STAT5 signaling. In some embodiments, increased cytotoxicity comprises increased killing of a target cell. In some embodiments, increased cytokine secretion comprises increased secretion of one or more of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-10 (IL-10), and TNFĮ. In some embodiments, increased cytotoxic protein secretion comprises increased secretion of one or more of granzyme A, granzyme B, granulysin and perforin. In some embodiments, increased viability comprises decreased cell death. [0125] In some embodiments, the immune cell is a lymphocyte. In some embodiments, the immune cell is an effector cell. In some embodiments, the immune cell is a T cell or NK cell. In some embodiments, the immune cell is a T cell and the T cell is a CD4+ T cell or a CD8+ T cell. In some embodiments, the immune cell is a cytotoxic T cell. In some embodiments, the immune cell is a NK cell. In some embodiments, the immune cell is a primary cell. BRIEF DESCRIPTION OF THE FIGURES [0126] FIG.1 shows the design of exemplary synthetic cytokine receptors. [0127] FIG.2 shows the expression of exemplary synthetic cytokine receptors on the surface of primary human CD3-positive T cells, including CD8+ T cells (left panel) or CD4+ T cells (right panel). [0128] FIGS.3A and 3B show the surface expression level of CD27 and CD45RA 5 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing the 9RC synthetic cytokine receptor or in primary human CD3- positive T cells that were untransduced (“UTD”). FIG.3A is a representative flow cytometry dot plot with CD45RA staining on the x-axis and CD27 staining on the y-axis. FIG.3B depicts the percent of T cells that were positive for CD45RA and CD27 in UTD T cells or in 9RC+ transduced T cells. [0129] FIG.4 shows the surface expression level of CD27 and CD45RA 12 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing an exemplary
30761-20002.40 synthetic cytokine receptor (9RC, 9RC2, or 9RD2) or in primary human CD3-positive T cells that were untransduced. [0130] FIG.5 shows the surface expression level of CD27 and Fas 16 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing an exemplary synthetic cytokine receptor (9RC, 9RC2, or 9RD2) or in primary human CD3-positive T cells that were untransduced. [0131] FIG.6A shows the surface expression level of CD27 and Fas among all cells 16 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing an exemplary synthetic cytokine receptor (9RC or 9RC2), the exemplary synthetic cytokine receptor 9RC co-expressed with a CAR separated by a 2A sequence (designated “40bbz-2a-CD34-9RC”), or in untransduced cells. FIG.6B shows the surface expression level of CD27 and Fas among Flag-positive cells 16 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing an exemplary synthetic cytokine receptor (9RC or 9RC2) or the exemplary synthetic cytokine receptor 9RC co-expressed with a CAR separated by a 2A cleavable linker (designated “40bbz-2a-CD34-9RC”). [0132] FIG.7A shows the surface expression level of CCR7 and CD45RA among all cells 16 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing an exemplary synthetic cytokine receptor (9RC or 9RC2), the exemplary synthetic cytokine receptor 9RC co-expressed with a CAR separated by a 2A cleavable linker (designated “40bbz-2a-CD34- 9RC”), or in untransduced cells. FIG.7B shows the surface expression level of CCR7 and CD45RA among Flag-positive cells 16 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing an exemplary synthetic cytokine receptor (9RC or 9RC2) or the exemplary synthetic cytokine receptor 9RC co-expressed with a CAR separated by a 2A cleavable linker (designated “40bbz-2a-CD34-9RC”). [0133] FIG.8 shows the surface expression level of CD27 in 9RC expressing primary human CD3-positive T cells based on geometric mean fluorescence intensity (gMFI) as determined by flow cytometry. [0134] FIGS.9A-9B show the surface expression level of CD57 in 9RC expressing primary human CD3-positive T cells, as determined by co-staining for the FLAG tag after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing the 9RC synthetic cytokine receptor or in primary human CD3- positive T cells that were untransduced (“UTD”). FIG.9A is a representative flow cytometry dot plot with CD57 staining on the y-axis and staining for FLAG on the x-axis. FIG.9B depicts the percent of CD8+ T cells that were high for CD57 expression (CD57hi) in UTD T cells or in 9RC+ transduced T cells. [0135] FIG.10 shows the surface expression level of TIM3 and LAG3 in primary human CD3- positive T cells that were co-transduced with lentiviral vectors expressing the 9RC synthetic cytokine receptor and a CAR separated by either a 2A sequence (40bbz-2a-FLAG-9RC”) or a IRES sequence
30761-20002.40 (40bbz-IRES-CD34-9RC), or in primary human CD3- positive T cells that were untransduced (“UTD”). A representative flow cytometry dot plot is shown with TIM3 staining on the y-axis and LAG3 staining on the x-axis. [0136] FIG.11 shows the surface expression level of CD4 and CD8 in primary human CD3- positive T cells 5 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing an exemplary synthetic cytokine receptor (9RC, 9RC2, or 9RD2) or in primary human CD3-positive T cells that were untransduced. [0137] FIG.12 illustrates the growth of primary human CD3-positive T cells expressing an exemplary synthetic cytokine receptor (9RC or 9RC2), the exemplary synthetic cytokine receptor 9RC co-expressed with a CAR (designated “40bbz-9RC”), or in untransduced (UTD) cells during extended in vitro culture. The results depict the day of culture post-activation and the fold growth at the respective day compared to the number of cells at the previous count for each condition. [0138] FIGS.13A-13E show the design and expression of additional exemplary synthetic cytokine receptors comprising an IL7R TMD and an IL-9R ICD. Each additional synthetic receptor construct had a different extracellular domain (ECD) as follows: GpA (construct “G-7-9R”; FIG. 13A), truncated GpA (construct “tG-7-9R”; FIG.13B), CD8 (construct “CD8-7-9R”; FIG.13C), truncated CD8 (construct “tCD8-7-9R”; FIG.13D), or Muc24 (construct “M-7-9R”; FIG.13E). [0139] FIGS.14A-14B show the design and expression of additional exemplary synthetic cytokine receptors comprising an IL-9R ICD and a transmembrane domain other than an IL7R TMD. FIG.14A depicts a synthetic cytokine construct with a Glycophorin A (GpA) ECD, a GpA TMD and an IL-9R ICD (“G-G-9R”). FIG.14B depicts a synthetic cytokine receptor with a Muc24 ECD, a Muc24 TMD and an IL-9R ICD (“M-M-9R”). [0140] FIGS.15A-15H show the designs and expression data of additional exemplary synthetic cytokine receptors comprising an IL9R ICD, and full-length or truncated ECD from GpA, CD8 or Muc24 and a TMD from GpA, truncated GpA (designated GpA*), TACI, CPT1 or DR5. The depicted constructs include an ECD, TMD and ICD as follows: truncated GpA ECD, GpA TMD and IL9R ICD (construct “tGpA-G-9R”; FIG.15A), CD8 ECD, GpA TMD and IL9R ICD (construct “CD8-G- 9R”; FIG.15B), truncated CD8 ECD, GpA TMD and IL9R ICD (construct “tCD8-G-9R”; FIG. 15C), Muc24 ECD, GpA TMD and IL9R ICD (construct “M24-G-9R”; FIG.15D), truncated CD8 ECD, truncated GpA TMD and IL9R ICD (construct “tCD8-G*-9R”; FIG.15E), truncated CD7 ECD, TACI TMD and IL9R ICD (construct “tCD8-T-9R”; FIG.15F), truncated CD8 ECD, CPT1 TMD and IL9R ICD (construct “tCD8-C-9R”; FIG.15G), and truncated CD8 ECD, DR5 TMD and IL9R ICD (construct “tCD8-D-9R”; FIG.15H). [0141] FIG.16 shows transduction efficiency of exemplary synthetic cytokine receptors in primary human CD3-positive T cells 5 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing the exemplary synthetic cytokine receptor or in
30761-20002.40 untransduced T cells (UTD). Transduction efficiency was determined by flow cytometry by cell surface staining for cells positive for the Flag tag. [0142] FIGS.17A-17C show the surface expression level of CD27 and CD45RA 5 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing the exemplary synthetic cytokine receptors. The figure provides representative flow cytometry contour plots with CD45RA staining on the x-axis and CD27 staining on the y-axis. [0143] FIG.18 shows in vivo survival of tumor-bearing mice administered primary human CD3- positive T cells co-expressing a CAR (40BBz) and an exemplary synthetic cytokine receptor, as compared to primary human CD3-positive T cells expressing the CAR only or that were un- transduced (UTD). [0144] FIG.19 shows STAT phosphorylation (pSTAT) in primary human CD3-positive T cells expressing exemplary synthetic cytokine receptors. pSTAT was measured at 0 hours, 20 minutes, 2 hours, and 24 hours post-stimulation with cytokine. [0145] FIG.20 shows the expression of an exemplary synthetic cytokine receptor on the surface of primary human CD3-positive T cells. [0146] FIG.21 shows STAT phosphorylation (pSTAT) of STAT1, STAT3, and STAT5 in primary human CD3-positive T cells expressing exemplary synthetic cytokine receptors. [0147] FIG.22 shows the number of HCT116 tumor cells per field of view (FOV) across time in a T cell-mediated killing assay. T cells were obtained from two healthy donors and cocultured with tumor cells at an effector:target (E:T) ratio of 1:2. [0148] FIG.23 shows the number of HCT116 tumor cells per field of view (FOV) across time in a T cell-mediated killing assay. T cells were obtained from a healthy donor and cocultured with tumor cells at an effector:target (E:T) ratio of 1:4. [0149] FIG.24 shows phenotype markers CD27 and CD45RA in primary T cells expressing exemplary synthetic cytokine receptors. [0150] FIG.25 shows the total number of T cells co-expressing a CAR (40BBz) and a synthetic cytokine receptor at plating and after two rounds of T cell-mediated killing. [0151] FIG.26 shows the percentage of CD8 T cells positive for cytokine expression after target stimulation. CD8 T cells either co-expressed a CAR (40BBz) and an exemplary synthetic cytokine receptor or a CAR alone. Measured cytokines included IL-2, IFN-Ȗ, and TNFĮ. [0152] FIG.27 shows the percentage of live T cells after two rounds of stimulation. T cells co- expressing a CAR (40BBz) and synthetic cytokine receptor were cocultured with K562 cells expressing an antigen recognized by the CAR (40BBz). [0153] FIGS.28A-28B show the percentage of T cells expressing a CAR (40BBz) and exemplary synthetic cytokine receptors post-transduction.9RC5 = C-7-9R(a); 9RC5.1 = CD8-7- 9R(c); 9RC5.2 = tCD8-7-9R(c).
30761-20002.40 [0154] FIG.29 shows STAT phosphorylation (pSTAT) in primary human CD3-positive T cells co-expressing a CAR (40BBz) and exemplary synthetic cytokine receptors. pSTAT1, pSTAT3 and pSTAT5 are represented as a percentage of the highest signal observed for each of the individual STATs. [0155] FIG.30 shows the percentage of CD8+ T cells expressing cytokines after 4-hour stimulation with target cells. CD8+ T cells either co-expressed a CAR (40BBz) and an exemplary synthetic cytokine receptor or only a CAR. Measured cytokines included IFN-Ȗ, IL-2, IL-10, and TNFĮ. [0156] FIG.31 shows cytokine and effector molecule secretion by CD8+ T cells after 24-hour stimulation with target cells. CD8+ T cells either co-expressed a CAR (40BBz) and an exemplary synthetic cytokine receptor or only a CAR. Cytokine and effector molecule secretion are displayed as a percentage relative to the highest expressor of each analyte. [0157] FIGS.32A-32B show CD45RA and CD27 staining of CD8+ T cells in a continuous co- culture with target cells. CD8+ T cells either co-expressed a CAR (40BBz) and an exemplary synthetic cytokine receptor or only a CAR. FIG.32A depicts CD45RA and CD27 expression across three weeks. FIG.32B depicts summary data for three technical replicates at the week three timepoint. [0158] FIGS.33A-33C show live CD8+ T cell counts in a continuous co-culture with target cells. CD8+ T cells either co-expressed a CAR (40BBz) and an exemplary synthetic cytokine receptor or only a CAR. FIG.33A depicts live CD8+ T cell counts across three weeks. FIG.33B depicts live CD8+ T cells at week three. FIG.33C depicts CD39 expression in CD8+ T cells at week three. [0159] FIG.34 shows expansion of CD8+ T cells cultured with or without IL-2 for four weeks. CD8+ T cells either co-expressed a CAR (40BBz) and an exemplary synthetic cytokine receptor or only a CAR. [0160] FIG.35 shows tumor growth in tumor-bearing mice administered primary human CD3- positive T cells co-expressing a CAR (40BBz) and an exemplary synthetic cytokine receptor, as compared to primary human CD3-positive T cells expressing the CAR only or that were un- transduced (UTD). Tumor-bearing mice were administered 1 x 106 cells. [0161] FIG.36 shows tumor growth in tumor-bearing mice administered primary human CD3- positive T cells co-expressing a CAR (40BBz) and an exemplary synthetic cytokine receptor, as compared to primary human CD3-positive T cells expressing the CAR only or that were un- transduced (UTD). Tumor-bearing mice were administered 3 x 105 cells. DETAILED DESCRIPTION [0162] The present disclosure provides synthetic cytokine receptors capable of driving interleukin-9 (IL-9) receptor (IL-9R) signaling in the absence of its cognate cytokine ligand (i.e.,
30761-20002.40 constitutively active). These synthetic cytokine receptors comprise an intracellular domain derived from the intracellular domain of human IL-9 receptor alpha (IL-9RĮ), which can activate STAT1, STAT3, and STAT5 and trigger a strong JAK/STAT signal cascade. These synthetic cytokine receptors also comprise various modified intracellular domains, transmembrane domains, and extracellular domains. Immune cells such as T cells expressing these synthetic cytokine receptors are advantageous because such T cells assume characteristics of stem memory T cells without exhibiting signs of malignant transformation. Such T cells can be used in adoptive immunotherapy. [0163] In some embodiments, the mechanism of constitutive activation comprises multimerization of the extracellular domain. In some embodiments, the mechanism of constitutive activation comprises multimerization of the transmembrane domain. In some embodiments, the mechanism of constitutive activation comprises multimerization of the intracellular domain. In some embodiments, the mechanism of constitutive activation comprises multimerization of at least two domains (e.g., the extracellular domain and the transmembrane domain). In some embodiments, the mechanism of constitutive activation comprises multimerization of three domains (i.e., the extracellular domain, the transmembrane domain, and the intracellular domain). [0164] Adoptively transferred genetically engineered immune cells (e.g., T cells) have shown substantial anti-tumor activity in patients with hematopoietic malignancies but have limited efficacy in solid tumors. This is due to the immunosuppressive environment of solid tumors, the inefficient infiltration of solid tumors, and chronic antigen stimulation, which leads to lack persistence and/or efficacy. In some cases, multipotency and replicative potential are also diminished. Thus, there is a need for compositions and/or methods that reduce or eliminate immune cell exhaustion, lack persistence, and/or decreased efficacy. [0165] The provided disclosure addresses these needs. The present disclosure demonstrates that the provided synthetic chimeric IL-9 receptors, even in the absence of ligand, have the ability to constitutively activate cells in which they are expressed, such as T cells. In some embodiments, the activation triggers a strong JAK/STAT signal cascade where STAT1, STAT3, and STAT5 can all be activated. In some embodiments, the activation has the ability to result in T cells that overcome T cell exhaustion by polarizing T cells toward a naiver phenotype and skewing the ratio of CD4:CD8, such as to a ratio of 2:1, without triggering malignant transformation of T cells. Thus, the present disclosure demonstrates that the constitutive IL-9R disclosed herein has the potential to reprogram and alter the phenotype of immune cells to overcome exhaustion, which can improve anti-tumor activity in solid tumors. I. Definitions [0166] Unless otherwise defined herein, technical, and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa unless the content
30761-20002.40 clearly dictates otherwise. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control. [0167] The terms “a”, “an”, and “the”, as used herein, include plural references unless the context clearly dictates otherwise. [0168] The term “about”, as used herein, in reference to a number or range of numbers, is understood to mean the stated number and numbers +/- 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range. [0169] The terms “or” and “and/or”, as used herein, include any, and all, combinations of one or more of the associated listed items. [0170] The terms “including”, “includes”, “included”, and other forms, as used herein, are not limiting. [0171] The terms “comprise” and its grammatical equivalents, as used herein, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. [0172] The term “administer”, “administration”, or “administering”, as used herein refers to the act of injecting or otherwise physically delivering a substance (e.g., a pharmaceutical composition provided herein) to a subject (e.g., human), such as by oral, mucosal, topical, intradermal, parenteral, intravenous, intravitreal, intraarticular, subretinal, intramuscular, intrathecal delivery and/or any other method of physical delivery described herein or known in the art. The delivery can be systemic or to a specific tissue. [0173] The term “binds” or “binding”, as used herein, refers to a covalent or non-covalent interaction between molecules (e.g., forming a complex by interactions). Exemplary non-covalent interactions include hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. With regard to terms such as “specific binding,” “specifically binds to,” or “is specific for” means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. [0174] The term “chimeric antigen receptor” or “CAR”, as used herein, refers to a genetically engineered receptor, which can be used to graft one or more antigen specificity onto immune effector cells, such as T cells and NK cells. CARs are also known as “artificial T-cell receptors,” “chimeric T cell receptors,” or “chimeric immune receptors.” “CAR-T cell” refers to a T cell that expresses a CAR. “CAR-NK cell” refers to an NK cell that expresses a CAR. [0175] The term “constitutively active”, as used in the context of a receptor, refers to a receptor capable of activating downstream signaling (e.g., interleukin-9 signaling) in the absence of the cognate ligand of the receptor.
30761-20002.40 [0176] The term “contact”, as used in the context of contacting a target cell with a compound or another cell, is intended to include incubating the target cell and the compound or the other cell together. [0177] The term “effective amount” or “therapeutically effective amount”, as used herein, refers to an amount of a therapeutic (e.g., a pharmaceutical composition provided herein) which is sufficient to treat, diagnose, prevent, delay the onset of, reduce and/or ameliorate the severity and/or duration of a given condition, disorder, or disease and/or a symptom related thereto. The term also encompasses an amount necessary for the reduction, slowing, or amelioration of the advancement or progression of a given disease, reduction, slowing, or amelioration of the recurrence, development or onset of a given disease, and/or to improve or enhance the prophylactic or therapeutic effect (s) of another therapy or to serve as a bridge to another therapy. [0178] The term “immune effector cell”, as used herein, refers to cells involved in mounting innate and adaptive immune responses, including but not limited to lymphocytes, natural killer (NK) cells, NKT cells, macrophages, monocytes, eosinophils, basophils, neutrophils, dendritic cells, and mast cells. In some embodiments, the immune effector cell is a lymphocyte. In some embodiments, the immune effector cell is a cytotoxic T cell, such as a CD4+ T cell, a CD8+ T cell (also referred to as a cytotoxic T cell or CTL), a regulatory T cell (Treg), a Th1 cell, a Th2 cell, or a Th17 cell. [0179] The term “leucine zipper domain”, as used herein, refers to an amphipathic Į helix containing heptad repeats of Leu residues on one face of the helix and serves as a dimerization module. In some embodiments, the leucine zipper domain is an active leucine zipper domain. On dimerization with another leucine zipper domain, the leucine-zipper Į helices form a parallel-coiled coil based on hydrophobic interfacial side-chain packing. In some embodiments, the leucine zipper domain is an inactive leucine zipper domain that cannot form a dimer with another leucine zipper domain. [0180] The term “naturally-occurring”, as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by a human in the laboratory is naturally occurring. [0181] The term “pharmaceutically acceptable excipient, carrier or diluent”, as used herein, refers to any substance formulated alongside the active ingredient of a pharmaceutical composition that allows the active ingredient to retain biological activity and is non-reactive with the subject’s immune system. Such a substance can be included for the purpose of long-term stabilization, bulking up solid formulations that contain potent active ingredients in small amounts, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating absorption, reducing viscosity, or enhancing solubility. The selection of appropriate substance can depend upon the route of administration and the dosage form, as well as the active ingredient and
30761-20002.40 other factors. Compositions having such substances can be formulated by well-known conventional methods (see, e.g., Remington, The Science and Practice of Pharmacy, 23rd edition, A. Adejare, ed., Academic Press, 2020). [0182] The term “pharmaceutical composition” or “therapeutic composition”, as used herein, refers to a composition capable of being administered to a subject for the treatment of a particular disease or disorder. [0183] The term “polynucleotide” or “nucleic acid”, as used herein, refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA/RNA hybrids. Polynucleotides may be single-stranded or double-stranded and either recombinant, synthetic, or isolated. Polynucleotides include but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotides can comprises modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction. [0184] The terms “polypeptide” and “peptide” and “protein”, as used herein, refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, including but not limited to, unnatural amino acids, as well as other modifications known in the art. [0185] The term “population” of cells, as used herein, refers to any number of cells greater than 1, but is preferably at least about 1x103 cells, at least about 1x104 cells, at least about 1x105 cells, at least about 1x106 cells, at least about 1x107 cells, at least about 1x108 cells, at least about 1x109 cells, at least about 1x1010 cells, at least about 1x1011 or more cells. A population of cells may refer to an in vitro population (e.g., a population of cells in culture) or an in vivo population (e.g., a population of cells residing in a particular tissue). [0186] The term “sequence identity”, as used herein, refers to the percentage of bases or amino acids between two polynucleotide or polypeptide sequences that are the same, and in the same relative position. As such one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. The term “reference sequence” refers to a molecule to which a test sequence is compared. Methods of sequence
30761-20002.40 alignment for comparison and determination of percent sequence identity and percent complementarity are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology), by use of algorithms know in the art including the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res.25:3389-3402; and Altschul et al., (1990) J. Mol. Biol.215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score l 00, word length-! 2 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, word length-3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res., 1997, 25:3389-402. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI BLAST programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:1117. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. [0187] The term “subject”, as used herein, refers to an “animal” and in particular a “mammal” such as a non-primate (e.g., mice, rats, bovines, horses, household cats, tigers and other large cats, dogs, pigs, rabbits, goats, deer, sheep, ferrets, gerbils, guinea pigs, hamsters, bats, and birds (e.g., chickens, turkeys, and ducks)) or a primate (e.g., monkeys, baboons, chimpanzees, and human). The term may be used interchangeably with the term “patient” or “individual”. In some embodiments, the subject is a mammal, e.g., a human, diagnosed with a disease or disorder provided herein. In some embodiments, the subject is a mammal, e.g., a human, at risk of developing a disease or disorder provided herein. In some embodiments, the subject is human. [0188] The term “synthetic”, as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that cannot be directly isolated from a source in nature. In some embodiments, the synthetic nucleic acid, polypeptide, cell, or organism is
30761-20002.40 substantially similar as compared to the corresponding natural-occurring one. In some embodiments, the synthetic nucleic acid, polypeptide, cell, or organism is altered or changed as compared to the corresponding natural-occurring one. In some embodiments, the synthetic nucleic acid, polypeptide, cell, or organism is produced by functionally linking or combining different fragments of sourcing nucleic acids, polypeptides, cells, or organisms together. For instance, a synthetic polypeptide can comprise different sourcing polypeptides functionally linked together. [0189] The terms “treatment” and “treating”, as used herein, refer to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made. [0190] The term “vector”, as used herein, refers to a substance that is used to carry or introduce a nucleic acid sequence (e.g., a nucleic acid sequence encoding a synthetic cytokine receptor as described herein) into a host cell. Vectors applicable for use include, for example, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes. A vector may include sequences that direct autonomous replication in a cell or may include sequences sufficient to allow integration into host cell DNA. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., both an antibody heavy and light chain or an antibody VH and VL), both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test
30761-20002.40 the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art. [0191] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001 ); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. II. Synthetic Cytokine Receptor [0192] Provided herein is a synthetic cytokine receptor. In some embodiments, the synthetic cytokine receptor is capable of interleukin 9 receptor (IL-9R) signaling. [0193] In some embodiments, the synthetic cytokine receptor comprises an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain. In some embodiments, the synthetic cytokine receptor provided herein can multimerize, typically as a homodimer, to facilitate downstream signaling in the absence of external stimuli of the receptor, such as in the absence of binding of a ligand to the extracellular domain. Thus, in some embodiments, the synthetic cytokine receptor is constitutively active. In some embodiments, multimerization (e.g, dimerization) of a synthetic cytokine receptor allows the receptor to be constitutively active by orienting polypeptide chains of the receptor so that JAK/STAT signaling molecules are recruited thereto for subsequent activation. Typically, IL-9R signaling requires binding of its cytokine to the IL-9R alpha chain to promote heterodimerization with the common gamma chain subunit, activation of JAK kinases (e.g., JAK1 and JAK3) for association with and phosphorylation of the receptor chains, and subsequent phosphorylation and recruitment of other signaling molecules such as STAT transcription factors, in particular STAT1, STAT3 and STAT5. In provided embodiments, synthetic receptor spontaneously form homo-multimers, such as homodimers, that constitutively activate JAK signaling resulting in activation of STAT1, STAT3 and/or STAT5, including translocation of homodimers of STAT-1, STAT-3 or STAT-5 as well as their heterodimers (e.g., STAT-1/STAT-3) to the nucleus for regulating gene expression. [0194] In some embodiments, engineered cells (e.g., T cells) expressing the synthetic cytokine receptors provided herein, and in some cases also expressing a chimeric antigen receptor (CAR), maintain the ability to proliferate even in the absence of a ligand of the extracellular domain (e.g., absence of a cytokine).
30761-20002.40 [0195] In some embodiments, the synthetic cytokine receptor comprises a multimer. In some embodiments, the multimer is a dimer. [0196] In some embodiments, the synthetic cytokine receptor multimerizes in higher orders (e.g., trimer, tetramer, or higher). In some embodiments, the synthetic cytokine receptor multimerizes as a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer or decamer. In some embodiments, the multimer is a trimer. In some embodiments, the multimer is a tetramer. In some embodiments, the multimer is a pentamer. In some embodiments, the multimer is a hexamer. In some embodiments, the multimer is a heptamer. In some embodiments, the multimer is an octamer. In some embodiments, the multimer is a nonamer. In some embodiments, the multimer is a decamer. In some embodiments, the multimer is an undecamer. In some embodiments, the multimer is a dodecamer. [0197] In some embodiments, the multimer comprises identical polypeptide chains, each comprising the extracellular domain, the transmembrane domain, and the IL-9R intracellular domain. In some embodiments, the multimer is a homo-multimer. In some embodiments, the homo-multimer is a homo-dimer. In some embodiments, the homo-multimer is a homo-trimer. In some embodiments, the homo-multimer is a homo-tetramer. In some embodiments, the homo-multimer is a homo- pentamer. In some embodiments, the homo-multimer is a homo-hexamer. In some embodiments, the homo-multimer is a homo-heptamer. In some embodiments, the homo-multimer is a homo-octamer. In some embodiments, the homo-multimer is a homo-nonamer. In some embodiments, the homo- multimer is a homo-decamer. In some embodiments, the homo-multimer is a homo-undecamer. In some embodiments, the homo-multimer is a homo-dodecamer. In some embodiments, the multimer is a hetero-multimer. [0198] In some embodiments, the synthetic cytokine receptor is multimerized through the extracellular domain, transmembrane domain, and/or IL-9R intracellular domain. In some embodiments, the synthetic cytokine receptor is multimerized through the extracellular domain. In some embodiments, the synthetic cytokine receptor is multimerized through the transmembrane domain. In some embodiments, the synthetic cytokine receptor is multimerized through the IL-9R intracellular domain. In some embodiments, the synthetic cytokine receptor is multimerized through both the extracellular domain and the transmembrane domain. In some embodiments, the synthetic cytokine receptor is multimerized through both the transmembrane domain and the IL-9R intracellular domain. In some embodiments, the synthetic cytokine receptor is multimerized through both the extracellular domain and the IL-9R intracellular domain. In some embodiments, the synthetic cytokine receptor is multimerized through the extracellular domain, the transmembrane domain, and the IL-9R intracellular domain. [0199] In some embodiments, the synthetic cytokine receptor is a constitutively active cytokine receptor. In some embodiments, the synthetic cytokine receptor is a multimer of polypeptide chains that is constitutively multimerized. In some embodiments, a constitutively multimerized synthetic cytokine receptor is constitutively active. In some embodiments, the multimerized synthetic cytokine
30761-20002.40 receptor is constitutively active because each polypeptide chain of the multimer permits multimerization without the need for external stimuli of the receptor. In some embodiments, the constitutively multimerized synthetic cytokine receptor elicits signaling through a STAT pathway. [0200] In some embodiments, an advantage of the synthetic cytokine receptors provided herein is that the receptors are constitutively active in the absence of ligand. That is, downstream IL-9R signaling is constitutive in the absence of ligand binding to the ECD of the synthetic cytokine receptor. However, ligand binding to the ECD of the synthetic cytokine receptors provided herein is not precluded. In some embodiments, the ECD of the synthetic cytokine receptors provided herein bind their cognate ligand. However, constitutive signaling of a provided synthetic cytokine receptor is not dependent on ECD ligand binding. Thus, in some embodiments, the synthetic cytokine receptors provided herein are constitutively active in the absence and presence of one or more ligands. In some embodiments, the synthetic cytokine receptors provided herein are constitutively active in the presence or absence of any ligand. In particular embodiments, when the ECD of the synthetic cytokine receptors provided herein is derived from a CD34 receptor, the synthetic cytokine receptor is constitutively active in the presence or absence of CD34 receptor ligand. In particular embodiments, when the ECD of the synthetic cytokine receptors provided herein is derived from a DAP12 receptor, the synthetic cytokine receptor is constitutively active in the presence or absence of DAP12 receptor ligand. In particular embodiments, when the ECD of the synthetic cytokine receptors provided herein is derived from a GpA receptor, the synthetic cytokine receptor is constitutively active in the presence or absence of GpA receptor ligand. In particular embodiments, when the ECD of the synthetic cytokine receptors provided herein is derived from a CD8 receptor, the synthetic cytokine receptor is constitutively active in the presence or absence of CD8 receptor ligand. In particular embodiments, when the ECD of the synthetic cytokine receptors provided herein is derived from a Muc24 receptor, the synthetic cytokine receptor is constitutively active in the presence or absence of Muc24 receptor ligand. In particular embodiments, the ECD does not substantially bind, such as does not bind specifically or with high affinity, the native ligand of the receptor from which the ECD is derived. In some embodiments, the native ligand of the receptor from which the ECD is derived does not bind the ECD of the synthetic cytokine receptor in a manner that could modulate intracellular signaling (e.g., STAT signaling) by the synthetic cytokine receptor. In particular embodiments, the synthetic cytokine receptors provided herein elicit IL-9R signaling in the absence of IL-9. In some embodiments, the synthetic cytokine receptors provided herein do not bind IL-9. [0201] In some embodiments, the constitutively multimerized synthetic cytokine receptor is a constitutively active cytokine receptor that elicits signaling through STAT1, STAT3 or STAT5 pathway. In some embodiments, the constitutively multimerized synthetic cytokine receptor elicits signaling through STAT1 pathway. In some embodiments, the multimerized synthetic cytokine receptor elicits signaling through STAT3 pathway. In some embodiments, the constitutively multimerized synthetic cytokine receptor elicits signaling through STAT5 pathway. In some
30761-20002.40 embodiments, the constitutively multimerized synthetic cytokine receptor elicits signaling through STAT1, STAT3, and STAT5 pathways at the same time. In some embodiments, the constitutively multimerized synthetic cytokine receptor elicits signaling through STAT1, STAT3, and STAT5 pathways sequentially in any order. Signaling through STAT1, STAT3, and/or STAT5 can be determined by any method known in the art. In some embodiments, signaling through STAT1, STAT3, and/or STAT5 is determined by detecting phosphorylated STAT1, STAT3 and/or STAT5. As an example, STAT1, STAT3 and/or STAT5 phosphorylation can be detected by Western blot with antibodies that specifically bind to phosphorylated STAT1, STAT3, and/or STAT5. [0202] In some embodiments, the synthetic cytokine receptor that multimerizes in higher orders (e.g, timer, tetramer or higher) elicits stronger signaling when compared to dimerized synthetic cytokine receptor. In some embodiments, the elicited signaling of the higher ordered multimerized synthetic cytokine receptor is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% stronger than the dimerized synthetic cytokine receptor. In some embodiments, the elicited signaling of the higher ordered multimerized synthetic cytokine receptor is at least about 1 time, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, or at least about 9 times stronger than the dimerized synthetic cytokine receptor. In some embodiments, the elicited signaling of a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, undecamer, and/or dodecamer synthetic cytokine receptor is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% stronger than the dimerized synthetic cytokine receptor. In some embodiments, the elicited signaling of a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, undecamer, and/or dodecamer synthetic cytokine receptor is at least about 1 time, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, or at least about 9 times stronger than the dimerized synthetic cytokine receptor. [0203] In some embodiments, stronger signaling can be demonstrated by sustained or prolonged STAT activation. In some embodiments, STAT activation can be shown by detecting phosphorylated STAT. In some embodiments, STAT1, STAT3, and/or STAT5 phosphorylation can be detected by Western blot with antibodies that specifically bind to phosphorylated STAT1, STAT3, and/or STAT5. In some embodiments, signaling through STAT1, STAT3, and/or STAT5 can be determined by any method known in the art. [0204] In some embodiments, synthetic cytokine receptor multimerization occurs through a covalent or noncovalent interaction. [0205] In some embodiments, synthetic cytokine receptor multimerization occurs through a covalent interaction. In some embodiments, each synthetic cytokine receptor is linked to each other
30761-20002.40 via one or more covalent links within the multimer. In some embodiments, the covalent link is a disulfide bond. In some embodiments, the disulfide bond is formed between cysteine residues. [0206] In some embodiments, the synthetic cytokine receptor comprises a self-assembly domain to promote multimerization of the receptor. Self-assembly is the spontaneous association of an ensemble of molecules into one or more supramolecular structures, driven by multiple noncovalent interactions. In some embodiments, synthetic cytokine receptor multimerization occurs through a noncovalent interaction. In some embodiments, the noncovalent interaction is promoted by an amino acid sequence motif. In some embodiments, the amino acid sequence motif promotes alpha-helix dimerization or multimerization. In some embodiments, the amino acid sequence motif promotes leucine-zipper dimerization or multimerization. [0207] It is understood that reference to amino acids, including to a specific sequence set forth as a SEQ ID NO used to describe domain organization (e.g. of a extracellular domain, transmembrane domain or intracellular domain) are for illustrative purposes and are not meant to limit the scope of the embodiments provided. It is understood that polypeptides and the description of domains thereof are theoretically derived based on homology analysis and alignments with similar molecules. Thus, the exact locus can vary, and is not necessarily the same for each protein. Hence, a specific domain can be several amino acids (such as one, two, three or four) longer or shorter. Typically, it will be understood that any transmembrane domain(s) identified for the polypeptides herein are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified. In some embodiments, the extracellular domain, when it is free of the transmembrane and cytoplasmic domains, is one that may be soluble (i.e. is not membrane bound). It is understood that the intracellular signaling domain is a IL-9R sequence or a variant or portion thereof that exhibits ability to elicit intracellular signaling such as by recruiting JAK and STAT molecules (e.g., STAT1, STAT3 and STAT5). [0208] In some embodiments, the different domains (i.e., extracellular domain, transmembrane domain and intracellular domain) of the synthetic cytokine receptor are operatively linked. In some embodiments, the different elements are directly linked to each other. In some embodiments, the different elements are linked via a peptide linker, such as a (GxS)n linker, wherein x can be an integer between 1 and 10, and n can be an integer between 2 and 20. Additional linkers are known to a skilled artisan. A. Extracellular Domain [0209] In some embodiments, the extracellular domain comprises the extracellular domain of a naturally occurring protein. In some embodiments, the extracellular domain comprises a fragment or a truncated portion of the extracellular domain of a naturally occurring protein. [0210] In some embodiments, the extracellular domain in a provided synthetic cytokine receptor allows the synthetic cytokine receptor to be constitutively active by permitting multimerization (e.g.,
30761-20002.40 dimerization) of its polypeptide chains to allow the downstream intracellular domains of the polypeptide chains to recruit JAK/STAT molecules in the absence of an external stimuli, such as in the absence of binding ligand to the extracellular domain. [0211] In some embodiments, the extracellular domain may or may not be from the same natural molecule as the transmembrane domain to which it is operably linked. In some embodiments, the extracellular domain is from the same molecule as its corresponding transmembrane domain. In some embodiments, the extracellular domain is not from the same molecule as its corresponding transmembrane domain. [0212] In some embodiments, the extracellular domain comprises one or more mutations compared to its corresponding wild-type protein. In some embodiments, a mutation may comprise a substitution, insertion, deletion, or any combination thereof. [0213] In some embodiments, the extracellular domain interacts with one or more additional extracellular domains to promote the multimerization of the synthetic cytokine receptor. [0214] In some embodiments, the extracellular domain stabilizes the synthetic cytokine receptor expressed on the surface of a cell. In some embodiments, the extracellular domain increases downstream signaling triggered by IL-9R intracellular domain. In some embodiments extracellular domain imparts a sink or ligand trap function, such as binding up of one or more molecules that would be harmful to the cells expressing the synthetic cytokine receptor. In some embodiments, the ligand is immunosuppressive. Examples of harmful ligands include TGF-beta, PD-Ll, IL-4, IL-13, IL-8, and IL-10. [0215] In some embodiments, the extracellular domain disclosed herein is capable of binding a ligand but the ligand’s signal is not transmitted. [0216] In some embodiments, the size of the extracellular domain is at least about 30 amino acids. In some embodiments, the size of the extracellular domain is at most about 300 amino acids in length. In some embodiments, the size of the extracellular domain is about 30 amino acids to about 300 amino acids. In some embodiments, the size of the extracellular domain is about 30 amino acids to about 50 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 110 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 210 amino acids, about 30 amino acids to about 250 amino acids, about 30 amino acids to about 300 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 110 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 190 amino acids, about 50 amino acids to about 210 amino acids, about 50 amino acids to about 250 amino acids, about 50 amino acids to about 300 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino
30761-20002.40 acids to about 130 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 170 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 210 amino acids, about 70 amino acids to about 250 amino acids, about 70 amino acids to about 300 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 150 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 210 amino acids, about 90 amino acids to about 250 amino acids, about 90 amino acids to about 300 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 210 amino acids, about 110 amino acids to about 250 amino acids, about 110 amino acids to about 300 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to about 170 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 210 amino acids, about 130 amino acids to about 250 amino acids, about 130 amino acids to about 300 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 210 amino acids, about 150 amino acids to about 250 amino acids, about 150 amino acids to about 300 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 210 amino acids, about 170 amino acids to about 250 amino acids, about 170 amino acids to about 300 amino acids, about 190 amino acids to about 210 amino acids, about 190 amino acids to about 250 amino acids, about 190 amino acids to about 300 amino acids, about 210 amino acids to about 250 amino acids, about 210 amino acids to about 300 amino acids, or about 250 amino acids to about 300 amino acids. In some embodiments, the size of the extracellular domain is about 30 amino acids, about 50 amino acids, about 70 amino acids, about 90 amino acids, about 110 amino acids, about 130 amino acids, about 150 amino acids, about 170 amino acids, about 190 amino acids, about 210 amino acids, about 250 amino acids, or about 300 amino acids. [0217] In some embodiments, the size of the extracellular domain is about 10 amino acids to about 260 amino acids in length. In some embodiments, the size of the extracellular domain is about 60 amino acids to 260 amino acids in length. In some embodiments, the size of the extracellular domain is about 69 amino acids in length. In some embodiments, the size of the extracellular domain is about 72 amino acids in length. In some embodiments, the size of the extracellular domain is about 139 amino acids in length. In some embodiments, the size of the extracellular domain is about 259 amino acids in length. [0218] In some embodiments, the extracellular domain comprises a dimerizing domain. In some embodiments, the dimerizing domain comprises a hinge region. In some embodiments, the extracellular domain promotes disulfide-linked dimerization. In some embodiments, disulfide-linked dimerization is caused or facilitated by one or more cysteine residues. In some embodiments, the extracellular domain comprises 1 to 6 cysteine residues. In some embodiments, the extracellular
30761-20002.40 domain comprises 1 cysteine residue. In some embodiments, the extracellular domain comprises 2 cysteine residues. In some embodiments, the extracellular domain comprises 3 cysteine residues. In some embodiments, the extracellular domain comprises 4 cysteine residues. In some embodiments, the extracellular domain comprises 5 cysteine residues. In some embodiments, the extracellular domain comprises 6 cysteine residues. In some embodiments, the disulfide-linked dimerization forms 1 to 4 disulfide bridges between polypeptide chains of the synthetic cytokine receptor. In some embodiments, the disulfide-linked dimerization forms 1 disulfide bridge between the polypeptide chains of the synthetic cytokine receptor. In some embodiments, the disulfide-linked dimerization forms 2 disulfide bridges between the polypeptide chains of the synthetic cytokine receptor. In some embodiments, the disulfide-linked dimerization forms 3 disulfide bridges between the polypeptide chains of the synthetic cytokine receptor. In some embodiments, the disulfide-linked dimerization domain forms 4 disulfide bridges between the polypeptide chains of the synthetic cytokine receptor. [0219] In some embodiments, the extracellular domain comprises an extracellular domain derived from CD8, CD34, Muc24, DAP12, or Glycophorin A (GpA). In some embodiments, the extracellular domain is derived from the extracellular domain of CD34. In some embodiments, the extracellular domain is derived from the extracellular domain of DAP12. In some embodiments, the extracellular domain is derived from the extracellular domain of Glycophorin A. In some embodiments, the extracellular domain is derived from the extracellular domain of CD8. In some embodiments, the extracellular domain is derived from the extracellular domain of Muc24. [0220] In some embodiments, the extracellular domain comprises an extracellular domain of CD8 or a truncated portion thereof. In some embodiments, the CD8 extracellular domain comprises at least one cysteine residue. [0221] In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 18. In some embodiments, the extracellular domain comprises an amino acid sequence of SEQ ID NO: 18. [0222] In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about
30761-20002.40 88% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the CD8 extracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 18. [0223] In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 19. In some embodiments, the extracellular domain comprises an amino acid sequence of SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about
30761-20002.40 90% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the CD8 extracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 185. In some embodiments, the CD8 extracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 185. In some embodiments, the CD8 extracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 185. [0224] In some embodiments, the extracellular domain comprises an extracellular domain of CD34 or a truncated portion thereof. In some embodiments, the CD34 extracellular domain comprises at least one cysteine residue. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 4. In some embodiments, the extracellular domain comprises an amino acid sequence of SEQ ID NO: 4. [0225] In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain
30761-20002.40 comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the CD34 extracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 4. [0226] In some embodiments, the extracellular domain is an extracellular domain of Muc24 or a truncated portion thereof. In some embodiments, the Muc24 extracellular domain comprises at least one cysteine residue. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 20. In some embodiments, the extracellular domain comprises an amino acid sequence of SEQ ID NO: 20. [0227] In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 20. In some embodiments, the Muc24
30761-20002.40 extracellular domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the Muc24 extracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 20. [0228] In some embodiments, the extracellular domain is an extracellular domain of DAP12 or a truncated portion thereof. In some embodiments, the DAP12 extracellular domain comprises at least one cysteine residue. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 5. In some embodiments, the extracellular domain comprises an amino acid sequence of SEQ ID NO: 5. [0229] In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 5. In some embodiments, the DAP12
30761-20002.40 extracellular domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the DAP12 extracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 5. [0230] In some embodiments, the extracellular domain is an extracellular domain of GpA or a truncated portion thereof. In some embodiments, the GpA extracellular domain comprises at least one cysteine residue. [0231] In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 16. In some embodiments, the extracellular domain comprises an amino acid sequence of SEQ ID NO: 16. [0232] In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino
30761-20002.40 acids that is at least about 86% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 16. In some embodiments, the GpA extracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the GpA extracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 16. [0233] In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 17. In some embodiments, the extracellular domain comprises an amino acid sequence of SEQ ID NO: 17. [0234] In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 87% identical to SEQ
30761-20002.40 ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 17. In some embodiments, the GpA extracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the GpA extracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 17. [0235] In some embodiments, the extracellular domain is an active leucine zipper domain of a transcription factor. In some embodiments, the transcription factor is a basic leucine zippers (bZIP). Non-limiting examples of bZIP transcription factors that can be used herein include ATF, JUN, CREB, Fos. In some embodiments, the extracellular domain is the leucine zipper domain of ATF. In some embodiments, the extracellular domain is the leucine zipper domain of JUN. In some embodiments, the extracellular domain is the leucine zipper domain of CREB. In some embodiments, the extracellular domain is the leucine zipper domain of Fos. [0236] In some embodiments, the leucine zipper comprises Leu-X6-Leu-X6-Leu-X6-Leu (SEQ ID NO: 72). In some embodiments, the leucine zipper comprises MKQIEDKLEEILSKLYHIENELARIKKLLGE (SEQ ID NO: 73). In some embodiments, the leucine zipper comprises MKQIEDKIEEILSKIYHIENEIARIKKLIGE (SEQ ID NO: 74). [0237] In some embodiments, the extracellular domain is an extracellular domain of Thrombopoietin Receptor (TpoR) or a truncated portion thereof. In some embodiments, the extracellular domain is a truncated portion of TpoR. In some embodiments, the truncated TpoR extracellular domain comprises deletion of up to 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino
30761-20002.40 acid residues from the N-terminus of wild-type TpoR extracellular domain set forth in SEQ ID NO: 178). [0238] In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 178. In some embodiments, the TpoR extracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 178. In some embodiments, the TpoR extracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 178. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 162. In some embodiments, the extracellular domain comprises an amino acid sequence of SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 162. In some
30761-20002.40 embodiments, the TpoR extracellular domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 162. In some embodiments, the TpoR extracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 162. [0239] In some embodiments, the extracellular domain provided herein can comprise any one of the extracellular domains set forth in Table 1. As indicated, the exact locus or residues corresponding to a given domain can vary, such as depending on the methods used to identify or classify the domain. Also, in some cases, adjacent N- and/or C-terminal amino acids of a given extracellular domain also can be included in a sequence of a synthetic cytokine receptor, so long as the resulting synthetic cytokine receptor is able to constitutively multimerize (e.g., dimerize) to result in constitutive activation of downstream signaling molecules. Thus, it is understood that the exemplification of the SEQ ID NOS in Table 1 is not to be construed as limiting. For example, the particular extracellular domain can be several amino acids longer or shorter, such as 1-10, e.g., 1, 2, 3, 4, 5, 6 or 7 amino acids longer or shorter, than the sequence of amino acids set forth in the respective SEQ ID NO. Also provided are variants of any such SEQ ID NO (e.g., sequence that exhibits at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, such as at least about 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NOS: 4, 5, 16, 17, 18, 19, 20, 73, 74, 162, 178 or 185), in which the resulting synthetic cytokine receptor containing such a variant extracellular domain is able to constitutively multimerize (e.g., dimerize) to result in constitutive activation of downstream signaling molecules.
B. Transmembrane Domain [0240] In some embodiments, the transmembrane domain of the synthetic cytokine receptor comprises the transmembrane domain of a naturally occurring protein. In some embodiments, the transmembrane domain comprises a fragment or a truncated portion of the transmembrane domain of a naturally occurring protein.
30761-20002.40 [0241] In some embodiments, the transmembrane domain in a provided synthetic cytokine receptor allows the synthetic cytokine receptor to be constitutively active by permitting multimerization (e.g., dimerization) of its polypeptide chains to allow the downstream intracellular domains of the polypeptide chains to recruit JAK/STAT molecules in the absence of an external stimuli, such as in the absence of binding ligand to the extracellular domain. [0242] In some embodiments, the transmembrane domain may or may not be from the same natural molecule as either the extracellular domain to which it is operably linked. In particular embodiments the transmembrane is not from the same natural molecule as the extracellular domain to which it is operably linked. [0243] In some embodiments, the transmembrane domain comprises one or more mutations compared to the transmembrane domain of its corresponding wild-type protein. In some embodiments, the one or more mutations promote, cause, or facilitate the multimerization of the transmembrane domain compared to its corresponding wildtype protein. In some embodiments, the transmembrane domain comprises one or more mutations that change the configuration of the downstream intracellular domain compared to its corresponding wildtype protein. In some embodiments, the transmembrane domain comprises one or more mutations that cause or facilitate the multimerization of the transmembrane domain and change the configuration of the downstream intracellular domain. Upon multimerization and/or change of configuration, the downstream IL-9R intracellular domain is oriented relative to each other in a manner that is conducive to signaling. [0244] The mutation(s) may be a substitution, insertion, deletion, or combination thereof. In some embodiments, the one or more mutations comprises at least one cysteine. In some embodiments, the one or more mutations comprises at least one proline. Methods of determining whether a particular mutation will trigger the downstream signaling is known to a skilled artisan (e.g., assaying for STAT1, STAT3, or STAT5 phosphorylation following growth of the cells harboring the receptor being tested in the absence of growth factors).In some embodiments, the one or more mutations of a transmembrane domain is introduction of at least one cysteine residue. In some embodiments, introduction of at least one cysteine residue, such as by amino acid substitution, induces disulfide bond formation in the transmembrane domain. In some embodiments, the one or more mutations does not include introduction of a cysteine. For example, a variant transmembrane domain may be utilized that does not have a cysteine insertion(s) or amino acid substitution (such as for a disulfide bond with an adjacent polypeptide chain) but that still signals and is constitutively active, for example because the mutation rendered the transmembrane domain conformationally changed compared to a natural version of the transmembrane domain, thereby allowing induction of signaling. For example, insertion or amino acid substitution of an amino acid such as a proline produces a "kink" that twists the transmembrane domain, which can induce signaling (see, e.g., Shochat et al, 2011, J Exp Med.2011 May 9;208(5):901-8; Zenatti et al, 2011, Nat Genet.2011 Sep 4;43(10):932-9). In some embodiments, the one or more mutations of a transmembrane domain is introduction of a proline residues. In some
30761-20002.40 embodiments, the mutation is, or comprises, the insertion or amino acid substitution of one or more cysteines, and/or one or more prolines. In some embodiments, the mutation is by introduction (e.g., insertion or amino acid substitution) of a trimer peptide of cysteine, proline, and another amino acid other than cystine or proline (e.g., threonine) into the transmembrane domain. In some embodiments, a trimer peptide confers the disulfide bond formation between the -SH (thiol) groups of cysteine residues of two molecules, allowing a homodimer to form between them (the proline immediately following the cysteine helps to twist the homodimer into the correct orientation, in specific embodiments). In some embodiments, the trimer peptide is a trimer peptide of cysteine, proline and threonine, in some cases a CPT insertion or in some cases a TCP insertion. [0245] In some embodiments, the size of the transmembrane domain is at least about 15 amino acids. In some embodiments, the size of the transmembrane domain is about 15 amino acids to about 45 amino acids. In some embodiments, the size of the transmembrane domain is at most about 45 amino acids. In some embodiments, the size of the transmembrane domain is about 15 amino acids to about 20 amino acids, about 15 amino acids to about 25 amino acids, about 15 amino acids to about 30 amino acids, about 15 amino acids to about 35 amino acids, about 15 amino acids to about 40 amino acids, about 15 amino acids to about 45 amino acids, about 20 amino acids to about 25 amino acids, about 20 amino acids to about 30 amino acids, about 20 amino acids to about 35 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 45 amino acids, about 25 amino acids to about 30 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 45 amino acids, about 30 amino acids to about 35 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 45 amino acids, about 35 amino acids to about 40 amino acids, about 35 amino acids to about 45 amino acids, or about 40 amino acids to about 45 amino acids. In some embodiments, the size of the transmembrane domain is about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, or about 45 amino acids. [0246] In some embodiments, the transmembrane domain is about 21 amino acids, about 22 amino acids, about 23 amino acids, about 24 amino acids, about 25 amino acids, about 26 amino acids, about 27 amino acids, about 28 amino acids, about 29 amino acids, about 30 amino acids, about 31 amino acids, about 32 amino acids or about 33 amino acids in length. In some embodiments, the transmembrane domain is 21 amino acids in length. In some embodiments, the transmembrane domain is 22 amino acids in length. In some embodiments, the transmembrane domain is 23 amino acids in length. In some embodiments, the transmembrane domain is 24 amino acids in length. In some embodiments, the transmembrane domain is 25 amino acids in length. In some embodiments, the transmembrane domain is 26 amino acids in length. In some embodiments, the transmembrane domain is 27 amino acids in length. In some embodiments, the transmembrane domain is 28 amino acids in length. In some embodiments, the transmembrane domain is 29 amino acids in length. In some embodiments, the transmembrane domain is 30 amino acids in length. In some embodiments,
30761-20002.40 the transmembrane domain is 31 amino acids in length. In some embodiments, the transmembrane domain is 32 amino acids in length. In some embodiments, the transmembrane domain is 33 amino acids in length. [0247] In some embodiments, the transmembrane domain promotes dimerization. [0248] In some embodiments, the transmembrane domain is derived from the transmembrane domain of interleukin 7 receptor. In some embodiments, the transmembrane domain is derived from the transmembrane domain of Glycophorin A. In some embodiments, the transmembrane domain is derived from the transmembrane domain of Muc24. [0249] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from Glycophorin A (GpA) Carnitine palmitoyltransferase 1 (CPT1), or a tumor necrosis factor receptor (TNFR) or Muc24. In some embodiments, the transmembrane domain is a variant or a truncated form of a transmembrane domain derived from Glycophorin A (GpA) Carnitine palmitoyltransferase 1 (CPT1), or a tumor necrosis factor receptor (TNFR) or Muc24. [0250] In some embodiments, the transmembrane domain promotes alpha-helix dimerization. In some embodiments, alpha-helix oligomerization, such as dimerization or trimerization, is driven by sequence motifs, which are simple recognizable amino acid sequences that promote lateral interaction. In some embodiments, the transmembrane domain contains a proline rich motif ĭPXĭ (SEQ ID NO: 75), in which ĭ represents a hydrophobic residue, P is proline and X can be any amino acid, typically an apolar residue except for proline and glycine. In some embodiments, the transmembrane domain contains a ĭTXXAĭ, in which ĭ represents a hydrophobic residue, T is threonine, X can be any amino acid, typically an apolar residue except for proline and glycine, and A is alanine. In some embodiments, the transmembrane domain contains a GXXXG motif (SEQ ID NO:68). In some embodiments, the transmembrane domain contains a GXXXA motif (SEQ ID NO:69). In some embodiments, the transmembrane domain contains an AXXXA motif (SEQ ID NO: 77). In some embodiments, the transmembrane domain contains an AXXXS motif (SEQ ID NO: 78). [0251] In some embodiments, the transmembrane domain comprises the motif GXXXG (SEQ ID NO: 68). In some embodiments, the transmembrane domain comprises the motif LIxxGVxxGVxxT (SEQ ID NO: 70). In some embodiments, the transmembrane domain comprises the motif GXXXG (SEQ ID NO: 68) and the motif LIxxGVxxGVxxT (SEQ ID NO: 70). [0252] In some embodiments, the transmembrane domain is a transmembrane domain derived from Glycophorin A (GpA) or a variant thereof. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from the transmembrane domain of Glycophorin A (GpA). In some embodiments, the transmembrane domain is a variant of the transmembrane domain of Glycophorin A (GpA). In some embodiments, the variant comprises one or more mutations, such as 1, 2, 3, 4, 5 or 6 mutations (e.g., amino acid substitutions), compared to a wild-type GpA transmembrane domain. In some embodiments, the one or more mutations promote alpha-helix dimerization. In some embodiments, the GpA transmembrane domain or the variant GpA
30761-20002.40 transmembrane domain comprises the motif GXXXG (SEQ ID NO: 68). In some embodiments, the GpA transmembrane domain or the variant GpA transmembrane domain comprises the motif LIxxGVxxGVxxT (SEQ ID NO: 70). In some embodiments, the GpA transmembrane domain or the variant GpA transmembrane domain comprises the motif GXXXG (SEQ ID NO: 68) and the motif LIxxGVxxGVxxT (SEQ ID NO: 70). [0253] In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 22. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 22. [0254] In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises a sequence of amino acids that is at least about 99% identical to
30761-20002.40 SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain comprises the amino acid sequence to set forth in SEQ ID NO: 22. In some embodiments, the GpA transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 22. [0255] In some embodiments, the transmembrane domain is a variant GpA transmembrane domain. In some embodiments, the transmembrane domain comprises a sequence of amino acids that exhibits at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 43. In some embodiments, the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 43.
30761-20002.40 [0256] In some embodiments, the transmembrane domain comprises motif GXXXG (SEQ ID NO: 68) and GXXXA (SEQ ID NO: 69). [0257] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from carnitine palmitoyltransferase 1 (CPT1). In some embodiments, the CPT1 transmembrane domain or a variant of the CPT1 transmembrane domain comprises the motif GXXXG (SEQ ID NO: 68) and GXXXA (SEQ ID NO: 69). [0258] In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that exhibits at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 45. In some embodiments, the CPT1 transmembrane domain comprises the amino acid sequence set forth in SEQ
30761-20002.40 ID NO: 45. In some embodiments, the CPT1 transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 45. [0259] In some embodiments, the transmembrane domain promotes disulfide-linked dimerization. In some embodiments, the transmembrane domain comprises 1 to 6 cysteine residues. In some embodiments, the transmembrane domain comprises 1 cysteine residue. In some embodiments, the transmembrane domain comprises 2 cysteine residues. In some embodiments, the transmembrane domain comprises 3 cysteine residues. In some embodiments, the transmembrane domain comprises 4 cysteine residues. In some embodiments, the transmembrane domain comprises 5 cysteine residues. In some embodiments, the transmembrane domain comprises 6 cysteine residues. In some embodiments, the disulfide-linked dimerization comprises a disulfide bridge. In some embodiments, the disulfide- linked dimerization comprises 1 to 4 disulfide bridges between polypeptide chains of the synthetic cytokine receptor. In some embodiments, the disulfide-linked dimerization forms 1 disulfide bridge between polypeptide chains of the synthetic cytokine receptor. In some embodiments, the disulfide- linked dimerization forms 2 disulfide bridges between polypeptide chains of the synthetic cytokine receptor. In some embodiments, the disulfide-linked dimerization forms 3 disulfide bridges between polypeptide chains of the synthetic cytokine receptor. In some embodiments, the disulfide-linked dimerization domain forms 4 disulfide bridges between polypeptide chains of the synthetic cytokine receptor. [0260] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from interleukin 7 receptor (IL-7R). In some embodiments, the IL-7R transmembrane domain is a variant transmembrane domain. In some embodiments, the variant transmembrane domain comprises one or more mutations compared to a wild-type IL-7R transmembrane domain. In some embodiments, the one or more mutations promote homodimerization of the synthetic cytokine receptor. In some embodiments, the one or more mutations promote disulfide-linked dimerization. In some embodiments, the one or more mutations introduces at least one cysteine into the transmembrane domain. In some embodiments, one or more mutations introduces at least one proline into the transmembrane domain. In some embodiments, the mutation is, or comprises, the insertion or amino acid substitution of one or more cysteines, and/or one or more prolines. In some embodiments, the mutation is by introduction (e.g., insertion or amino acid substitution) of a trimer peptide of cysteine, proline, and another amino acid other than cystine or proline (e.g., threonine) into the IL-7R transmembrane domain. In some embodiments, the trimer peptide is a trimer peptide of cysteine, proline and threonine, such as in some cases a CPT insertion or a TCP insertion. In some embodiments, the transmembrane domain is a transmembrane domain from IL-7R or a portion thereof that comprises one or more mutations compared to a wild-type IL-7R transmembrane domain PILLTISILSFFSVALLVILACVLW (SEQ ID NO: 71) or a contiguous sequence thereof of at least 21, 22, 23, 24, 25, 26, 27, or 28 amino acids. In some embodiments, the transmembrane domain also may contain 1, 2, 3 or 4 additional N-terminal or C-terminal amino acids compared to the sequence
30761-20002.40 set froth in SEQ ID NO:71. In some embodiments, the transmembrane domain is a transmembrane domain from IL-7R or a portion thereof that comprises one or more mutations compared to a wild- type IL-7R transmembrane domain PILLTISILSFFSVALLVILACVLW (SEQ ID NO: 71). In some embodiments, the variant transmembrane domain that contains one or more mutations compared to the wild-type IL-7R transmembrane domain has a sequence that is less than 100% identical to SEQ ID NO:71 and at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95% identical to SEQ ID NO: 71. [0261] In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 6. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 6. [0262] In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a
30761-20002.40 sequence of amino acids that is at least about 97% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 6. In some embodiments, the variant IL-7R transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 6. [0263] In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 7. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 7. [0264] In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a
30761-20002.40 sequence of amino acids that is at least about 97% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the variant IL-7R transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 7. [0265] In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 21. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 21. [0266] In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 21. In some
30761-20002.40 embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain comprises the amino acid sequence to set forth in SEQ ID NO: 21. In some embodiments, the variant IL-7R transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 21. [0267] In some embodiments, the transmembrane domain is a transmembrane domain from a TNF receptor (TNFR), which naturally contain a transmembrane domain helix that can oligomerize, such as dimerize or trimerize. In some embodiments, the transmembrane domain derived from the TNFR is a transmembrane domain from TACI, Death Receptor 5 (DR5), p75NTR, Fas,TNFR1, TNFR2 or OX40. In some embodiments, inclusion of such transmembrane domains in provided synthetic cytokine receptors allows constitutive clustering of the intracellular domains that recruits and activates downstream signaling proteins such as JAKs and STATs. In some embodiments, the transmembrane domain contains a motif that supports oligomerization, such as dimerization and/or trimerization (see e.g., Zhao et al., 2020, Frontiers in Cell and Developmental Biology, 8.569684. 10.3389/fcell.2020.569684), incorporated by reference herein). In some embodiments, the transmembrane domain derived from a TNFR contains a proline rich motif ĭPXĭ (SEQ ID NO: 75), in which ĭ represents a hydrophobic residue, P is proline and X can be any amino acid, typically an apolar residue except for proline and glycine. In some embodiments, the transmembrane domain derived from a TNFR contains a ĭTXXAĭ, in which ĭ represents a hydrophobic residue, T is threonine, X can be any amino acid, typically an apolar residue except for proline and glycine, and A is alanine. In some embodiments, the transmembrane domain derived from a TNFR contains a GXXXG motif (SEQ ID NO:68). In some embodiments, the transmembrane domain derived from a TNFR contains a GXXXA motif (SEQ ID NO:69). In some embodiments, the transmembrane domain derived from a TNFR contains an AXXXA motif (SEQ ID NO: 77). In some embodiments, the transmembrane domain derived from a TNFR contains an AXXXS motif (SEQ ID NO: 78). [0268] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from DR5. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that exhibits at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain
30761-20002.40 comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the DR5 transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, any of such DR5 transmembrane domain comprises the motif ĭTXXAĭ (SEQ ID NO: 76) and/or the motif GXXXG (SEQ ID NO:68). In some embodiments, any of such DR5 transmembrane domain comprises the motif ĭTXXAĭ (SEQ ID NO: 76). In some embodiments, any of such DR5 transmembrane domain comprises the motif GXXXG (SEQ ID NO:68). In some embodiments, any of such DR5 transmembrane domain comprises the motif ĭTXXAĭ (SEQ ID NO: 76) and the motif GXXXG (SEQ ID NO:68). [0269] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from TACI. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that exhibits at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 44. In some
30761-20002.40 embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 44. In some embodiments, the TACI transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 44. In some embodiments, any of such TACI transmembrane domain comprises the motif AXXXS (SEQ ID NO:78). [0270] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from Muc24. [0271] In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 23. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 23. [0272] In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 23. In some embodiments, the Muc24
30761-20002.40 transmembrane domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain comprises the amino acid sequence to set forth in SEQ ID NO: 23. In some embodiments, the Muc24 transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 23. [0273] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from Thrombopoietin Receptor (TpoR). In some embodiments, the TpoR transmembrane domain is a variant transmembrane domain. In some embodiments, the variant transmembrane domain comprises one or more mutations compared to a wild-type TpoR transmembrane domain (ISLVTALHLVLGLSAVLGLLLL; SEQ ID NO: 169). In some embodiments, the one or more mutations promote homodimerization of the synthetic cytokine receptor. In some embodiments, the one or more mutations promote alpha-helix dimerization. In some embodiments, the variant transmembrane domain that contains one or more mutations compared to the wild-type TpoR transmembrane domain has a sequence that is less than 100% identical to SEQ ID NO: 169 and at
30761-20002.40 least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 169. In some embodiments, the one or more mutations replaces at least one amino acid residue in the wild-type transmembrane domain with a leucine residue. In some embodiments, the one or more mutations replaces at least one amino acid residue in the wild-type transmembrane domain with an asparagine residue. In some embodiments, the one or more mutations replaces one or more amino acid residues in the wild-type transmembrane domain with a leucine residue and an asparagine residue. In some embodiments, the TpoR TMD comprises amino acid substitution H499L with reference to wild-type TpoR (SEQ ID NO: 168) or H8L with reference to wild-type TpoR transmembrane domain (SEQ ID NO: 169). In some embodiments, the TpoR TMD comprises amino acid substitution S505N with reference to wild-type TpoR or S14N with reference to wild-type TpoR transmembrane domain . In some embodiments, the TpoR TMD comprises amino acid substitution H499L and S505N with reference to wild-type TpoR or H8L and S14N with reference to wild-type TpoR transmembrane domain. [0274] In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 169. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 169. [0275] In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 93% identical to
30761-20002.40 SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain comprises the amino acid sequence to set forth in SEQ ID NO: 169. In some embodiments, the TpoR transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 169. [0276] In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 163. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 163. [0277] In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 163. In some embodiments, the TpoR
30761-20002.40 transmembrane domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain comprises the amino acid sequence to set forth in SEQ ID NO: 163. In some embodiments, the TpoR transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 163. [0278] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from wild-type IL-9R. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 125. In some embodiments, the wild-type IL-9R transmembrane domain comprises the amino acid sequence to set forth in SEQ ID NO: 125. In some embodiments, the wild- type IL-9R transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 125. [0279] In some embodiments, the transmembrane domain provided herein can comprise any one of the transmembrane domains set forth in Table 2. As indicated, the exact locus or residues corresponding to a given domain can vary, such as depending on the methods used to identify or classify the domain. Also, in some cases, adjacent N- and/or C-terminal amino acids of a given transmembrane domain also can be included in a sequence of a synthetic cytokine receptor, so long as the resulting receptor is able to constitutively multimerize (e.g., dimerize) to result in constitutive activation of downstream signaling molecules. Thus, it is understood that the exemplification of the SEQ ID NOS in Table 2 is not to be construed as limiting. For example, the particular transmembrane domain can be several amino acids longer or shorter, such as 1-10, e.g., 1, 2, 3, 4, 5, 6 or 7 amino acids longer or shorter, than the sequence of amino acids set forth in the respective SEQ ID NO. Also provided are variants of any such SEQ ID NO (e.g., sequence that exhibits at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, such as at least about 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NOS: 6, 7, 21, 22, 23, 43, 44, 45, 46, 125, 163 or 169), in which the resulting synthetic cytokine receptor containing such a variant transmembrane domain is able to constitutively multimerize (e.g., dimerize) to result in constitutive activation of downstream signaling molecules.
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C. Intracellular Domain [0280] Provided embodiments relate to a synthetic cytokine receptor that comprises an extracellular domain (e.g., any as described in Section II.A), a transmembrane domain (e.g., any as described in Section II.B), and an intracellular domain capable of interleukin 9 receptor (IL-9R) signaling. In some embodiments, the intracellular domain comprises an IL-9R intracellular domain or a variant thereof. In some embodiments, the intracellular domain comprises a chimeric JAK/STAT fusion domain. 1. IL-9R intracellular domain or Variant Thereof [0281] In some embodiments, the intracellular domain comprises the intracellular domain of a naturally occurring IL-9R (e.g., set forth in SEQ ID NO:8). In some embodiments, the intracellular domain comprises one or more mutations compared to a naturally occurring IL-9R (e.g., one or more mutations compared to the sequence set forth in SEQ ID NO:8). The one or more mutation(s) may be a substitution, insertion, deletion, or combination thereof. In some embodiments, the one or more mutation(s) promote downstream signaling. Methods of determining whether a particular mutation will trigger the downstream signaling is known to a skilled artisan (e.g., assaying for STAT1, STAT3, or STAT5 phosphorylation following growth of the cells harboring the receptor being tested in the absence of growth factors). [0282] In some embodiments, the intracellular domain is at least about 150 amino acids in length. In some embodiments, the intracellular domain is at most about 250 amino acids in length. In some embodiments, the intracellular domain is about 150 amino acids to about 250 amino acids in length. In some embodiments, the intracellular domain is about 169, 220, 223, or 230 amino acids in length. In some embodiments, the intracellular domain is about 169 amino acids in length. In some embodiments, the intracellular domain is about 220 amino acids in length. In some embodiments, the intracellular domain is about 223 amino acids in length. In some embodiments, the intracellular
30761-20002.40 domain is about 230 amino acids in length. In some embodiments, the intracellular domain is 169 amino acids in length. In some embodiments, the intracellular domain is 220 amino acids in length. In some embodiments, the intracellular domain is 223 amino acids in length. In some embodiments, the intracellular domain is 230 amino acids in length. [0283] In some embodiments, the IL-9R intracellular domain is wild-type IL-9R intracellular domain or is a variant thereof that comprises one or more mutations compared to the wild-type IL-9R intracellular domain set forth in SEQ ID NO: 8. In some embodiments, the one or more mutations comprise one or more amino acid insertions, deletions, and/or substitutions. In some embodiments, the one or more mutations promote signaling through STAT1, STAT3, and/or STAT5 pathways. [0284] In some embodiments, the IL-9R intracellular domain or variant thereof comprises a BOX1 motif and/or a BOX2 motif. In some embodiments, the IL-9R intracellular domain or variant thereof comprises a BOX2 motif. [0285] In some embodiments, the IL-9R intracellular domain or variant thereof comprises a BOX 1 motif, which is defined as proline, any amino acid residue, and proline preceded by hydrophobic sequences. In some embodiments, the IL-9R intracellular domain, including a variant IL- 9R intracellular domain, contains the proline rich BOX1 motif that allows JAK molecules to bind. In some embodiments, the BOX1 motif is the sequence FYQNVPSPA (SEQ ID NO:79; corresponding to positions 10-18 of the sequence set forth in SEQ ID NO:8). [0286] In some embodiments, the IL-9R intracellular domain or variant thereof comprises a BOX 2 motif, which is defined as a cluster of hydrophobic amino acids followed by positively charged amino acids. In some embodiments, the IL-9R intracellular domain, including a variant IL-9R intracellular domain, contains the BOX2 motif that allows STAT molecules to bind. [0287] In some embodiments, the IL-9R intracellular domain, including a variant IL-9R intracellular domain, contains a conserved tyrosine residue at the position corresponding to position 116 in the sequence set forth in SEQ ID NO:8. In some embodiments, the IL-9R intracellular domain, including a variant IL-9R intracellular domain, additionally contains a conserved proline at position 118 and a conserved glutamine at position 119, each with reference to positions corresponding to positions set forth in SEQ ID NO:8. In some embodiments, the IL-9R intracellular domain, including a variant IL-9R intracellular domain, contains a conserved glutamine residue at the position corresponding to position 118 in the sequence set forth in SEQ ID NO: 8. [0288] In some embodiments, the IL-9R intracellular domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises an amino acid sequence of SEQ ID NO: 8. [0289] In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 8. In some embodiments, the IL-9R
30761-20002.40 intracellular domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 8. [0290] In some embodiments, the IL-9R intracellular domain comprises deletion of contiguous amino acids compared to wild-type IL-9R intracellular domain. In some embodiments, the mutation is a deletion and the variant is a deleted IL-9R intracellular signaling domain lacking one or more regions of the wild-type IL-9R intracellular signaling domain (e.g., lacking one or more regions of the sequence set forth in SEQ ID NO:8). In some embodiments, the IL-9R lacks up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous amino acids present within a wild-type IL- 9R intracellular domain (e.g., SEQ ID NO:8). In some embodiments, the IL-9R lacks a region of 10 contiguous amino acids with reference to a wild-type IL-9R intracellular domain. In some
30761-20002.40 embodiments, the region that is deleted is a sequence that is not necessary for functional activity of the IL-9R intracellular domain such that IL-9R-mediated signaling is retained. [0291] In some embodiments, the IL-9R intracellular domain, including a variant IL-9R intracellular domain, contains deletion of amino acids NGNFQTWMGA (SEQ ID NO:82; corresponding to deletion of amino acids 29-38 with reference to positions set forth in SEQ ID NO:8). In some embodiments, the IL-9R intracellular domain, including a variant IL-9R intracellular domain, contains deletion of amino acids HGAGVLLSQD (SEQ ID NO:81; corresponding to deletion of amino acids 39-48 with reference to positions set forth in SEQ ID NO:8). In some embodiments, the IL-9R intracellular domain, including a variant IL-9R intracellular domain, contains deletion of amino acids TCGPARPWKS (SEQ ID NO:83; corresponding to deletion of amino acids 69-78 with reference to positions set forth in SEQ ID NO: 8). In some embodiments, the IL-9R intracellular domain, including a variant IL-9R intracellular domain, contains deletion of amino acids ALGCYGGWHL (SEQ ID NO: 80; corresponding to deletion of amino acids 154-163 with reference to positions set forth in SEQ ID NO: 8). In some embodiments, one or more additional mutation can be present in any of such truncated IL-9R intracellular domain. [0292] In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that exhibits at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least
30761-20002.40 about 94% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, the IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 51. [0293] In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that exhibits at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain
30761-20002.40 comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, the IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 52. [0294] In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that exhibits at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 53. In some
30761-20002.40 embodiments, the IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, the IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 53. [0295] In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that exhibits at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, the IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 54.
30761-20002.40 [0296] In some embodiments, the mutation is a deletion and the variant is a truncated IL-9R intracellular signaling domain that is truncated by deletion of one or more amino acid residues at one or both of the N- and C-terminus of a wild-type IL-9R intracellular domain. In some embodiments, the truncated IL-9R is a contiguous sequence of amino acids of at least 150 amino acids in length from the wild-type IL-9R intracellular signaling domain set forth in SEQ ID NO:8 that is truncated by deletion of one or more amino acid residues at one or both of the N- and C-terminus of SEQ ID NO:8. In some embodiments, the truncated IL-9R signaling domain is a contiguous sequence of from 150 amino acids to 229 amino acids of the sequence set forth in SEQ ID NO:8. In some embodiments, the variant IL-9R is an N-terminal truncation of up to 80 amino acids. In some embodiments, the variant IL-9R is an N-terminal truncation of up to 70 amino acids. In some embodiments, the variant IL-9R is an N-terminal truncation of up to680 amino acids. In some embodiments, the variant IL-9R is an N-terminal truncation of up to 50 amino acids. In some embodiments, the variant IL-9R is an N- terminal truncation of up to 40 amino acids. In some embodiments, the variant IL-9R is an N- terminal truncation of up to 30 amino acids. In some embodiments, the variant IL-9R is an N-terminal truncation of up to 20 amino acids. In some embodiments, the variant IL-9R is an N-terminal truncation of up to 10 amino acids. In some embodiments, the variant IL-9R is an N-terminal truncation of up to 7 amino acids. In some embodiments, the variant IL-9R is an N-terminal truncation of up to 5 amino acids. In some embodiments, one or more additional mutation can be present in any of such truncated IL-9R intracellular domain. [0297] In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that exhibits at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 55. In some
30761-20002.40 embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 94% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 55. [0298] In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that exhibits at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 75% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 80% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 85% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 86% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 87% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 88% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 89% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 90% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 91% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 92% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 93% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least
30761-20002.40 about 94% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 95% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 96% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 97% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 98% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises a sequence of amino acids that is at least about 99% identical to SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, the IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 56.Exemplary of any of such intracellular domains, including wild-type or variant IL-9R intracellular domains or chimeric JAK/STAT fusion intracellular domains are described in the subsections that follow below. [0299] In some embodiments, the intracellular domain provided herein can comprise any one of the intracellular domains set forth in Table 3. In some cases, adjacent N- and/or C-terminal amino acids of a given intracellular domain also can be included or deleted in a sequence of a synthetic cytokine receptor, so long as the resulting receptor is able to constitutively multimerize (e.g., dimerize) to result in constitutive activation of downstream signaling molecules. Thus, it is understood that the exemplification of the SEQ ID NOS in Table 3 is not to be construed as limiting. For example, the particular intracellular domain can be several amino acids longer or shorter, such as 1-10, e.g., 1, 2, 3, 4, 5, 6 or 7 amino acids longer or shorter, than the sequence of amino acids set forth in the respective SEQ ID NO. Also provided are variants of any such SEQ ID NO (e.g., sequence that exhibits at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, such as at least about 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NOS: 8 or 51-56), in which the resulting synthetic cytokine receptor containing such a variant IL-9R intracellular domain is able to constitutively multimerize (e.g., dimerize) to result in constitutive activation of downstream signaling molecules.
a. Wild-type IL-9R Intracellular Domain (ICD) [0300] In some embodiments, the intracellular domain is capable of interleukin 9 receptor (IL- 9R) signaling. In some embodiments, the intracellular domain capable of IL-9R signaling comprises
30761-20002.40 an intracellular domain of IL-9R. In some embodiments, the IL-9R intracellular domain comprises a naturally occurring IL-9R (i.e., wild-type IL-9R). In some embodiments, the IL-9R intracellular domain comprises a mammalian IL-9R. In some embodiments, the IL-9R intracellular domain comprises a human IL-9R. In some embodiments, the IL-9R intracellular domain comprises a wild- type human IL-9R. [0301] In some embodiments, the IL-9R intracellular domain is 230 amino acids in length. In some embodiments, the wild-type IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 8. In some embodiments, the wild-type IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the wild-type IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 8. b. Variant IL-9R Intracellular Domain (ICD) [0302] In some embodiments, the intracellular domain is a variant IL-9R intracellular domain capable of interleukin 9 receptor (IL-9R) signaling. In some embodiments, the variant IL-9R intracellular domain comprises one or more mutations compared to a naturally occurring IL-9R. The one or more mutation(s) may be a substitution, insertion, deletion, or combination thereof. In some embodiments, the one or more mutation(s) promote downstream signaling. Methods of determining whether a particular mutation will trigger the downstream signaling is known to a skilled artisan (e.g., assaying for STAT1, STAT3, or STAT5 phosphorylation following growth of the cells harboring the receptor being tested in the absence of growth factors). In some embodiments, the variant IL-9R intracellular domain elicits signaling through STAT1, STAT3, and/or STAT5 pathways. [0303] In some embodiments, STAT1 signaling by variant IL-9R is increased compared to STAT1 signaling by wild-type IL-9R. In some embodiments, STAT3 signaling by variant IL-9R is increased compared to STAT3 signaling by wild-type IL-9R. In some embodiments, STAT5 signaling by variant IL-9R is increased compared to STAT5 signaling by wild-type IL-9R. In some embodiments, signaling through the STAT1, STAT3, and/or STAT5 pathways is sustained for a longer period of time compared to STAT1, STAT3, and/or STAT5 via wild-type IL-9R. In some embodiments, the period of time is about 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or longer. In some embodiments, the period of time is about 24 hours. In some embodiments, signaling through STAT1, STAT3, and/or STAT5 is sustained for as long as the cell expresses any of the synthetic cytokine receptors provided herein. In some embodiments, sustained STAT1, STAT3, and/or STAT5 signaling is determined by phosphorylation status of STAT1, STAT3, and/or STAT5. [0304] In some embodiments, the IL-9R intracellular domain comprises deletions of a region such as those outlined in Table 4. In some embodiments, the IL-9R intracellular domain comprises deletions of contiguous amino acids compared to wild-type IL-9R intracellular domain. In some
30761-20002.40 embodiments, the IL-9R intracellular domain comprises a deletion of a region, such as those outlined in Table 4, at the C terminus of wild-type IL-9R intracellular domain. In some embodiments, the IL- 9R intracellular domain comprises a deletion of a region at the N terminus of wild-type IL-9R intracellular domain. In some embodiments, the IL-9R intracellular domain comprises a deletion of a region of contiguous amino acids within the wild-type IL-9R intracellular domain. In some embodiments, the IL-9R intracellular domain comprises deletions of non-contiguous amino acids compared to the wild-type IL-9R intracellular domain. In some embodiments, the IL-9R intracellular domain comprises deletions of a plurality of regions, such as those outlined in Table 4, of the wildtype IL-9R intracellular domain. Exemplary deleted regions from IL-9R intracellular domain are shown in Table 4A. Table 4A: Exemplary Deletion Mutations
[0305] Exemplary deleted regions from the IL-9R intracellular domain are shown in Table 4B. In some embodiments, the IL-9R intracellular domain is deleted for contiguous amino acids within or including contiguous amino acids 29-38, 39-48, 69-78 or 154-163, with reference to numbering of amino acids in SEQ ID NO:8, or any combination thereof. Table 4B: Exemplary Deleted Regions
30761-20002.40
[0306] In some embodiments, the IL-9R intracellular domain comprises deletions of a plurality of regions, of the wildtype IL-9R intracellular domain. Exemplary intracellular domains with a plurality of deleted regions from IL-9R intracellular domain are shown in Table 4C. Table 4C: Exemplary Deletions of Multiple Regions
[0307] In some embodiments, the variant IL-9R intracellular domain is a truncated IL-9R that lacks a contiguous sequence of amino acids between the N-terminus and C-terminus of wild-type IL- 9R intracellular domain. In some embodiments, the variant IL-9R intracellular domain is a truncated IL-9R that lacks a noncontiguous sequence of amino acids between the N-terminus and C-terminus of wild-type IL-9R intracellular domain. In some embodiments, the variant IL-9R intracellular domain is a truncated IL-9R that lacks a contiguous sequence of amino acids at the C-terminus of wild-type IL-
30761-20002.40 9R intracellular domain. In some embodiments, the variant IL-9R intracellular domain is a truncated IL-9R that lacks a contiguous sequence of amino acids at the N-terminus of wild-type IL-9R intracellular domain. In some embodiments, the variant IL-9R intracellular domain is a truncated IL- 9R that lacks a noncontiguous sequence of amino acids at the C-terminus of wild-type IL-9R intracellular domain. In some embodiments, the variant IL-9R intracellular domain is a truncated IL- 9R that lacks a noncontiguous sequence of amino acids at the N-terminus of wild-type IL-9R intracellular domain. In some of any of such embodiments, the truncation is at the C-terminus of wild-type IL-9R intracellular domain set forth in SEQ ID NO: 8. In some of any of such embodiments, the truncation is at the N-terminus of wild-type IL-9R intracellular domain set forth in SEQ ID NO: 8. [0308] In some embodiments, the truncated IL-9R intracellular domain or variant thereof is truncated by between 62 and 99 contiguous amino acids from the C-terminus of wild-type IL-9R intracellular domain. In some embodiments, the truncated IL-9R intracellular domain or variant thereof is truncated by 61 contiguous amino acids from the C-terminus of wild-type IL-9R intracellular domain. In some embodiments, the truncated IL-9R intracellular domain or variant thereof is truncated by 62 contiguous amino acids from the C-terminus of wild-type IL-9R intracellular domain. In some embodiments, the truncated IL-9R intracellular domain or variant thereof is truncated by 99 contiguous amino acids from the C-terminus of wild-type IL-9R intracellular domain. [0309] In some embodiments, the variant IL-9R intracellular domain is a truncated IL-9R that lacks amino acids 132 to 230 of SEQ ID NO:8. In some embodiments, the variant IL-9R intracellular domain lacks amino acids 134 to 230 of SEQ ID NO:8. [0310] In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 84. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 85. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 51. In some embodiments, the variant
30761-20002.40 IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 52. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 86. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 86. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 86. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 87. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 87. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 87. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 53. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 88. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 88. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 88. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 89. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 89. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 89. In some embodiments, the variant IL-9R intracellular domain comprises a sequence
30761-20002.40 of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 90. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 90. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 90. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 91. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 91. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 91. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 92. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 93. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 93. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 93. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 94. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 94. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 94. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 54. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 95. In some embodiments, the variant IL-9R intracellular domain comprises
30761-20002.40 the amino acid sequence set forth in SEQ ID NO: 95. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 95. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 96. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 97. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 98. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 98. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 98. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 99. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 99. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 99. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 100. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 100. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 100. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 55. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at
30761-20002.40 least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 56. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 103. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 104. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 104. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 104. [0311] In some embodiments, the variant IL-9R intracellular domain comprises one or more amino acid deletions and insertions. In some embodiments, the IL-9R intracellular domain comprises deletion of a contiguous sequence of amino acids and insertion of a contiguous sequence of amino acids. In some embodiments, the length of the deleted contiguous sequence of amino acids and length of the inserted contiguous sequence of amino acids are the same. In some embodiments, the length of the deleted contiguous sequence of amino acids and length of the inserted contiguous sequence of amino acids are the different. [0312] In some embodiments, the variant IL-9R intracellular domain comprises replacement of a contiguous sequence of amino acids with one or more STAT binding domains. In some embodiments, the contiguous sequence of amino acids is replaced with one STAT binding domain. In some embodiments, the STAT binding domain is derived from a Type I cytokine receptor. In some embodiments, the Type I cytokine receptor is selected from the group consisting of interleukin 2 receptor (IL-2R), interleukin 4 receptor (IL-4R), interleukin 7 receptor (IL-7R), interleukin 13 receptor (IL-13R), interleukin 15 receptor (IL-15R), and interleukin 2 receptor (IL-21R). In some embodiments, the Type I cytokine receptor is IL-2R. [0313] In some embodiments, the contiguous sequence of amino acids is replaced with one or more STAT binding domains. In some embodiments, the contiguous sequence of amino acids is replaced with one STAT binding domain. In some embodiments, the contiguous sequence of amino acids is replaced with two STAT binding domains. In some embodiments, the contiguous sequence of amino acids is replaced with three STAT binding domains. In some embodiments, the contiguous sequence of amino acids is replaced with four STAT binding domains. In some embodiments, the
30761-20002.40 contiguous sequence of amino acids is replaced with five STAT binding domains. In some embodiments, replacement of the contiguous sequence of amino acids with one, two, three, four or five STAT binding domains increases STAT binding and signaling. [0314] In some embodiments, the variant IL-9R intracellular domain comprises one or more YLPQ, YRPQ, YLPL, or YLKQ STAT binding domains. In some embodiments, the variant IL-9R intracellular domain comprises two or more YLPQ, YRPQ, YLPL, or YLKQ STAT binding domains. In some embodiments, the variant IL-9R intracellular domain comprises three or more YLPQ, YRPQ, YLPL, or YLKQ STAT binding domains. In some embodiments, the variant IL-9R intracellular domain comprises four or more YLPQ, YRPQ, YLPL, or YLKQ STAT binding domains. In some embodiments, the variant IL-9R intracellular domain comprises five or more YLPQ, YRPQ, YLPL, or YLKQ STAT binding domains. [0315] In some embodiments, the one or more STAT binding domains comprises YLPQ (SEQ ID NO: 171). In some embodiments, the one or more STAT binding domains comprises YRPQ (SEQ ID NO: 172). In some embodiments, the one or more STAT binding domains comprises YLPL (SEQ ID NO: 173). In some embodiments, the one or more STAT binding domain comprises YLKQ (SEQ ID NO: 174).In some embodiments, the STAT binding domain comprises a STAT1 binding domain. In some embodiments, the STAT binding domain comprises a STAT3 binding domain. In some embodiments, the STAT binding domain comprises a STAT5 binding domain. [0316] In some embodiments, the STAT5 binding domain is 11 amino acids in length. In some embodiments, the STAT5 binding domain comprises amino acid sequence LNTDAYLSLQE (SEQ ID NO: 120). In some embodiments, the contiguous sequence of amino acids that is replaced with the one or more STAT binding domains comprises 11 amino acid residues. In some embodiments, the contiguous sequence of amino acids that is replaced with the one or more STAT binding domains comprises SNNNNYCALGC (SEQ ID NO: 170). Exemplary insertion mutations of one, two, and three STAT binding domains are shown in Table 5. Table 5: Exemplary Insertion Mutations
[0317] In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 110. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 110. In some embodiments, the variant
30761-20002.40 IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 110. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 111. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 111. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 111. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 112. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 112. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 112. [0318] In some embodiments, the variant IL-9R intracellular domain comprises one or more amino acid substitutions with reference to wild-type IL-9R intracellular domain (SEQ ID NO:8). In some embodiments, the variant IL9R intracellular domain comprises a STAT binding motif. In some embodiments, the one or more amino acid substitutions are located in the STAT binding motif of the IL-9R intracellular domain. In some embodiments, the STAT binding motif comprises a STAT1, STAT3, and/or STAT5 binding motif. [0319] In some embodiments, the STAT binding motif comprises YLPQ (SEQ ID NO: 171). In some embodiments, the STAT binding motif comprises a variant STAT binding motif. In some embodiments, the variant STAT binding motif comprises one or more amino acid substitutions with reference to SEQ ID NO: 171. In some embodiments, the amino acid substitution comprises substitution of a leucine residue for an arginine residue with reference to SEQ ID NO: 171. In some embodiments, the amino acid substitution comprises substitution of a glutamine residue for a leucine residue with reference to SEQ ID NO: 171. In some embodiments, the amino acid substitution comprises substitution of a proline with a lysine with reference to SEQ ID NO: 171. [0320] In some embodiments, the STAT binding motif comprises YRPQ (SEQ ID NO: 172). In some embodiments, the STAT binding motif comprises YLPL (SEQ ID NO: 173). In some embodiments, the STAT binding motif comprises YLKQ (SEQ ID NO: 174). [0321] In some embodiments, STAT1, STAT3, and/or STAT5 bind YLPQ. In some embodiments, STAT1, STAT3, and STAT5 bind YLPQ. In some embodiments, STAT1 and/or STAT3 bind YRPQ. In some embodiments, STAT1 and STAT3 bind YRPQ. In some embodiments, STAT5 binds YLPL. In some embodiments, STAT1, STAT3, and/or STAT5 bind YLKQ. In some embodiments, STAT1, STAT3, and STAT5 bind YLKQ. [0322] In some embodiments, the one or more amino acid substitutions of the STAT binding motif with reference to SEQ ID NO: 171 (YLPQ) are shown Table 6.
30761-20002.40 Table 6: Exemplary Substitution Mutations
[0323] In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 107. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 107. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 107. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 108. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 108. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 108. In some embodiments, the variant IL-9R intracellular domain comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 109. In some embodiments, the variant IL-9R intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, the variant IL-9R intracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, the variant IL-9R intracellular domain provided herein can comprise any one of the variant IL-9R intracellular domains set forth in Table 7. In some embodiments, the variant IL-9R intracellular domain can comprise any combination of the variant IL- 9R intracellular domains set forth in Table 7. In some embodiments, the variant IL-9R intracellular domain comprises one or more amino acid deletions with reference to a wild-type IL-9R intracellular domain and one or more amino acid substitutions with reference to a wild-type IL-9R intracellular domain. In some embodiments, the variant IL-9R intracellular domain comprises one or more amino acid deletions with reference to a wild-type IL-9R intracellular domain and one or more amino acid insertions with reference to a wild-type IL-9R intracellular domain. [0324] In particular embodiments, the variant IL-9R intracellular domain comprises IL9R(Large D2) (SEQ ID NO: 103), IL9R(Large D2’) (SEQ ID NO: 104) or IL9R(d15) (SEQ ID NO: 54) and one
30761-20002.40 or amino acid substitutions as represented by IL9R(Mut6YRPQ) (SEQ ID NO: 107), IL9R(Mut7YLPL) (SEQ ID NO: 108), or IL9R(Mut9YLKQ) (SEQ ID NO: 109).
2. Chimeric JAK/STAT Fusion Intracellular Domain (ICD) [0325] In some embodiments, the intracellular domain is a chimeric fusion domain capable of interleukin 9 receptor (IL-9R) signaling. In some embodiments, the chimeric fusion domain comprises a JAK binding domain derived from IL-7R fused to a STAT binding domain derived from IL-9R. In Some embodiments, the chimeric fusion domain comprises a JAK binding domain derived from TpoR fused to a STAT binding domain derived from IL-9R. [0326] In some embodiments, the chimeric JAK/STAT fusion domain promotes downstream signaling. Methods of determining whether the chimeric JAK/STAT fusion domain will trigger the downstream signaling is known to a skilled artisan (e.g., assaying for STAT1, STAT3, or STAT5 phosphorylation following growth of the cells harboring the receptor being tested in the absence of growth factors). In some embodiments, the chimeric JAK/STAT fusion domain elicits signaling through STAT1, STAT3, and/or STAT5 pathways. [0327] In some embodiments, STAT1 signaling by the chimeric JAK/STAT fusion domain is increased compared to STAT1 signaling by wild-type IL-9R. In some embodiments, STAT3 signaling by the chimeric JAK/STAT fusion domain is increased compared to STAT3 signaling by wild-type IL-9R. In some embodiments, STAT5 signaling by the chimeric JAK/STAT fusion domain is increased compared to STAT5 signaling by wild-type IL-9R. In some embodiments, STAT1, STAT3, and/or STAT5 signaling is sustained for a longer period of time by the chimeric JAK/STAT fusion domain compared to STAT1, STAT3, and/or STAT5 signaling via wild-type IL-9R. In some embodiments, the period of time is about 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours. In some embodiments, the period of time is about 24 hours. In some embodiments, sustained STAT1, STAT3, and/or STAT5 signaling is determined by phosphorylation status of STAT1, STAT3, and/or STAT5. [0328] In some embodiments, the chimeric JAK/STAT fusion domain comprises a JAK binding domain from a type I cytokine receptor and a STAT binding domain from an IL-9R intracellular domain. [0329] In some embodiments, the IL-9R STAT binding domain comprises amino acid residues 73 to 230 of SEQ ID NO: 8. In some embodiments, the IL-9R STAT binding domain is 59 to 158 amino acids in length and comprises an IL-9R STAT binding motif. In some embodiments, the IL-9R
30761-20002.40 STAT binding motif comprises YLPQ (SEQ ID NO: 171). In some embodiments, the IL-9R STAT binding domain comprises one or more amino acid deletions with reference to the IL-9R ICD set forth in SEQ ID NO: 8. In some embodiments, the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks a contiguous sequence of amino acids at the N-terminus of SEQ ID NO: 8. In some embodiments, the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks a contiguous sequence of amino acids at the C-terminus of SEQ ID NO: 8. In some embodiments, the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks amino acids at positions 1 to 72 of SEQ ID NO: 8. In some embodiments, the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks amino acids at positions 132 to 230 of SEQ ID NO: 8. In some embodiments, the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks amino acids at positions 1 to 72 and 132 to 230 of SEQ ID NO: 8. Exemplary STAT binding domains from a IL-9R receptors are shown in Table 8. Table 8: Exemplary JAK binding domains
[0330] In some embodiments, the IL-9R STAT binding domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 122. In some embodiments, the IL-9R STAT binding domain comprises an amino acid sequence of SEQ ID NO: 122. In some embodiments, the IL-9R STAT binding domain consists of the amino acid sequence set forth in SEQ ID NO: 122. In some embodiments, the IL-9R STAT binding domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 123. In some embodiments, the IL-9R STAT binding domain comprises an amino acid sequence of SEQ ID NO: 123. In some embodiments, the IL-9R STAT binding domain consists of the amino acid sequence set forth in SEQ ID NO: 123. [0331] In some embodiments, the type I cytokine receptor is selected from the group consisting of interleukin 2 receptor (IL-2R), interleukin 4 receptor (IL-4R), interleukin 7 receptor (IL-7R), interleukin 13 receptor (IL-13R), interleukin 15 receptor (IL-15R), interleukin 2 receptor (IL-21R), and thrombopoietin receptor (TpoR). In some embodiments, the type I cytokine receptor is IL-7R. In some embodiments, the type I cytokine receptor is TpoR. Exemplary JAK binding domains from a type I cytokine receptors are shown in Table 9. Table 9: Exemplary JAK binding domains
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[0332] In some embodiments, the IL-7R JAK binding domain is up to 65 amino acids in length. In some embodiments, the IL-7R JAK binding domain is 65 amino acids in length. In some embodiments, the IL-7R JAK binding domain comprises a box 1 motif. In some embodiments, the IL- 7R JAK binding domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 119. In some embodiments, the IL-7R JAK binding domain comprises an amino acid sequence of SEQ ID NO: 119. In some embodiments, the IL-7R JAK binding domain consists of the amino acid sequence set forth in SEQ ID NO: 119. In some embodiments, the IL-7R JAK binding domain comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 121. In some embodiments, the IL-7R JAK binding domain comprises an amino acid sequence of SEQ ID NO: 121. In some embodiments, the IL-7R JAK binding domain consists of the amino acid sequence set forth in SEQ ID NO: 121. [0333] In some embodiments, the chimeric JAK/STAT fusion intracellular domain comprises a fusion of any of the JAK and STAT binding domains described above. In some embodiments, the chimeric JAK/STAT fusion intracellular domain comprises about 223 amino acids. In some embodiments, the JAK/STAT fusion intracellular domain is a truncated version of the 223 amino acid sequence. In some embodiments, the JAK/STAT fusion intracellular domain is truncated at the C terminus. In some embodiments, 99 amino acids are truncated from the C terminus. In other embodiments, 97 amino acids are truncated from the C terminus. In some embodiments, the chimeric JAK/STAT fusion intracellular domain provided herein can comprise any one of the intracellular domains set forth in Table 10.
[0334] In some embodiments, the chimeric JAK/STAT fusion comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 114. In some embodiments, the chimeric JAK/STAT fusion comprises the amino acid
30761-20002.40 sequence set forth in SEQ ID NO: 114. In some embodiments, the chimeric JAK/STAT fusion consists of the amino acid sequence set forth in SEQ ID NO: 114. In some embodiments, the chimeric JAK/STAT fusion comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 116. In some embodiments, the chimeric JAK/STAT fusion comprises the amino acid sequence set forth in SEQ ID NO: 116. In some embodiments, the chimeric JAK/STAT fusion consists of the amino acid sequence set forth in SEQ ID NO: 116. In some embodiments, the TpoR JAK binding domain comprises a variant TpoR JAK binding domain. In some embodiments, the variant TpoR JAK binding domain comprises one or more mutations compared to a wild-type TpoR JAK binding domain set forth in SEQ ID NO: 177 (RKQFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEILPKSSE RTPLPL). In some embodiments, the one or more mutations promote homodimerization of the synthetic cytokine receptor. In some embodiments, the one or more mutations promote alpha-helix dimerization. In some embodiments, the one or more mutations replaces at least one amino acid residue in the wild-type TpoR JAK binding domain with an arginine residue. In some embodiments, the TpoR JAK binding domain is up to 70 amino acids in length. In some embodiments, the TpoR JAK binding domain is 69 amino acids in length. In some embodiments, the TpoR JAK binding domain comprises a box 1 motif. In some embodiments, the TpoR JAK binding domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 175. In some embodiments, the TpoR JAK binding domain comprises an amino acid sequence of SEQ ID NO: 175. In some embodiments, the TpoR JAK binding domain consists of the amino acid sequence set forth in SEQ ID NO: 175. In some embodiments, the box 1 motif comprises an amino acid sequence of LWPSLPDLH (SEQ ID NO: 176). D. Variants of Synthetic Cytokine Receptors [0335] Amino acid sequence modification(s) of the synthetic cytokine receptors provided herein are contemplated. For example, it may be desirable to improve the signaling elicited by the synthetic cytokine receptors; it may also be desirable to improve other biological properties of the synthetic cytokine receptors, including but not limited to thermostability, expression level, or solubility. Thus, in addition to the synthetic cytokine receptors described herein, it is contemplated that variants can be prepared. [0336] In some embodiments, the synthetic cytokine receptors provided herein are chemically modified, for example, by the covalent attachment of any type of molecule to the synthetic cytokine receptors. Exemplary non-limiting modifications include glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Additionally, the synthetic cytokine receptors may contain one or more non-classical amino acids.
30761-20002.40 [0337] In some embodiments, variations may be a substitution, deletion, or insertion of one or more codons encoding the synthetic cytokine receptors that results in a change in the amino acid sequence as compared with the original sequence. [0338] Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which results in amino acid substitutions. [0339] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains ( e.g., aspartic acid, glutamic acid), uncharged polar side chains ( e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g, tyrosine, phenylalanine, tryptophan, histidine). Naturally occurring residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined. [0340] Substantial modifications in the biological properties of the synthetic cytokine receptor are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. [0341] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. [0342] Amino acid sequence deletions include amino- and/or carboxyl-terminal deletions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence deletions of single or multiple amino acid residues. E. Exemplary Synthetic Cytokine Receptors
30761-20002.40 [0343] In some embodiments, the synthetic cytokine receptor provided herein can comprise any one of the extracellular, transmembrane, and intracellular domains provided herein. In some embodiments, the synthetic cytokine receptor provided herein can comprise one extracellular domain from Table 1, one transmembrane domain from Table 2, and one intracellular domain from any one of Tables 3-5, 7 and 10. In some embodiments, the synthetic cytokine receptor can comprise any combination of one extracellular domain, one transmembrane domain, and one intracellular domain. Various combinations of extracellular, transmembrane and intracellular domains are provided in the exemplary synthetic cytokine receptors are set forth in Table 11. [0344] In some embodiments, the synthetic cytokine receptor can comprise an extracellular domain derived from CD34 receptor. In some embodiments, the synthetic cytokine receptor can comprise an extracellular domain derived from DAP12 receptor. In some embodiments, the synthetic cytokine receptor can comprise an extracellular domain derived from a GpA receptor. In some embodiments, the extracellular domain derived from GpA comprises the sequence of amino acids in SEQ ID NO: 16. In some embodiments, the synthetic cytokine receptor can comprise an extracellular domain derived from a GpA receptor that is further truncated (i.e., tGpA in Table 11). In some embodiments, the truncated GpA extracellular domain comprises the sequence of amino acids in SEQ ID NO: 17. In some embodiments, the synthetic cytokine receptor can comprise an extracellular domain derived from a CD8 receptor. In some embodiments, the extracellular domain derived from CD8 comprises the sequence of amino acids in SEQ ID NO: 185. In some embodiments, the synthetic cytokine receptor can comprise an extracellular domain derived from a CD8 receptor that is further truncated (i.e., tCD8 in Table 11). In some embodiments, the truncated CD8 extracellular domain comprises the sequence of amino acids in SEQ ID NO: 19. In some embodiments, the synthetic cytokine receptor can comprise an extracellular domain derived from Muc24 receptor. [0345] In some embodiments, the synthetic cytokine receptor can comprise a transmembrane domain derived from IL7R receptor. In some embodiments, the transmembrane domain derived from IL7R comprises a variant of wild-type IL7R (e.g., IL7R* and IL7R*2 in Table 11) with reference to SEQ ID NO: 71. In some embodiments, the variant of wild-type IL7R (SEQ ID NO: 71) comprises one or more amino acid insertions. In some embodiments, the variant of wild-type IL-7R comprises a trimer peptide. In some embodiments, the trimer comprises a cysteine. In some embodiments, the trimer comprises a proline. In some embodiments, the trimer comprises a threonine. In some embodiments, the trimer peptide comprises CPT. In some embodiments, the variant of IL7R comprises the sequence of amino acids set forth in SEQ ID NO: 6 (i.e., IL7R* in Table 11). In some embodiments, the variant of IL7R further comprises one or more amino acid substitutions and insertions at the C-terminus domain with reference to SEQ ID NO: 6 or SEQ ID NO: 71. In some embodiments, the one or more amino acid substitutions comprises a W to K substitution. In some embodiments, the one or more amino acid insertions comprises insertion of a K. In some embodiments, the one or more amino acid insertions comprises insertion of a R. In some
30761-20002.40 embodiments, the one or more amino acid insertions comprises insertion of a I. In some embodiments, the one or more amino acid insertions comprises insertion of a K, R and I. In some embodiments, the variant of IL7R comprises the sequence of amino acids set forth in SEQ ID NO: 7 (i.e., IL7R*2 in Table 11). [0346] In some embodiments, the synthetic cytokine receptor can comprise a transmembrane domain derived from Muc24 receptor. [0347] In some embodiments, the synthetic cytokine receptor can comprise a transmembrane domain derived from TACI receptor. [0348] In some embodiments, the synthetic cytokine receptor can comprise a transmembrane domain derived from CPT1 receptor. [0349] In some embodiments, the synthetic cytokine receptor can comprise a transmembrane domain derived from DR5 receptor. [0350] In some embodiments, the synthetic cytokine receptor can comprise a transmembrane domain derived from GpA receptor. [0351] In some embodiments, the synthetic cytokine receptor can comprise a transmembrane domain derived from a GpA receptor that is modified (i.e., tGpA* in Table 11). In some embodiments, the modified GpA transmembrane domain comprises one or more amino acid deletions. In some embodiments, the modified GpA transmembrane domain comprises one or more amino acid deletions at the C-terminus. In some embodiments, the GpA transmembrane domain comprises the sequence of amino acids in SEQ ID NO: 22. In some embodiments, the modified GpA transmembrane domain (i.e., tGPA in Table 11) comprises the sequence of amino acids in SEQ ID NO: 43. [0352] In some embodiments, the synthetic cytokine receptor can comprise an intracellular domain derived from wild-type IL9R receptor. In some embodiments, the synthetic cytokine receptor can comprise an intracellular domain derived from wild-type IL9R that is further modified. In some embodiments, the modification comprises a truncation or deletion of one or more amino acid residues. In some embodiments, the modification can comprise a insertion of one or more amino acid residues. As discussed in Section II.C.1.a., in some embodiments, the synthetic cytokine receptor can comprise a intracellular domain derived from wild-type IL9R (i.e., SEQ ID NO: 8 in Table 11). As discussed in Section II.C.1.b., in some embodiments, the synthetic cytokine receptor can comprise an intracellular domain derived from wild-type IL9R, which is further truncated (i.e., SEQ ID NOS: 51-56 in Table 11). As discussed in Section II.C.2., in some embodiments, the synthetic cytokine receptor can comprise a chimeric JAK/STAT fusion (i.e., SEQ ID NOS: 114, 116 and 181 in Table 11). Also provided are variants of any such SEQ ID NO (e.g., sequence that exhibits at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, such as at least about 96% identity, 97% identity, 98% identity, or 99% identity to any one of SEQ ID NOS: 31-42, 47-50, and 58-64), in which the resulting synthetic
30761-20002.40 cytokine receptor containing such a variant IL-9R intracellular domain is able to constitutively multimerize (e.g., dimerize) to result in constitutive activation of downstream signaling molecules. [0353] In some embodiments, any of such sequence set forth by a respective SEQ ID NO also can contain an N-terminal signal sequence. In some embodiments, the signal sequence is a heterologous signal sequence that is not naturally contiguous in a wild-type sequence. In some embodiments, the signal sequence is a natural signal sequence contiguous to the extracellular domain sequence. In some embodiments, the signal sequence is a CD34 signal peptide MLVRRGARAGPRMPRGWTALCLLSLLPSGFM (SEQ ID NO:1). In some embodiments, the signal sequence is a DAP12 signal peptide MGGLEPCSRLLLLPLLLAVSGLRPVQA (SEQ ID NO:12). [0354] In some embodiments, any of such sequence set forth by a respective SEQ ID NO may further include a tag or other sequence to facilitate detection of an expressed protein. In some embodiments, the sequence is a fluorescent moiety or is a peptide tag sequence. For instance a peptide tag may include a Flag tag, a Rho1D4-tag, a Myc tag, a His tag, CL7 tag, an HA tag or a V5 tag. In some embodiments, the sequence is a Flag Tag (DYKDDDDK; SEQ ID NO:3).
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30761-20002.40 [0355] In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 31. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 9. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 9. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 65. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 65. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 65. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 10. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 10. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 66. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 66. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 66. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 11. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 11. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 67. In
30761-20002.40 some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 12. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 12. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 32. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 24. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 24. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 33. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 25. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 25.In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 34. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%,
30761-20002.40 about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 26. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 26. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 35. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 27. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 36. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 28. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 28. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 38. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 29. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 29. In some
30761-20002.40 embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 37. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 30. In some embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 39. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 40. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 41. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 42. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 47. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in
30761-20002.40 SEQ ID NO: 47. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 48. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 49. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 50. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 50. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 50. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 58. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 59. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 59. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 59. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 60. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about
30761-20002.40 98%, or about 99% identical to SEQ ID NO: 61. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 61. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 61. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 62. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 63. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 63. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 63. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 113. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 113. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 113. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 115. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 115. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 115. In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 117. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 117. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 117. [0356] In particular embodiments, the synthetic cytokine receptors provided herein comprise a CD34 extracellular domain as described herein, an IL7R transmembrane domain as described herein and a wild-type IL-9R intracellular domain as described herein. In particular embodiments, the synthetic cytokine receptors provided herein comprise a CD34 extracellular domain as described herein, an IL7R transmembrane domain as described herein and a chimeric JAK/STAT fusion intracellular domain as described herein. In particular embodiments, the synthetic cytokine receptors
30761-20002.40 provided herein comprise a truncated CD8 extracellular domain as described herein, an IL7R transmembrane domain as described herein and a wild-type IL-9R intracellular domain as described herein. In particular embodiments, the synthetic cytokine receptors provided herein comprise a truncated CD8 extracellular domain as described herein, an IL7R transmembrane domain as described herein and a chimeric JAK/STAT fusion intracellular domain as described herein. [0357] In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 180. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 180. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 180. [0358] In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 182. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 182. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 182. [0359] In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 179. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 179. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 179. [0360] In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 183. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 183. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 183. [0361] In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 188. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 188. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 188. [0362] In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about
30761-20002.40 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 113. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 113. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 113. [0363] In some embodiments, the synthetic cytokine receptor comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 184. In some embodiments, the synthetic cytokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 184. In some embodiments, the synthetic cytokine receptor consists of the amino acid sequence set forth in SEQ ID NO: 184. III. Polynucleotides [0364] Provided herein are polynucleotides encoding the synthetic cytokine receptors disclosed herein. [0365] Polynucleotides disclosed herein can be at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 1000, at least about 5000, at least about 10000, or at least about 15000 or more nucleotides in length, as well as all intermediate lengths. It will be readily understood that “intermediate lengths,” in this context, means any length between the quoted values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc. [0366] The present disclosure further relates to variants of the polynucleotides disclosed herein. The polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. [0367] In some embodiments, a polynucleotide variant comprising a nucleotide sequence that is at least about 75 %, about 80 %, about 85 %, about 90 %, about 91 %, about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98%, or about 99 % identical to the nucleotide sequence of a polynucleotide disclosed herein. [0368] In some embodiments, a polynucleotide variant contains substitutions, additions, or deletions that alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant contains silent substitutions, additions, or deletions that does not alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.
30761-20002.40 [0369] In some embodiments, polynucleotides are codon-optimized. As used herein, the term “codon-optimized” refers to substituting codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability and/or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, (x) systematic variation of codon sets for each amino acid, (xi) isolated removal of spurious translation initiation sites and/or (xii) elimination of fortuitous polyadenylation sites otherwise leading to truncated RNA transcripts. [0370] It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide, or fragment of variant thereof, as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated in particular embodiments, for example polynucleotides that are optimized for human and/or primate codon selection. Further, alleles of the genes comprising the polynucleotide sequences provided herein may also be used. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. [0371] The polynucleotides contemplated herein, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters and/or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), termination codons, transcriptional termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or as known in the art, such that their overall length may vary considerably. It is therefore contemplated that a polynucleotide fragment of almost any length may be employed in particular embodiments, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. [0372] Polynucleotides can be prepared, isolated, purified, manipulated, and/or expressed using any of a variety of well-established techniques known and available in the art.
30761-20002.40 IV. Vectors [0373] Provided herein are vectors comprising the polynucleotides or nucleic acid molecules encoding the synthetic cytokine receptors disclosed herein. [0374] In order to express the synthetic cytokine receptors described herein in a cell, an expression cassette encoding the synthetic cytokine receptors can be inserted into a nucleic acid vector. The “expression cassette” contains the gene of interest. The cassette is positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein or a polypeptide, undergo appropriate post- translational modifications required for activity in the host cell, and be translocated to the appropriate compartment for biological activity by targeting to appropriate intracellular compartments or secretion into extracellular compartments. Preferably, the cassette has its 3’ and 5’ ends adapted for ready insertion into a vector, e.g., it has restriction endonuclease sites at each end. The cassette can be removed and inserted into a plasmid or viral vector as a single unit. [0375] In some embodiments, vectors include, without limitation, plasmids, phagemids, cosmids, transposons, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. In some embodiments, the coding sequences of the synthetic cytokine receptors disclosed herein can be ligated into such vectors for expression in mammalian cells. [0376] In some embodiments, non-viral vectors are used to deliver one or more polynucleotides contemplated herein. In some embodiments, the recombinant vector comprising a polynucleotide encoding the synthetic cytokine receptors described herein is a plasmid. Numerous suitable plasmid expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are provided by way of example; for eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other plasmid vector may be used so long as it is compatible with the host cell. [0377] In some embodiments, viral vectors are used to deliver one or more polynucleotides contemplated herein. Suitable viral vectors include, but are not limited to, viral vectors based on adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:25432549, 1994; Borras et al., Gene Ther 6:515524, 1999; Li and Davidson, PNAS 92:77007704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191 ; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., U.S. Patent No.7,078,387; Ali et al., Hum Gene Ther 9:8186, 1998, Flannery et al„ PNAS 94:69166921 , 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997; Jomary et al., Gene Ther 4:683690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al„ Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); alphaviruses; arenaviruses; baculovirus; herpes simplex virus; human
30761-20002.40 immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:1031923, 1997; Takahashi et al., J Virol 73:78127816, 1999); poliovirus; poxvirus; retrovirus (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); SV40; vaccinia virus; and the like. Examples of vectors are pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DEST™, pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. [0378] In some embodiments, the vector is a non-integrating vector, including but not limited to, an episomal vector or a vector that is maintained extrachromosomally. As used herein, the term “episomal” refers to a vector that is able to replicate without integration into host’s chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally. The vector is engineered to harbor the sequence coding for the origin of DNA replication or “ori” from a lymphotrophic herpes virus or a gamma herpesvirus, an adenovirus, SV40, a bovine papilloma virus, or a yeast, specifically a replication origin of a lymphotrophic herpes virus or a gamma herpesvirus corresponding to oriP of EBV. In some embodiments, the lymphotrophic herpes virus may be Epstein Barr virus (EBV), Kaposi’s sarcoma herpes virus (KSHV), Herpes virus saimiri (HS), or Marek’s disease virus (MDV). Epstein Barr virus (EBV) and Kaposi’s sarcoma herpes virus (KSHV) are also examples of a gamma herpesvirus. A viral vector delivered by such viruses or viral particles may be referred to by the type of virus to deliver the viral vector (e.g., a lentiviral vector is a viral vector that is to be delivered by a lentivirus). A viral vector can contain viral elements (e.g., nucleotide sequences) necessary for packaging of the viral vector into the virus or viral particle, replicating the virus, or other desired viral activities. A virus containing a viral vector may be replication competent, replication deficient or replication defective. [0379] In some embodiments, the vector is an integrating vector. In some embodiments, a polynucleotide is introduced into a target or host cell using a transposon vector system. In some embodiments, the transposon vector system comprises a vector comprising transposable elements and a polynucleotide contemplated herein; and a transposase. In some embodiments, the transposon vector system is a single transposase vector system, see, e.g., WO 2008/027384. Exemplary transposases include, but are not limited to: piggyBac, Sleeping Beauty, Mos1, Tc1/mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince, and derivatives thereof. The piggyBac transposon and transposase are described, for example, in U.S. Patent 6,962,810, which is incorporated herein by reference in its entirety. The Sleeping Beauty transposon and transposase are described, for example, in Izsvak et al., J. Mol. Biol.302: 93-102 (2000), which is incorporated herein by reference in its entirety. The Tol2 transposon which was first isolated from the medaka fish Oryzias latipes and belongs to the hAT family of transposons is described in Kawakami et al. (2000). Mini-Tol2 is a variant of Tol2 and is described in Balciunas et al. (2006). The Tol2 and Mini-Tol2 transposons facilitate integration of a
30761-20002.40 transgene into the genome of an organism when co-acting with the Tol2 transposase. The Frog Prince transposon and transposase are described, for example, in Miskey et al., Nucleic Acids Res.31:6873- 6881 (2003). [0380] In some embodiments, a polynucleotide sequence encoding the synthetic cytokine receptors disclosed herein is operably linked to one or more control elements that allow expression of the polynucleotide in both prokaryotic and eukaryotic cells. “Control elements” refer those non- translated regions of the vector which interact with host cellular proteins to carry out transcription and translation. Non-limiting examples of control elements include origin of replication, selection cassettes, constitutive and inducible promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, transcription terminators, 5’ and 3’ untranslated regions. See e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544) Such elements may vary in their strength and specificity. The transcriptional control element may be functional in either a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., bacterial, or archaeal cell). [0381] In some embodiments, polynucleotides encoding the synthetic cytokine receptors described herein are operably linked to a promoter and/or an enhancer. The term “promoter” as used herein refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds. An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter. In some embodiments, a promoter operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and/or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide. The term “enhancer” refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence. An enhancer can function cooperatively or additively with promoters and/or other enhancer elements. [0382] Non-limiting examples of suitable eukaryotic promoters (promoters functional in a eukaryotic cell) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, a viral simian virus 40 (SV40) (e.g., early and late SV40), a spleen focus forming virus (SFFV) promoter, long terminal repeats (LTRs) from retrovirus (e.g., a Moloney murine leukemia virus (MoMLV) LTR promoter or a Rous sarcoma virus (RSV) LTR), a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, an elongation factor 1-alpha (EF1Į) promoter, early growth response 1 (EGR1) promoter, a ferritin H (FerH) promoter, a ferritin L (FerL) promoter, a Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, a eukaryotic translation initiation factor 4A1 (EIF4A1) promoter, a heat shock 70kDa protein 5 (HSPA5) promoter, a heat shock protein 90kDa beta, member 1 (HSP90B1) promoter, a heat shock protein 70kDa (HSP70) promoter, a ȕ-kinesin (ȕ-KIN) promoter, the human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), a Ubiquitin C (UBC) promoter, a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirus enhancer/chicken ȕ-actin
30761-20002.40 (CAG) promoter, a ȕ-actin promoter and a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) promoter, and mouse metallothionein-l. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. [0383] In some embodiments, a polynucleotide sequence encoding the synthetic cytokine receptors described herein is operably linked to a constitutive promoter. In such embodiments, the polynucleotides encoding the synthetic cytokine receptors described herein are constitutively and/or ubiquitously expressed in a cell. [0384] In some embodiments, a polynucleotide sequence encoding the synthetic cytokine receptors described herein is operably linked to an inducible promoter. In such embodiments, polynucleotides encoding the synthetic cytokine receptors described herein are conditionally expressed. As used herein, “conditional expression” may refer to any type of conditional expression including, but not limited to, inducible expression; repressible expression; expression in cells or tissues having a particular physiological, biological, or disease state (e.g., cell type or tissue specific expression) etc. Illustrative examples of inducible promoters/systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the “GeneSwitch” mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67), the cumate inducible gene switch (WO 2002/088346), tetracycline-dependent regulatory systems, etc. [0385] In some embodiments, the vectors described herein further comprise a transcription termination signal. Elements directing the efficient termination and polyadenylation of the heterologous nucleic acid transcripts increases heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal. In some embodiments, vectors comprise a polyadenylation sequence 3’ of a polynucleotide encoding a polypeptide to be expressed. The term “polyA site” or “polyA sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by addition of a polyA tail to the 3’ end of the coding sequence and thus, contribute to increased translational efficiency. Cleavage and polyadenylation are directed by a poly(A) sequence in the RNA. The core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking a cleavage- polyadenylation site. Typically, an almost invariant AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5’ cleavage product. In some embodiments, the core poly(A) sequence is an ideal polyA sequence (e.g., AATAAA, ATTAAA, AGTAAA). In some embodiments, the poly(A) sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbit ȕ-globin polyA sequence
30761-20002.40 (rȕgpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art. [0386] In some embodiments, a vector may also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, mitochondrial localization), fused to the polynucleotide encoding the synthetic cytokine receptors. For example, a vector may comprise a nuclear localization sequence (e.g., from SV40) fused to the polynucleotide encoding the synthetic cytokine receptors. In some embodiments, the signal peptide is an Igk signal peptide. In some embodiments, the signal peptide is a CD8 signal peptide. [0387] The expression vector may also include nucleotide sequences encoding protein tags (e.g., Flag tag, 6xHis tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to the site- directed modifying polypeptide, thus resulting in a chimeric polypeptide. [0388] Methods of introducing polynucleotides and recombinant vectors into a host cell are known in the art. Suitable methods include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome- mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al., Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9), microfluidics delivery methods (See e.g., International PCT Publication No. WO 2013/059343), and the like. [0389] In some embodiments, delivery via electroporation comprises mixing the cells with the polynucleotides encoding the synthetic cytokine receptors in a cartridge, chamber, or cuvette and applying one or more electrical impulses of defined duration and amplitude. In some embodiments, cells are mixed with polynucleotides encoding the synthetic cytokine receptors in a vessel connected to a device (e.g., a pump) which feeds the mixture into a cartridge, chamber, or cuvette wherein one or more electrical impulses of defined duration and amplitude are applied, after which the cells are delivered to a second vessel. Illustrative examples of polynucleotide delivery systems suitable for use in particular embodiments contemplated include, but are not limited to, those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, NeonTM Transfection Systems, and Copernicus Therapeutics Inc. Lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient lipofection of polynucleotides have been described in the literature. See e.g., Liu et al. (2003) Gene Therapy.10:180–187; and Balazs et al. (2011) Journal of Drug Delivery.2011:1-12. [0390] In some embodiments, polynucleotides encoding the synthetic cytokine receptors described herein are introduced to a cell in a non-viral delivery vehicle, such as a transposon, a nanoparticle (e.g., a lipid nanoparticle), a liposome, an exosome, an attenuated bacterium, or a virus- like particle. In some embodiments, the vehicle is an attenuated bacterium (e.g., naturally, or artificially engineered to be invasive but attenuated to prevent pathogenesis including Listeria
30761-20002.40 monocytogenes, certain Salmonella strains, Bifidobacterium longum, and modified Escherichia coli), bacteria having nutritional and tissue-specific tropism to target specific cells, and bacteria having modified surface proteins to alter target cell specificity. In some embodiments, the vehicle is a genetically modified bacteriophage (e.g., engineered phages having large packaging capacity, less immunogenicity, containing mammalian plasmid maintenance sequences and having incorporated targeting ligands). In some embodiments, the vehicle is a biological liposome. For example, the biological liposome is a phospholipid-based particle derived from human cells (e.g., erythrocyte ghosts, which are red blood cells broken down into spherical structures derived from the subject and wherein tissue targeting can be achieved by attachment of various tissue or cell-specific ligands), secretory exosomes, or subject derived membrane-bound nanovescicles (30 -100 nm) of endocytic origin (e.g., can be produced from various cell types and can therefore be taken up by cells without the need for targeting ligands). [0391] In some embodiments, vectors comprising polynucleotides encoding the synthetic cytokine receptors described herein are introduced to cells by viral delivery methods, e.g., by viral transduction. A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The heterologous nucleic acid can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the engineered mammalian cell in vitro or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In some embodiments, lentivirus vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying the immunomodulator (such as immune checkpoint inhibitor) coding sequence and/or self-inactivating lentiviral vectors carrying chimeric antigen receptors can be packaged with protocols known in the art. The resulting lentiviral vectors can be used to transduce a mammalian cell (such as primary human T cells) using methods known in the art. Vectors derived from retroviruses such as lentivirus are suitable tools to achieve long-term gene transfer, because they allow long-term, stable integration of a transgene and its propagation in progeny cells. Lentiviral vectors also have low immunogenicity and can transduce nonproliferating cells. [0392] In some embodiments, the vehicle is a mammalian virus-like particle. For example, modified viral particles can be generated (e.g., by purification of the “empty” particles followed by ex vivo assembly of the virus with the desired cargo). V. Engineered Cells and Population of Cells [0393] Provided herein are cells engineered by vectors comprising the polynucleotides or nucleic acid molecules encoding the synthetic cytokine receptors. Also provided herein are a population of cells comprising at least one such engineered cell.
30761-20002.40 [0394] In some aspects, provided herein is an engineered cell expressing one or more synthetic cytokine receptors, wherein the synthetic cytokine receptor comprises an extracellular domain (e.g., any as described in Section II.A), a transmembrane domain (e.g., any as described in Section II.B), and an interleukin 9 receptor (IL-9R) intracellular domain (e.g., any as described in Section II.C. [0395] In some embodiments, the synthetic cytokine receptor expressed by the cell is a constitutively active cytokine receptor. In some embodiments, the synthetic cytokine receptor elicits signaling through STAT1, STAT3, and STAT5 pathways. [0396] In some embodiments, the engineered cell is an immune effector cell. In some embodiments, the immune effector cell is a cytotoxic T cell. In some embodiments, the immune effector cell is a natural killer cell. In some embodiments, the immune effector cell is an animal cell or is derived from an animal cell, including invertebrate animals and vertebrate animals (e.g., fish, amphibian, reptile, bird, or mammal). In some embodiments, the immune effector cell is a mammalian cell or is derived from a mammalian cell (e.g., a pig, a cow, a goat, a sheep, a rodent, a non-human primate, a human, etc.). In some embodiments, the immune effector cell is a human cell or is derived from a human cell. [0397] In some aspects, provided herein is a population of cells comprising at least one of the engineered cells provided herein. [0398] In some aspects, provided herein is a method of engineering a cell to express any of the synthetic cytokine receptors provided herein. In some embodiments, the method comprises contacting the cell with any of the polynucleotides or vectors provided herein. [0399] Producing the engineered cells comprising obtaining a population of unengineered cells from a subject and contacting the cells with polynucleotides encoding the synthetic cytokine receptors disclosed herein or vectors comprising such polynucleotides. In some embodiments, the population of unengineered cells are autologous. The term “autologous” in this context refers to cells that have been derived from the same subject to which they are administered. For example, immune effector cells may be obtained from a subject, engineered ex vivo, and then administered to the same subject in order to treat a disease. In some embodiments, the population of unengineered cells are allogeneic. The term “allogenic” in this context refers to cells that have been derived from one subject and are administered to another subject. For example, immune effector cells may be obtained from a first subject, engineered ex vivo, and then administered to a second subject in order to treat a disease such as a cancer. [0400] In some embodiments, the engineered cells described herein further comprise an exogenous transgene encoding a detectable tag. Examples of detectable tags include but are not limited to, FLAG tags, poly-histidine tags (e.g.6xHis), SNAP tags, Halo tags, cMyc tags, glutathione- S-transferase tags, avidin, enzymes, fluorescent proteins, luminescent proteins, chemiluminescent proteins, bioluminescent proteins, and phosphorescent proteins. In some embodiments the fluorescent protein is selected from the group consisting of blue/UV proteins (such as BFP, TagBFP, mTagBFP2,
30761-20002.40 Azurite, EBFP2, mKalama1, Sirius, Sapphire, and T-Sapphire); cyan proteins (such as CFP, eCFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, and mTFP1); green proteins (such as: GFP, eGFP, meGFP (A208K mutation), Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, and mNeonGreen); yellow proteins (such as YFP, eYFP, Citrine, Venus, SYFP2, and TagYFP); orange proteins (such as Monomeric Kusabira-Orange, mKO^, mKO2, mOrange, and mOrange2); red proteins (such as RFP, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, and mRuby2); far-red proteins (such as mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP); near-infrared proteins (such as TagRFP657, IFP1.4, and iRFP); long stokes shift proteins (such as mKeima Red, LSS-mKate1, LSS-mKate2, and mBeRFP); photoactivatible proteins (such as PA-GFP, PamCherry1, and PATagRFP); photoconvertible proteins (such as Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), mEos3.2 (green), mEos3.2 (red), PsmOrange, and PsmOrange); and photoswitchable proteins (such as Dronpa). In some embodiments, the detectable tag can be selected from AmCyan, AsRed, DsRed2, DsRed Express, E2-Crimson, HcRed, ZsGreen, ZsYellow, mCherry, mStrawberry, mOrange, mBanana, mPlum, mRasberry, tdTomato, DsRed Monomer, and/or AcGFP, all of which are available from Clontech. [0401] In some embodiments, the engineered cells described herein further comprise an exogenous transgene encoding a safety-switch system. Safety-switch systems (also referred to in the art as suicide gene systems) comprise exogenous transgenes encoding for one or more proteins that enable the elimination of an engineered immune effector cell after the cell has been administered to a subject. Examples of safety-switch systems are known in the art. For example, safety-switch systems include genes encoding for proteins that convert non-toxic pro-drugs into toxic compounds such as the Herpes simplex thymidine kinase (Hsv-tk) and ganciclovir (GCV) system (Hsv-tk/GCV). Hsv-tk converts non-toxic GCV into a cytotoxic compound that leads to cellular apoptosis. As such, administration of GCV to a subject that has been treated with the engineered cells comprising a transgene encoding the Hsv-tk protein can selectively eliminate the engineered cells while sparing endogenous immune effector cells. (See e.g., Bonini et al., Science, 1997, 276(5319):1719-1724; Ciceri et al., Blood, 2007, 109(11):1828-1836; Bondanza et al., Blood 2006, 107(5):1828-1836). [0402] Additional safety-switch systems include genes encoding for cell-surface markers, enabling elimination of engineered cells by administration of a monoclonal antibody specific for the cell-surface marker via ADCC. In some embodiments, the cell-surface marker is CD20 and the engineered cells can be eliminated by administration of an anti-CD20 monoclonal antibody such as Rituximab (See e.g., Introna et al., Hum Gene Ther, 2000, 11(4):611-620; Serafini et al., Hum Gene Ther, 2004, 14, 63-76; van Meerten et al., Gene Ther, 2006, 13, 789-797). Similar systems using EGF-R and Cetuximab or Panitumumab are described in International PCT Publication No. WO 2018006880. Additional safety-switch systems include transgenes encoding pro-apoptotic molecules comprising one or more binding sites for a chemical inducer of dimerization (CID), enabling
30761-20002.40 elimination of engineered cells by administration of a CID which induces oligomerization of the pro- apoptotic molecules and activation of the apoptosis pathway. In some embodiments, the pro-apoptotic molecule is Fas (also known as CD95) (Thomis et al., Blood, 2001, 97(5), 1249-1257). In some embodiments, the pro-apoptotic molecule is caspase-9 (Straathof et al., Blood, 2005, 105(11), 4247- 4254). [0403] In some embodiments, the engineered cells described herein further expresses at least one different type of engineered receptor. In some embodiments, the at least one different type of engineered receptor is an engineered antigen-specific receptor (engineered antigen receptor) recognizing a protein target expressed by a target cell, such as a tumor cell or an antigen presenting cell (APC). [0404] In some embodiments, the at least one different type of engineered receptor is a chimeric antigen receptor (CAR). In some embodiments, the at least one different type of engineered receptor is an engineered T-cell receptor (TCR). [0405] In some embodiments, the extracellular domain of the CAR or the TCR binds to an antigen expressed on a cancer cell. In some embodiments, the extracellular domain of the CAR or the TCR binds to an idiotype of an antibody. In some embodiments, the antibody is against an antigen expressed on a cancer cell. In some embodiments, the antigen is selectively expressed or overexpressed on a cancer cell, as compared to normal or non-targeted cells or tissues. In some embodiments, the cancer cell is a blood cancer cell or a solid tumor cancer cell. [0406] In some embodiments, the antigen selected from a cluster of differentiation molecule, such as CD3, CD4, CD8, CD16, CD24, CD25, CD33, CD34, CD45, CD64, CD71, CD78, CD80 (also known as B7-1), CD86 (also known as B7-2), CD96, CD116, CD117, CD123, CD133, and CD138, CD371 (also known as CLL1); a tumor-associated surface antigen, such as 5T4, BCMA (also known as CD269 and TNFRSF17, UniProt# Q02223), carcinoembryonic antigen (CEA), carbonic anhydrase 9 (CAIX or MN/CAIX), CD19, CD20, CD22, CD30, CD40, disialogangliosides such as GD2, ELF2M, ductal-epithelial mucin, ephrin B2, epithelial cell adhesion molecule (EpCAM), ErbB2 (HER2/neu), FCRL5 (UniProt# Q68SN8), FKBP11 (UniProt# Q9NYL4), glioma-associated antigen, glycosphingolipids, gp36, GPRC5D (UniProt# Q9NZD1), mut hsp70-2, intestinal carboxyl esterase, IGF-I receptor, ITGA8 (UniProt# P53708), KAMP3, LAGE-1a, MAGE, mesothelin, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, PAP, prostase, prostate-carcinoma tumor antigen-1 (PCTA-1), prostate specific antigen (PSA), PSMA, prostein, RAGE-1, ROR1, RU1 (SFMBT1), RU2 (DCDC2), SLAMF7 (UniProt# Q9NQ25), survivin, TAG-72, and telomerase; a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope; tumor stromal antigens, such as the extra domain A (EDA) and extra domain B (EDB) of fibronectin; the A1 domain of tenascin-C (TnC A1) and fibroblast associated protein (FAP); cytokine receptors, such as epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), TFGȕ-R or components thereof such as endoglin; a major histocompatibility complex (MHC) molecule; a virus-specific surface antigen such
30761-20002.40 as an HIV-specific antigen (such as HIV gp120); an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen, a Lassa virus-specific antigen, an Influenza virus-specific antigen as well as any derivate or variant of these surface antigens. [0407] In some embodiments, the at least one different type of engineered receptor is a chimeric antigen receptor. In some embodiments, the extracellular domain of the chimeric antigen receptor binds to an antigen expressed on a cancer cell. In some embodiments, the extracellular domain of the chimeric antigen receptor binds to an idiotype of an antibody. In some embodiments, the antibody is against an antigen expressed on a cancer cell. In some embodiments, the cancer cell is a blood cancer cell or a solid tumor cancer cell. In some embodiments, the cancer cell is a blood cancer cell. In some embodiments, the cancer cell is a solid tumor cancer cell. [0408] A CAR generally includes an extracellular antigen binding domain, such as a portion of an antibody molecule, generally a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment. [0409] In some embodiments, the CAR is constructed with a specificity for the particular antigen, such as an antigen expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer marker, and/or an antigen intended to induce a dampening response, such as an antigen expressed on a normal or non-diseased cell type. Thus, the CAR typically includes in its extracellular portion one or more antigen binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable domains, and/or antibody molecules. In some embodiments, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb). [0410] In some embodiments, the antibody or antigen-binding portion thereof is expressed on cells as part of a recombinant engineered receptor, such as a chimeric receptor (e.g. CAR), that binds, such as specifically binds, to the antigen. Among the antigens targeted by the chimeric receptors are those expressed in the context of a disease, condition, or cell type to be targeted via the adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and/or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas. [0411] A CAR comprises an extracellular antigen binding domain fused via hinge and transmembrane domains to a cytoplasmic domain comprising a signaling domain. [0412] In some embodiments, the CAR extracellular domain binds to an antigen expressed by a target cell in an MHC-independent manner. In some embodiments, the extracellular domain of a CAR recognizes a tag fused to an antibody or antigen-binding fragment thereof. In such embodiments, the antigen-specificity of the CAR is dependent on the antigen-specificity of the labeled antibody, such that a single CAR construct can be used to target multiple different antigens by substituting one
30761-20002.40 antibody for another (See e.g., US Patent Nos.9,233,125 and 9,624,279; US Patent Application Publication Nos.20150238631 and 20180104354). In some embodiments, the extracellular domain of a CAR may comprise an antigen binding fragment derived from an antibody. Antigen binding domains that are useful in the present disclosure include, for example, scFvs; antibodies; antigen binding regions of antibodies; variable regions of the heavy/light chains; and single chain antibodies. [0413] In some embodiments, the intracellular signaling domain of a CAR may be derived from the TCR complex zeta chain (such as CD3^ signaling domains), FcȖRIII, FcİRI, or the T-lymphocyte activation domain. In some embodiments, the intracellular signaling domain of a CAR further comprises a costimulatory domain, for example a 4-1BB, CD28, CD40, MyD88, or CD70 domain. In some embodiments, the intracellular signaling domain of a CAR comprises two costimulatory domains, for example any two of 4-1BB, CD28, CD40, MyD88, or CD70 domains. Exemplary CAR structures and intracellular signaling domains are known in the art (See e.g., WO 2009/091826; US 20130287748; WO 2015/142675; WO 2014/055657; and WO 2015/090229, incorporated herein by reference). [0414] CARs specific for a variety of tumor antigens are known in the art, for example CD171- specific CARs (Park et al., Mol Ther (2007) 15(4):825-833), EGFRvIII-specific CARs (Morgan et al., Hum Gene Ther (2012) 23(10):1043-1053), EGF-R-specific CARs (Kobold et al., J Natl Cancer Inst (2014) 107(1):364), carbonic anhydrase K-specific CARs (Lamers et al., Biochem Soc Trans (2016) 44(3):951-959), FR-Į-specific CARs (Kershaw et al., Clin Cancer Res (2006) 12(20):6106-6015), HER2-specific CARs (Ahmed et al., J Clin Oncol (2015) 33(15)1688-1696;Nakazawa et al., Mol Ther (2011) 19(12):2133-2143; Ahmed et al., Mol Ther (2009) 17(10):1779-1787; Luo et al., Cell Res (2016) 26(7):850-853; Morgan et al., Mol Ther (2010) 18(4):843-851; Grada et al., Mol Ther Nucleic Acids (2013) 9(2):32), CEA-specific CARs (Katz et al., Clin Cancer Res (2015) 21(14):3149- 3159), IL13RĮ2-specific CARs (Brown et al., Clin Cancer Res (2015) 21(18):4062-4072), GD2- specific CARs (Louis et al., Blood (2011) 118(23):6050-6056; Caruana et al., Nat Med (2015) 21(5):524-529), ErbB2-specific CARs (Wilkie et al., J Clin Immunol (2012) 32(5):1059-1070), VEGF-R-specific CARs (Chinnasamy et al., Cancer Res (2016) 22(2):436-447), FAP-specific CARs (Wang et al., Cancer Immunol Res (2014) 2(2):154-166), MSLN-specific CARs (Moon et al, Clin Cancer Res (2011) 17(14):4719-30), NKG2D-specific CARs (VanSeggelen et al., Mol Ther (2015) 23(10):1600-1610), CD19-specific CARs (Axicabtagene ciloleucel (Yescarta®) and Tisagenlecleucel (Kymriah®). See also¸ Li et al., J Hematol and Oncol (2018) 11(22), reviewing clinical trials of tumor-specific CARs. [0415] In some embodiments, the CAR may include, but is not limited to a CAR engineered into cells of idecabtagene vicleucel (ABECMA®), Orvacabtagene Autoleucel (JCARH125), ciltacabtagene autoleucel (CARVYKTI™, also called NJ-68284528; Janssen/Legend), P-BCMA-101 (Poseida), PBCAR269A (Poseida), P-BCMA- Allo1 (Poseida), Allo-715 (Pfizer/Allogene), CT053 (Carsgen), Descartes-08 (Cartesian), PHE885 (Novartis), CTX120 (CRISPR Therapeutics);
30761-20002.40 axicabtagene ciloleucel (YESCARTA®), tisagenlecleucel (KYMRIAH®), brexucabtagene autoleucel (TECARTUS®), or lisocabtagene maraleucel (BREYANZI®). [0416] Engineered TCRs comprise TCRĮ and/or TCRȕ chains that have been isolated and cloned from T cell populations recognizing a particular target antigen. For example, TCRĮ and/or TCRȕ genes (i.e., TRAC and TRBC) can be cloned from T cell populations isolated from individuals with particular malignancies or T cell populations that have been isolated from humanized mice immunized with specific tumor antigens or tumor cells. Engineered TCRs recognize antigen through the same mechanisms as their endogenous counterparts (e.g., by recognition of their cognate antigen presented in the context of major histocompatibility complex (MHC) proteins expressed on the surface of a target cell). This antigen engagement stimulates endogenous signal transduction pathways leading to activation and proliferation of the TCR-engineered cells. [0417] Engineered TCRs specific for tumor antigens are known in the art, for example WT1- specific TCRs (JTCR016, Juno Therapeutics; WT1-TCRc4, described in US Patent Application Publication No.20160083449), MART-1 specific TCRs (including the DMF4T clone, described in Morgan et al., Science 314 (2006) 126-129); the DMF5T clone, described in Johnson et al., Blood 114 (2009) 535-546); and the ID3T clone, described in van den Berg et al., Mol. Ther.23 (2015) 1541-1550), gp100-specific TCRs (Johnson et al., Blood 114 (2009) 535-546), CEA-specific TCRs (Parkhurst et al., Mol Ther.19 (2011) 620-626), NY-ESO and LAGE-1 specific TCRs (1G4T clone, described in Robbins et al., J Clin Oncol 26 (2011) 917-924; Robbins et al., Clin Cancer Res 21 (2015) 1019-1027; and Rapoport et al., Nature Medicine 21 (2015) 914-921), and MAGE-A3-specific TCRs (Morgan et al., J Immunother 36 (2013) 133-151) and Linette et al., Blood 122 (2013) 227- 242). (See also, Debets et al., Seminars in Immunology 23 (2016) 10-21). VI. Compositions and Kits [0418] Provided herein are pharmaceutical composition comprising an engineered cell expressing the synthetic cytokine receptors disclosed herein or a population of cells comprising at least one such engineered cell an engineered cell expressing the synthetic cytokine receptors disclosed herein or a population of cells comprising at least one such engineered cell. [0419] In some embodiments, the pharmaceutical composition further comprises a pharmaceutical acceptable carrier. [0420] Pharmaceutically acceptable carrier, diluent or excipient includes, without limitation, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, and/or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans and/or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc;
30761-20002.40 cocoa butter; waxes; animal and vegetable fats; paraffins; silicones; bentonites; silicic acid; zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical formulations. Except insofar as any conventional media and/or agent is incompatible with the agents of the present disclosure, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. [0421] Pharmaceutically acceptable salt includes both acid and base addition salts. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10- sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2- hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, ptoluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
30761-20002.40 [0422] Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. [0423] Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. [0424] Further guidance regarding formulations that are suitable for various types of administration can be found in Remington’s Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990). [0425] Also provided herein are kits for carrying out a method described herein. In some embodiments, a kit comprises an engineered cell expressing the synthetic cytokine receptors disclosed herein or a population of cells comprising at least one such engineered cell. [0426] In some embodiments, a kit further comprises instructions for using the components of the kit to practice the methods of the present disclosure. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert or in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging). In some embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate. [0427] In some embodiments, pharmaceutical compositions disclosed herein are for use in treating a cancer in a subject. Details of using pharmaceutical compositions disclosed herein for treating cancer are disclosed in the “Therapeutic Methods and Applications” section below. VII. Therapeutic Methods and Applications [0428] In some aspects, provided herein is a method of treating a disease or condition. In some embodiments, the disease or condition is a disease or condition that can be targeted by any of the engineered cells provided herein. For instance, in some embodiments provided engineered cell express an engineered receptor (e.g., CAR or TCR) that targets an antigen expressed by cells (also called “target cells”) of the disease or condition, such as an antigen expressed by tumor cells. In some
30761-20002.40 embodiments, the methods result in targeted killing of a target cell, such as a tumor cell. In some embodiments, the provided methods and uses treat a disease or condition in the subject. Also provided herein are compositions for use and methods relating to the provided engineered cell compositions, including any as described herein, for use in treating diseases or conditions in a subject. Such methods and uses include therapeutic methods and uses, for example, involving administration of the therapeutic cells, or compositions containing the same, to a subject having a disease or condition. In some embodiments, the co-expression with a provided synthetic cytokine receptor improves the persistence and function of the engineered cells when administered in vivo to a subject. [0429] In some embodiments, the disease or condition comprises a cancer. In some embodiments, the disease or condition comprises a solid tumor. [0430] Provided herein are methods of treating a cancer in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. [0431] In some embodiments, the method described herein may be used in the treatment of a cell-proliferative disorder, such as a cancer. In some embodiments, the cancer is blood cancer. In some embodiments, the cancer is solid tumor. For example, cancers that may be treated using the compositions and methods disclosed herein include, but are not limited to, leukemia, lymphoma, myeloma, carcinoma, sarcoma, or brain and spinal cord cancer. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL), B cell acute lymphocytic leukemia (B-ALL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), non-Hodgkin’s lymphoma (NHL), diffuse large cell lymphoma (DLCL), diffuse large B cell lymphoma (DLBCL), Hodgkin’s lymphoma, multiple myeloma, renal cell carcinoma (RCC), hepatocellular carcinoma, melanoma, mesothelioma, colorectal cancer, bladder cancer, breast cancer, colorectal cancer, ovarian cancer, prostate cancer, lung cancer, esophageal cancer, pancreatic cancer, head and neck cancer, liver cancer, cervical cancer, breast cancer, astrocytoma, medulloblastoma, and neuroblastoma. In some embodiments, the cancer is insensitive or resistant. [0432] In some embodiments, the subject is human. In some embodiments, the subject is non- human primates (e.g., monkeys, baboons, and chimpanzees), mice, rats, bovines, horses, household cats, tigers and other large cats, dogs, pigs, rabbits, goats, deer, sheep, ferrets, gerbils, guinea pigs, hamsters, bats, and birds (e.g., chickens, turkeys, and ducks). In some embodiments, the subject may be a neonate, a juvenile, or an adult. [0433] In some embodiments, treating refers to the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting disease development or preventing disease progression; (b) relieving the disease, i.e., causing regression of the disease state or relieving one or more symptoms of the disease; and (c) curing the disease, i.e., remission of one or more disease symptoms. In some embodiments, treatment results in an improvement or remediation of the symptoms of the disease. In some embodiments, treatment results in a reduction of tumor burden, e.g., tumor volume. In some embodiments, treatment may refer to a short-term (e.g., temporary and/or
30761-20002.40 acute) and/or a long-term (e.g., sustained) improvement or remediation in one or more disease symptoms. In some embodiments, the improvement is an observable or measurable improvement. In some embodiments, the improvement is an improvement in the general feeling of well-being of the subject. In some embodiments, the improvement is an increase in survival of the subject. In some embodiments, treating refers to increasing the likelihood of survival of the subject. [0434] Administration of the pharmaceutical compositions can occur by infusion (e.g., continuous or bolus), injection, irrigation, inhalation, consumption, electro-osmosis, hemodialysis, iontophoresis, and other methods known in the art. [0435] In some embodiments administration route is intraarterial, intracranial, intradermal, intraduodenal, intrammamary, intrameningeal, intraperitoneal, intrathecal, intratumoral, intravenous, intravitreal, ophthalmic, parenteral, spinal, subcutaneous, ureteral, urethral, vaginal, or intrauterine. In some embodiments, administration route is local or systemic. [0436] The effective amount of the pharmaceutical compositions administered to a particular subject will depend on a variety of factors, several of which will differ from patient to patient including the disorder being treated and the severity of the disorder; activity of the specific agent(s) employed; the age, body weight, general health, sex and diet of the patient; the timing of administration, route of administration; the duration of the treatment; drugs used in combination; the judgment of the prescribing physician; and like factors known in the medical arts. Dosage amount and interval can be adjusted individually to provide plasma levels of the compound(s) which are sufficient to maintain therapeutic or prophylactic effect. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of active compound(s) cannot be related to plasma concentration. Skilled artisans will be able to optimize effective local dosages without undue experimentation. [0437] In some embodiments, the effective amount of the pharmaceutical compositions may be the number of engineered cells required to result in at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, or more fold decrease in tumor mass or volume, decrease in the number of tumor cells, or decrease in the number of metastases. In some embodiments, the effective amount of the pharmaceutical compositions may be the number of engineered cells required to achieve an increase in life expectancy, an increase in progression-free or disease-free survival, or amelioration of various physiological symptoms associated with the disease being treated. [0438] In some embodiments, at least about 1 x 103 engineered cells expressing the synthetic cytokine receptors disclosed herein on the surface of the cells are administered to a subject. In some embodiments, at least about 5 x 103 such cells, about 1 x 104 such cells, about 5 x 104 such cells, about 1 x 105 such cells, about 5 x 105 such cells, about 1 x 106, about 2 x 106, about 3 x 106, about 4 x 106, about 5 x 106, about 1 x 107, about 1 x 108, about 5 x 108, about 1 x 109, about 5 x 109, about 1 x 1010, about 5 x 1010, about 1 x 1011, about 5 x 1011, about 1 x 1012, about 5 x 1012, or more such cells
30761-20002.40 are administered to a subject. In some embodiments, between about 1 x 107 and about 1 x 1012 such cells are administered to a subject. In some embodiments, between about 1 x 108 and about 1 x 1012 such cells are administered to a subject. In some embodiments, between about 1 x 109 and about 1 x 1012 such cells are administered to a subject. In some embodiments, between about 1 x 1010 and about 1 x 1012 such cells are administered to a subject. In some embodiments, between about 1 x 1011 and about 1 x 1012 such cells are administered to a subject. In some embodiments, between about 1 x 107 and about 1 x 1011 such cells are administered to a subject. In some embodiments, between about 1 x 107 and about 1 x 1010 such cells are administered to a subject. In some embodiments, between about 1 x 107 and about 1 x 109 such cells are administered to a subject. In some embodiments, between about 1 x 107 and about 1 x 108 such cells are administered to a subject. [0439] The number of administrations of treatment to a subject may vary. In some embodiments, introducing the pharmaceutical compositions into the subject may be a one-time event. In some embodiments, such treatment may require an on-going series of repeated treatments (e.g., once per day, once per week, or multiple times per day or week). In some embodiments, multiple administrations of the pharmaceutical compositions may be required before an effect is observed. The exact protocols depend upon the disease or condition, the stage of the disease and parameters of the individual subject being treated. [0440] In some embodiments, the pharmaceutical compositions disclosed herein are administered in combination with additional therapeutic composition(s). In some embodiments, the pharmaceutical compositions disclosed herein and the additional therapeutic composition(s) are administered simultaneously. In some embodiments, the pharmaceutical compositions disclosed herein is administered before the additional therapeutic composition(s). In some embodiments, the pharmaceutical compositions disclosed herein is administered after the additional therapeutic composition(s). [0441] In some embodiments, administration of the pharmaceutical compositions disclosed herein in combination with the additional therapeutic composition(s) results in an enhanced therapeutic effect in a cancer than is observed by treatment with either the pharmaceutical compositions disclosed herein or the additional therapeutic composition alone. In some embodiments, the cancer is resistant, refractory, or insensitive to treatment by the additional therapeutic composition alone. In some embodiments, the cancer is partially resistant, partially refractory, or partially insensitive to treatment by the additional therapeutic composition alone. [0442] In some embodiments, the additional therapeutic composition is an immune checkpoint inhibitor. Several immune checkpoint inhibitors are known in the art and have received FDA approval for the treatment of one or more cancers. For example, FDA-approved PD-L1 inhibitors include Atezolizumab (Tecentriq®, Genentech), Avelumab (Bavencio®, Pfizer), and Durvalumab (Imfinzi®, AstraZeneca); FDA-approved PD-1 inhibitors include Pembrolizumab (Keytruda®, Merck) and Nivolumab (Opdivo®, Bristol-Myers Squibb); and FDA-approved CTLA4 inhibitors include
30761-20002.40 Ipilimumab (Yervoy®, Bristol-Myers Squibb). Additional inhibitory immune checkpoint molecules that may be the target of future therapeutics include A2AR, B7-H3, B7-H4, BTLA, IDO, LAG3 (e.g., BMS-986016, under development by BSM), KIR (e.g., Lirilumab, under development by BSM), TIM3, TIGIT, and VISTA. [0443] In other embodiments, the additional therapeutic composition is an oncolytic virus. An oncolytic virus is a virus that preferentially infects and kills a cancer cell. In some embodiments, the oncolytic virus infects the cancer cell. In some embodiments, the oncolytic virus infects and kills the cancer cell. In some embodiments, the oncolytic virus infects but does not kill the cancer cell. In some embodiments, the oncolytic virus is engineered to express a synthetic protein in the cancer cell. In some embodiments, the engineered oncolytic virus expresses the synthetic protein in the cancer cell. In some embodiments, the cancer cell expresses the synthetic protein on its surface. In some embodiments, the cell surface expressed synthetic protein is recognized by an engineered receptor, such as a chimeric antigen receptor (CAR), expressed by engineered cells described herein. [0444] In some aspects, provided herein are methods of improving efficacy of an immune cell therapy (i.e., cell immunotherapy). Various immunotherapy methods are available for treating diseases and conditions. For example, adoptive cell therapies (including those involving the administration of cells expressing chimeric receptors specific for a disease or disorder of interest, such as chimeric antigen receptors (CARs) and/or other recombinant antigen receptors, such as the synthetic cytokine receptors provided herein, as well as other adoptive immune cell and adoptive T cell therapies) can be beneficial in the treatment of cancer or other diseases or disorders. In some embodiments, improving efficacy of a cell immunotherapy includes improving immune cell activation. In some embodiments, improving efficacy of a cell immunotherapy includes decreasing toxicity caused by immune cell therapies. [0445] In some embodiments, provided herein are methods for improving immune cell function. In some embodiments, improving immune cell function comprises increased STAT signaling in a cell expressing any of the synthetic cytokine receptors provided herein compared to an immune cell expressing wild-type IL-9R. In some embodiments, increased STAT signaling can be increased STAT1, STAT3, and/or STAT5 signaling. [0446] In some embodiments, provided herein are methods for improving cytotoxicity of an immune cell. In some embodiments, improving cytotoxicity of the immune cell comprises increased target cell killing by a cell expressing any of the synthetic cytokine receptors provided herein compared to an immune cell expressing wild-type IL-9R. [0447] In some embodiments, provided herein are methods for improving viability of an immune cell. In some embodiments, improving viability of an immune cell comprises decreased cell death for an immune cell expressing any of the synthetic cytokine receptors provided herein compared to an immune cell expressing wild-type IL-9R.
30761-20002.40 [0448] In some embodiments, provided herein are methods for improving cytokine secretion of an immune cell. In some embodiments, improving cytokine secretion comprises increased secretion of a proinflammatory cytokine. In some embodiments, the proinflammatory cytokine is selected from the group consisting of IFN-Ȗ, IL-1ȕ, IL-6, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, , TNF-Į, or TNF-ȕ. In some embodiments, the proinflammatory cytokine is IFN-Ȗ. [0449] In some embodiments, the methods described above first require introducing any of the polynucleotides or vectors provided herein to the immune cell. [0450] In some embodiments, the immune cell comprises any cell involved in mounting an innate or adaptive immune response. In some embodiments, the immune cell comprises a lymphocyte, a natural killer (NK) cell, a NKT cell, a macrophage, a monocyte, an eosinophil, a basophil, a neutrophil, a dendritic cell, or a mast cell. In some embodiments, the immune effector cell is a lymphocyte. In some embodiments, the immune effector cell is a cytotoxic T cell, such as a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), a Th1 cell, a Th2 cell, or a Th17 cell. In some embodiments, the immune effector cell is a natural killer cell. In some embodiments, the immune effector cell is a myeloid cell. In some embodiments, the immune effector cell is a monocyte. In some embodiments, immune effector cell is a macrophage. [0451] In some embodiments, the immune cell further comprises a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the extracellular domain binds to an antigen expressed on a cell or tissue associated with a disease or condition. In some embodiments, the extracellular domain binds to an antigen expressed on a cancer cell or tumor. VIII. EXEMPLARY EMBODIMENTS [0452] This invention provides the following non-limiting embodiments. Embodiment 1. A synthetic cytokine receptor that is a homodimer of identical polypeptide chains each comprising an extracellular domain, a transmembrane domain, and an intracellular domain capable of interleukin 9 receptor (IL-9R) signaling. Embodiment 2. The synthetic cytokine receptor of embodiment 1, wherein the intracellular domain capable of IL-9R signaling comprises an IL-9R intracellular domain or a variant thereof. Embodiment 3. The synthetic cytokine receptor of embodiment 1 or embodiment 2, wherein the intracellular domain capable of IL-9R signaling comprises a chimeric JAK/STAT fusion domain. Embodiment 4. A synthetic cytokine receptor, comprising an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain or a
30761-20002.40 variant thereof, wherein the synthetic cytokine receptor is a constitutively active cytokine receptor. Embodiment 5. The synthetic cytokine receptor of any of embodiments 1 to 4, wherein the synthetic cytokine receptor is a multimer. Embodiment 6. The synthetic cytokine receptor of any of embodiments 1 to 4 that is a multimer of identical polypeptide chains each comprising the extracellular domain, the transmembrane domain and the IL-9R intracellular domain or variant thereof. Embodiment 7. The synthetic cytokine receptor of embodiment 5 or embodiment 6, wherein the multimer is a dimer. Embodiment 8. The synthetic cytokine receptor of embodiment 7, wherein the dimer is a homodimer. Embodiment 9. The synthetic cytokine receptor of any of embodiments 5 to 8, wherein each polypeptide chain is constitutively multimerized. Embodiment 10. The synthetic cytokine receptor of any of embodiments 1 to 9, wherein the synthetic cytokine receptor comprises at least one self-assembly domain. Embodiment 11. The synthetic cytokine receptor of embodiment 10, wherein the at least one self-assembly domain is the extracellular domain and/or the transmembrane domain. Embodiment 12. The synthetic cytokine receptor of any of embodiments 1 to 11, wherein the synthetic cytokine receptor is multimerized through the transmembrane domain and/or the extracellular domain. Embodiment 13. The synthetic cytokine receptor of any of embodiments 1 to 12, wherein the synthetic cytokine receptor is multimerized through the transmembrane domain and the extracellular domain. Embodiment 14. A synthetic cytokine receptor that is a homodimer of identical polypeptide chains each comprising an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain or a variant thereof. Embodiment 15. The synthetic cytokine receptor of any of embodiments 1 to 14, wherein the extracellular domain and/or the transmembrane domain are heterologous to the IL-9R. Embodiment 16. The synthetic cytokine receptor of any of embodiments 1 to 15, wherein the transmembrane domain and extracellular domain are the transmembrane domain and extracellular domain from the same protein.
30761-20002.40 Embodiment 17. The synthetic cytokine receptor of any of embodiments 1 to 15, wherein the transmembrane domain and extracellular domain are the transmembrane domain and extracellular domain from different proteins. Embodiment 18. The synthetic cytokine receptor of any of embodiments 1 to 17, wherein the transmembrane domain is 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 amino acids in length. Embodiment 19. The synthetic cytokine receptor of any of embodiments 1 to 18, wherein the transmembrane domain comprises a transmembrane domain derived from Glycophorin A (GpA) Carnitine palmitoyltransferase 1 (CPT1), a tumor necrosis factor receptor (TNFR) or Muc24. Embodiment 20. The synthetic cytokine receptor of any of embodiments 1 to 18, wherein the transmembrane domain comprises a transmembrane domain derived from Thrombopoietin receptor. Embodiment 21. The synthetic cytokine receptor of any of embodiments 1 to 20, wherein the transmembrane domain promotes alpha-helix dimerization. Embodiment 22. The synthetic cytokine receptor of any of embodiments 1 to 21, wherein the transmembrane domain comprises the motif GXXXG (SEQ ID NO: 68). Embodiment 23. The synthetic cytokine receptor of any of embodiments 1 to 21, wherein the transmembrane domain comprises the motif LIxxGVxxGVxxT (SEQ ID NO: 70). Embodiment 24. The synthetic cytokine receptor of any of embodiments 1 to 23, wherein the transmembrane domain is a transmembrane domain derived from Glycophorin A (GpA) or a variant thereof that comprises one or more mutations (e.g, 1, 2, 3, 4, 5 or 6 mutations) compared to a wild-type GpA transmembrane domain, wherein the variant GpA is sufficient to promote alpha-helix dimerization. Embodiment 25. The synthetic cytokine receptor of any of embodiments 1 to 24, wherein the transmembrane domain comprises a transmembrane domain derived from Glycophorin A (GpA). Embodiment 26. The synthetic cytokine receptor of any of embodiments 1 to 25, wherein the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 22 or SEQ ID NO:43. Embodiment 27. The synthetic cytokine receptor of any of embodiments 1 to 26, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 43.
30761-20002.40 Embodiment 28. The synthetic cytokine receptor of any of embodiments 1 to 22, wherein the transmembrane domain comprises GXXXG (SEQ ID NO: 68) and GXXXA (SEQ ID NO: 69) motifs. Embodiment 29. The synthetic cytokine receptor of any of embodiments 1 to 22 and 28, wherein the transmembrane domain comprises a transmembrane domain derived from Carnitine palmitoyltransferase 1 (CPT1). Embodiment 30. The synthetic cytokine receptor of any of embodiments 1 to 22, 28 and 29, wherein the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 45. Embodiment 31. The synthetic cytokine receptor of any of embodiments 1 to 22 and 28 to 30, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 45. Embodiment 32. The synthetic cytokine receptor of any of embodiments 1 to 21, wherein the synthetic cytokine receptor comprises the motif AXXXA (SEQ ID NO:77) or AXXXS (SEQ ID NO: 78). Embodiment 33. The synthetic cytokine receptor of any of embodiments 1 to 22 or 32, wherein the synthetic cytokine receptor comprises the motif ĭPXĭ (SEQ ID NO:75) or ĭTXXAĭ (SEQ ID NO: 76). Embodiment 34. The synthetic cytokine receptor of any of embodiments 1 to 22, 32 or 33, wherein the transmembrane domain is derived from a TNFR. Embodiment 35. The synthetic cytokine receptor of embodiment 34, wherein the TNFR is TACI, DR5, p75NTR, Fas, TNFR1, TNFR2 or OX40. Embodiment 36. The synthetic cytokine receptor of any of embodiments 1 to 22, and 32 to 35, wherein the transmembrane domain comprises a transmembrane domain derived from DR5. Embodiment 37. The synthetic cytokine receptor of any of embodiments 1 to 22, and 32 to 36, wherein the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 46. Embodiment 38. The synthetic cytokine receptor of any of embodiments 1 to 22 and 32 to 37, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 46. Embodiment 39. The synthetic cytokine receptor of any of embodiments 1 to 22, and 32 to 36, wherein the transmembrane domain comprises a transmembrane domain derived from TACI.
30761-20002.40 Embodiment 40. The synthetic cytokine receptor of any of embodiments 1 to 22, 32 to 36 and 39, wherein the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 44. Embodiment 41. The synthetic cytokine receptor of any of embodiments 1 to 23, 32 to 36, 39 and 40, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 44. Embodiment 42. The synthetic cytokine receptor of any of embodiments 1 to 19, wherein the transmembrane domain comprises 1 to 6 cysteine residues. Embodiment 43. The synthetic cytokine receptor of any of embodiments 1 to 19 and 42, wherein the transmembrane domain promotes disulfide-linked dimerization. Embodiment 44. The synthetic cytokine receptor of embodiment 43, wherein the disulfide-linked dimerization forms 1 to 4 disulfide bridges between polypeptide chains of the synthetic cytokine receptor. Embodiment 45. The synthetic cytokine receptor of any of embodiments 1 to 19 and 42- 44, wherein the transmembrane domain is a variant transmembrane domain that comprises one or more mutations compared to a wild-type transmembrane domain to promote homodimerization of the receptor polypeptide. Embodiment 46. The synthetic cytokine receptor of embodiment 45, wherein the one or more mutations promote alpha-helix dimerization or disulfide-linked dimerization. Embodiment 47. The synthetic cytokine receptor of embodiment 45 or embodiment 46, wherein the one or more mutations introduces at least one cysteine into the transmembrane domain. Embodiment 48. The synthetic cytokine receptor of any of embodiments 45 to 47, wherein the one or more mutations introduces a proline into the transmembrane domain. Embodiment 49. The synthetic cytokine receptor of any of embodiments 45 to 47, wherein the one or more mutations introduces a threonine into the transmembrane domain. Embodiment 50. The synthetic cytokine receptor of any of embodiments 45 to 49, wherein the one or more mutations introduces a trimer peptide of cysteine, proline, and another amino acid other than cysteine or proline into the transmembrane domain. Embodiment 51. The synthetic cytokine receptor of any of embodiments 45-50, wherein the one or more mutations introduces a trimer peptide of cysteine, proline, threonine (CPT or TCP) into the transmembrane domain. Embodiment 52. The synthetic cytokine receptor of any of embodiments 45-51, wherein the transmembrane domain is a variant IL-7R transmembrane domain and the one or more
30761-20002.40 mutations is in the wild-type transmembrane sequence PILLTISILSFFSVALLVILACVLW (SEQ ID NO: 71). Embodiment 53. The synthetic cytokine receptor of any of embodiments 1 to 19 and 42 to 52, wherein the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 6, SEQ ID NO:7 or SEQ ID NO: 21. Embodiment 54. The synthetic cytokine receptor of any of embodiments 1 to 19 and 42 to 53, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:21. Embodiment 55. The synthetic cytokine receptor of any of embodiments 1 to 19, wherein the transmembrane domain comprises a transmembrane domain derived from Muc24. Embodiment 56. The synthetic cytokine receptor of any of embodiments 1 to 19 and 55, wherein the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 23. Embodiment 57. The synthetic cytokine receptor of any of embodiments 1 to 19, 55 or 56, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 23. Embodiment 58. The synthetic cytokine receptor of any of embodiments 1 to 57, wherein the extracellular domain is between about 150 to 260 amino acids in length. Embodiment 59. The synthetic cytokine receptor of any of embodiments 1 to 58, wherein the extracellular domain is a dimerizing domain. Embodiment 60. The synthetic cytokine receptor of embodiment 59, wherein the dimerizing domain comprises a hinge region. Embodiment 61. The synthetic cytokine receptor of any of embodiments 1 to 60, wherein the extracellular domain promotes disulfide-linked dimerization. Embodiment 62. The synthetic cytokine receptor of any of embodiments 1 to 61, wherein the extracellular domain comprises 1 to 6 cysteine residues. Embodiment 63. The synthetic cytokine receptor of embodiment 61 or embodiment 62, wherein the disulfide-linked dimerization forms 1 to 4 disulfide bridges between polypeptide chains of the synthetic cytokine receptor. Embodiment 64. The synthetic cytokine receptor of any of embodiments 1 to 63, wherein the extracellular domain is derived from the extracellular domain of CD34, DAP12, Glycophorin A, CD8, or Muc24.
30761-20002.40 Embodiment 65. The synthetic cytokine receptor of any of embodiments 1 to 63, wherein the extracellular domain comprises an extracellular domain derived from Thrombopoietin receptor. Embodiment 66. The synthetic cytokine receptor of any of embodiments 1 to 65, wherein the extracellular domain comprises an extracellular domain of CD8 or a truncated portion thereof comprising at least one cysteine residue. Embodiment 67. The synthetic cytokine receptor of any of embodiments 1 to 66, wherein the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 18 or SEQ ID NO: 19. Embodiment 68. The synthetic cytokine receptor of any of embodiments 1 to 67, wherein the extracellular domain comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19. Embodiment 69. The synthetic cytokine receptor of any of embodiments 1 to 65, wherein the extracellular domain comprises an extracellular domain of CD34 or a truncated portion thereof comprising at least one cysteine residue. Embodiment 70. The synthetic cytokine receptor of any of embodiments 1 to 65 and 69, wherein the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 4. Embodiment 71. The synthetic cytokine receptor of any of embodiments 1 to 65, 69 and 70, wherein the extracellular domain comprises the amino acid sequence of SEQ ID NO: 4. Embodiment 72. The synthetic cytokine receptor of any of embodiments 1 to 65, wherein the extracellular domain is an extracellular domain of Muc24 or a truncated portion thereof comprising at least one cysteine residue. Embodiment 73. The synthetic cytokine receptor of any of embodiments 1 to 65 and 72, wherein the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 20. Embodiment 74. The synthetic cytokine receptor of any of embodiments 1 to 65, 72 and 73, wherein the extracellular domain comprises the amino acid sequence of SEQ ID NO: 20. Embodiment 75. The synthetic cytokine receptor of any of embodiments 1 to 65, wherein the extracellular domain is an extracellular domain of DAP12 or a truncated portion thereof comprising at least one cysteine residue. Embodiment 76. The synthetic cytokine receptor of any of embodiments 1 to 65 and 75, wherein the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 5.
30761-20002.40 Embodiment 77. The synthetic cytokine receptor of any of embodiments 1 to 65, 75 and 76, wherein the extracellular domain comprises the amino acid sequence of SEQ ID NO: 5. Embodiment 78. The synthetic cytokine receptor of any of embodiments 1 to 65, wherein the extracellular domain is an extracellular domain of Glycophorin A (GpA) or a truncated portion thereof comprising at least one cysteine residue. Embodiment 79. The synthetic cytokine receptor of any of embodiments 1 to 65 and 78, wherein the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 16 or SEQ ID NO: 17. Embodiment 80. The synthetic cytokine receptor of any of embodiments 1 to 65, 78 and 79, wherein the extracellular domain comprises the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17. Embodiment 81. The synthetic cytokine receptor of any of embodiments 2 to 80, wherein the IL-9R intracellular domain or variant thereof is about 100 to 260 amino acids in length. Embodiment 82. The synthetic cytokine receptor of any of embodiments 2 to 81, wherein the IL-9R intracellular domain or variant thereof comprises a BOX1 motif and/or a BOX2 motif. Embodiment 83. The synthetic cytokine receptor of any of embodiments 2 to 83, wherein the IL-9R intracellular domain or variant thereof comprises a BOX2 motif. Embodiment 84. The synthetic cytokine receptor of any of embodiments 2 and 4 to 80, wherein the IL-9R intracellular domain or variant thereof is 230 amino acids in length. Embodiment 85. The synthetic cytokine receptor of any of embodiments 1 to 84, wherein the IL-9R intracellular domain or variant thereof is wild-type IL-9R intracellular domain or a variant thereof that comprises one or more mutations compared to the wild-type IL-9R intracellular domain set forth in SEQ ID NO: 8. Embodiment 86. The synthetic cytokine receptor of embodiment 85, wherein the one or more mutations comprises one or more amino acid insertions, deletions, and/or substitutions. Embodiment 87. The synthetic cytokine receptor of embodiment 85 or embodiment 86, wherein the one or more mutations promote signaling through STAT1, STAT3, and/or STAT5 pathways. Embodiment 88. The synthetic cytokine receptor of any of embodiments 1 to 87, wherein the IL-9R intracellular domain or variant thereof comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 8.
30761-20002.40 Embodiment 89. The synthetic cytokine receptor of any of embodiments 1 to 88, wherein the IL-9R intracellular domain or variant thereof comprises an amino acid sequence of SEQ ID NO: 8. Embodiment 90. The synthetic cytokine receptor of any of embodiments 1 to 87, wherein the IL-9R intracellular domain or variant thereof comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56. Embodiment 91. The synthetic cytokine receptor of any of embodiments 1 to 87 and 90, wherein the IL-9R intracellular domain or variant thereof comprises an amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56. Embodiment 92. The synthetic cytokine receptor of any of embodiments 1 to 91, wherein the synthetic cytokine receptor comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66 or SEQ ID NO: 67. Embodiment 93. The synthetic cytokine receptor of any of embodiments 1 to 92, wherein the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66 or SEQ ID NO: 67. Embodiment 94. The synthetic cytokine receptor of any of embodiments 2 to 87, wherein the IL-9R intracellular domain or variant thereof comprises one or more amino acid deletions with reference to wild-type IL-9R intracellular domain (SEQ ID NO: 8). Embodiment 95. The synthetic cytokine receptor of any of embodiments 2 to 87, wherein the IL-9R intracellular domain or variant thereof is a truncated IL-9R that lacks a contiguous sequence of amino acids at the C-terminus of wild-type IL-9R intracellular domain.
30761-20002.40 Embodiment 96. The synthetic cytokine receptor of embodiment 95, wherein the truncated IL-9R intracellular domain or variant thereof is truncated by between 62 and 99 contiguous amino acids from the C-terminus of wild-type IL-9R intracellular domain. Embodiment 97. The synthetic cytokine receptor of any of embodiments 2 to 87, wherein the IL-9R intracellular domain or variant thereof is a truncated IL-9R that lacks amino acids 132 to 230 of SEQ ID NO:8 or lacks amino acids 134 to 230 of SEQ ID NO:8. Embodiment 98. The synthetic cytokine receptor of any of embodiments 2 to 97, wherein the IL-9R intracellular domain or variant thereof comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 54, SEQ ID NO: 103, or SEQ ID NO: 104. Embodiment 99. The synthetic cytokine receptor of any of embodiments 2 to 98, wherein the IL-9R intracellular domain or variant thereof comprises an amino acid sequence of SEQ ID NO: 54, SEQ ID NO: 103, or SEQ ID NO: 104. Embodiment 100. The synthetic cytokine receptor of any of embodiments 2 to 99, wherein the synthetic cytokine receptor comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63. Embodiment 101. The synthetic cytokine receptor of any of embodiments 2 to 100, wherein the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63. Embodiment 102. The synthetic cytokine receptor of any of embodiments 2 to 87, wherein the IL-9R intracellular domain or variant thereof comprises one or more amino acid substitutions with reference to wild-type IL-9R intracellular domain (SEQ ID NO: 8). Embodiment 103. The synthetic cytokine receptor of embodiment 102, wherein the IL-9R intracellular domain or variant thereof comprises a STAT binding motif or a variant thereof. Embodiment 104. The synthetic cytokine receptor of embodiment 103, wherein the STAT binding motif comprises a STAT1, STAT3, and/or STAT5 binding motif. Embodiment 105. The synthetic cytokine receptor of embodiment 103 or embodiment 104, wherein the STAT binding motif comprises YLPQ (SEQ ID NO: 171). Embodiment 106. The synthetic cytokine receptor of any of embodiments 103 to 105, wherein the STAT binding motif comprises a variant STAT binding motif. Embodiment 107. The synthetic cytokine receptor of any of embodiments 103 to 106, wherein the variant STAT binding motif comprises YRPQ (SEQ ID NO: 172). Embodiment 108. The synthetic cytokine receptor of any of embodiments 103 to 106, wherein the variant STAT binding motif comprises YLPL (SEQ ID NO: 173).
30761-20002.40 Embodiment 109. The synthetic cytokine receptor of any of embodiments 103 to 106, wherein the variant STAT binding motif comprises YLKQ (SEQ ID NO: 174). Embodiment 110. The synthetic cytokine receptor of any of embodiments 2 to 109, wherein the variant IL-9R intracellular domain or variant thereof comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 107 or SEQ ID NO: 108, or SEQ ID NO: 109. Embodiment 111. The synthetic cytokine receptor of any of embodiments 2 to 110, wherein the variant IL-9R intracellular domain comprises an amino acid sequence of SEQ ID NO: 107 or SEQ ID NO: 108, or SEQ ID NO: 109. Embodiment 112. The synthetic cytokine receptor of embodiment 3, wherein the chimeric JAK/STAT fusion domain comprises a JAK binding domain from a type I cytokine receptor and a STAT binding domain from an IL-9R intracellular domain. Embodiment 113. The synthetic cytokine receptor of embodiment 112, wherein the IL-9R STAT binding domain comprises amino acid residues 73 to 230 of SEQ ID NO: 8. Embodiment 114. The synthetic cytokine receptor of embodiment 112 or embodiment 113, wherein the STAT binding domain is 59 to 158 amino acids in length and comprises an IL-9R STAT binding motif. Embodiment 115. The synthetic cytokine receptor of embodiment 114, wherein the IL-9R STAT binding motif comprises YLPQ (SEQ ID NO: 171). Embodiment 116. The synthetic cytokine receptor of any of embodiments 112 to 115, wherein the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks a contiguous sequence of amino acids at the N-terminus of SEQ ID NO: 8. Embodiment 117. The synthetic cytokine receptor of any of embodiments 112 to 116, wherein the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks a contiguous sequence of amino acids at the C-terminus of SEQ ID NO: 8. Embodiment 118. The synthetic cytokine receptor of any of embodiments 112 to 117, wherein the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks amino acids at positions 1 to 72 and/or 132 to 230 of SEQ ID NO: 8. Embodiment 119. The synthetic cytokine receptor of any of embodiments 112 to 118, wherein the IL-9R STAT binding domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 122 or SEQ ID NO: 123. Embodiment 120. The synthetic cytokine receptor of any of embodiments 112 to 119, wherein the IL-9R STAT binding domain comprises an amino acid sequence of SEQ ID NO: 122 or SEQ ID NO: 123.
30761-20002.40 Embodiment 121. The synthetic cytokine receptor of any of embodiments 112 to 120, wherein the type I cytokine receptor is selected from the group consisting of interleukin 2 receptor (IL-2R), interleukin 4 receptor (IL-4R), interleukin 7 receptor (IL-7R), interleukin 13 receptor (IL-13R), interleukin 15 receptor (IL-15R), and interleukin 2 receptor (IL-21R). Embodiment 122. The synthetic cytokine receptor of any of embodiments 112 to 121, wherein the type I cytokine receptor is IL-7R. Embodiment 123. The synthetic cytokine receptor of any of embodiments 112 to 122, wherein the IL-7R JAK binding domain is 65 amino acids in length and comprises a box 1 motif. Embodiment 124. The synthetic cytokine receptor of any of embodiments 112 to 123, wherein the IL-7R JAK binding domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 121. Embodiment 125. The synthetic cytokine receptor of embodiment 112 to 124, wherein the IL-7R JAK binding domain comprises an amino acid sequence of SEQ ID NO: 121. Embodiment 126. The synthetic cytokine receptor of any of embodiments 112 to 125, wherein the chimeric JAK/STAT fusion domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 114 or SEQ ID NO: 116. Embodiment 127. The synthetic cytokine receptor of any of embodiments 112 to 126, wherein the chimeric JAK/STAT fusion domain comprises an amino acid sequence of SEQ ID NO: 114 or SEQ ID NO: 116. Embodiment 128. The synthetic cytokine receptor of any of embodiments 1 to 127, wherein the synthetic cytokine receptor comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 113, SEQ ID NO: 115, or SEQ ID NO: 117. Embodiment 129. The synthetic cytokine receptor of any of embodiments 1 to 128, wherein the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 113, SEQ ID NO: 115, or SEQ ID NO: 117. Embodiment 130. The synthetic cytokine receptor of any of embodiments 1 to 3 and 14 to 129, wherein the synthetic cytokine receptor is a constitutively active cytokine receptor. Embodiment 131. The synthetic cytokine receptor of any of embodiments 1 to 130, wherein the synthetic cytokine receptor elicits signaling through STAT1, STAT3, and/or STAT5 pathways. Embodiment 132. The synthetic cytokine receptor of embodiment 131, wherein signaling through STAT1, STAT3, and/or STAT5 is increased compared to STAT1, STAT3, and/or STAT5 signaling via wild-type IL-9R.
30761-20002.40 Embodiment 133. The synthetic cytokine receptor of embodiment 131 or embodiment 132, wherein signaling through STAT1, STAT3, and/or STAT5 is sustained for a longer period of time compared to STAT1, STAT3, and/or STAT5 signaling via wild-type IL-9R. Embodiment 134. The synthetic cytokine receptor of embodiment 133, wherein sustained STAT1, STAT3, and/or STAT5 signaling is determined by phosphorylation status of STAT1, STAT3, and/or STAT5. Embodiment 135. A polynucleotide encoding the synthetic cytokine receptor of any of embodiments 1 to 134. Embodiment 136. A vector, comprising the polynucleotide of embodiment 135. Embodiment 137. A method of engineering an isolated cell, comprising contacting the cell with the polynucleotide of embodiment 135 or the vector of embodiment 136. Embodiment 138. An engineered cell expressing the synthetic cytokine receptor of any of embodiments 1 to 137. Embodiment 139. An engineered cell expressing a synthetic cytokine receptor that is a homodimer of identical polypeptide chains each comprising an extracellular domain, a transmembrane domain, and an intracellular domain capable of interleukin 9 receptor (IL-9R) signaling. Embodiment 140. The engineered cell of embodiment 139, wherein the intracellular domain capable of IL-9R signaling comprises an IL-9R intracellular domain or a variant thereof. Embodiment 141. The engineered cell of embodiment 139 or embodiment 140, wherein the intracellular domain capable of IL-9R signaling comprises a chimeric JAK/STAT fusion domain. Embodiment 142. An engineered cell expressing a synthetic cytokine receptor, wherein the synthetic cytokine receptor comprises an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain a variant thereof, wherein the synthetic cytokine receptor is a constitutively active cytokine receptor. Embodiment 143. The engineered cell of any of embodiments 139 to 142, wherein the synthetic cytokine receptor is a multimer. Embodiment 144. The engineered cell of any of embodiments 139 to 142, wherein the synthetic cytokine receptor is a multimer of identical polypeptide chains each comprising the extracellular domain, the transmembrane domain and the IL-9R intracellular domain or a variant thereof.
30761-20002.40 Embodiment 145. The engineered cell of embodiment 143 or embodiment 144 , wherein the multimer is a dimer. Embodiment 146. The engineered cell of embodiment 145, wherein the dimer is a homodimer. Embodiment 147. The engineered cell of any of embodiments 143 to 146, wherein each polypeptide chain is constitutively multimerized Embodiment 148. The engineered cell of any of embodiments 139 to 147, wherein the synthetic cytokine receptor comprises at least one self-assembly domain. Embodiment 149. The engineered cell of embodiment 148, wherein the at least one self- assembly domain is the extracellular domain and/or the transmembrane domain. Embodiment 150. The engineered cell of any of embodiments 139 to 149, wherein the synthetic cytokine receptor is multimerized through the transmembrane domain and/or the extracellular domain. Embodiment 151. The engineered cell of any of embodiments 139 to 150, wherein the synthetic cytokine receptor is multimerized through the transmembrane domain and the extracellular domain. Embodiment 152. An engineered cell expressing a synthetic cytokine receptor that is a homodimer of identical polypeptide chains each comprising an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain or a variant thereof. Embodiment 153. The engineered cell of any of embodiments 139 to 152, wherein the extracellular domain and/or the transmembrane domain are heterologous to the IL-9R. Embodiment 154. The engineered cell of any of embodiments 139 to 153, wherein the transmembrane domain and extracellular domain are the transmembrane domain and extracellular domain from the same protein. Embodiment 155. The engineered cell of any of embodiments 139 to 153, wherein the transmembrane domain and extracellular domain are the transmembrane domain and extracellular domain from different proteins. Embodiment 156. The engineered cell of any of embodiments139 to 155, wherein the transmembrane domain is 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 amino acids in length. Embodiment 157. The engineered cell of any of embodiments 139 to 156, wherein the transmembrane domain comprises a transmembrane domain derived from Glycophorin A
30761-20002.40 (GpA) Carnitine palmitoyltransferase 1 (CPT1), a tumor necrosis factor receptor (TNFR) or Muc24. Embodiment 158. The engineered cell of any of embodiments 139 to 156, wherein the transmembrane domain comprises a transmembrane domain derived from Thrombopoietin receptor. Embodiment 159. The engineered cell of any of embodiments 139 to 158, wherein the transmembrane domain promotes alpha-helix dimerization. Embodiment 160. The engineered cell of any of embodiments 139 to 159, wherein the transmembrane domain comprises the motif GXXXG (SEQ ID NO: 68). Embodiment 161. The engineered cell of any of embodiments 139 to 159, wherein the transmembrane domain comprises the motif LixxGVxxGVxxT (SEQ ID NO: 70). Embodiment 162. The engineered cell of any of embodiments 139 to 161, wherein the transmembrane domain is a transmembrane domain derived from Glycophorin A (GpA) or a variant thereof that comprises one or more mutations (e.g, 1, 2, 3, 4, 5 or 6 mutations) compared to a wild-type GpA transmembrane domain, wherein the variant GpA is sufficient to promote alpha-helix dimerization. Embodiment 163. The engineered cell of any of embodiments 139 to 162, wherein the transmembrane domain comprises a transmembrane domain derived from Glycophorin A (GpA). Embodiment 164. The engineered cell of any of embodiments 139 to 163, wherein the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 22 or SEQ ID NO:43. Embodiment 165. The engineered cell of any of embodiments 139 to 164, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 43. Embodiment 166. The engineered cell of any of embodiments 139 to 160, wherein the transmembrane domain comprises GXXXG (SEQ ID NO: 68) and GXXXA (SEQ ID NO: 69) motifs. Embodiment 167. The engineered cell of any of embodiments 139 to 160 and 166, wherein the transmembrane domain comprises a transmembrane domain derived from Carnitine palmitoyltransferase 1 (CPT1). Embodiment 168. The engineered cell of any of embodiments 139 to 160, 166 and 167, wherein the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 45.
30761-20002.40 Embodiment 169. The engineered cell of any of embodiments 139 to 160 and 166 to 168, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 45. Embodiment 170. The engineered cell of any of embodiments 139 to 169, wherein the synthetic cytokine receptor comprises the motif AXXXA (SEQ ID NO:77) or AXXXS (SEQ ID NO: 78). Embodiment 171. The engineered cell of any of embodiments 139 to 160 or 170, wherein the synthetic cytokine receptor comprises the motif ĭPXĭ (SEQ ID NO:75) or ĭTXXAĭ (SEQ ID NO: 76). Embodiment 172. The engineered cell of any of embodiments 139 to 160, 170 or 171, wherein the transmembrane domain is derived from a TNFR. Embodiment 173. The engineered cell of embodiment 172, wherein the TNFR is TACI, DR5, p75NTR, Fas, TNFR1, TNFR2 or OX40. Embodiment 174. The engineered cell of any of embodiments 139 to 160, and 170 to 173, wherein the transmembrane domain comprises a transmembrane domain derived from DR5. Embodiment 175. The engineered cell of any of embodiments 139 to 160 and 170 to 174, wherein the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 46. Embodiment 176. The engineered cell of any of embodiments 139 to 160 and 170 to 175, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 46. Embodiment 177. The engineered cell of any of embodiments 139 to 160 and 170 to 174, wherein the transmembrane domain comprises a transmembrane domain derived from TACI. Embodiment 178. The engineered cell of any of embodiments 139 to 160, 170 to 174 and 177, wherein the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 44. Embodiment 179. The engineered cell of any of embodiments 39 to 161, 170 to 174, 177 and 178, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 44. Embodiment 180. The engineered cell of any of embodiments 139 to 158, wherein the transmembrane domain comprises 1 to 6 cysteine residues. Embodiment 181. The engineered cell of any of embodiments 139 to 158 and 180, wherein the transmembrane domain promotes disulfide-linked dimerization.
30761-20002.40 Embodiment 182. The engineered cell of embodiment 181, wherein the disulfide-linked dimerization forms 1 to 4 disulfide bridges between polypeptide chains of the synthetic cytokine receptor. Embodiment 183. The engineered cell of any of embodiments 139 to 158 and 180 to 182, wherein the transmembrane domain is a variant transmembrane domain that comprises one or more mutations compared to a wild-type transmembrane domain to promote homodimerization of the receptor polypeptide. Embodiment 184. The engineered cell of embodiment 183, wherein the one or more mutations promote alpha-helix dimerization or disulfide-linked dimerization. Embodiment 185. The engineered cell of embodiment 183 or embodiment 184, wherein the one or more mutations introduces at least one cysteine into the transmembrane domain. Embodiment 186. The engineered cell of any of embodiments 183 to 185, wherein the one or more mutations introduces a proline into the transmembrane domain. Embodiment 187. The engineered cell of any of embodiments 183 to 185, wherein the one or more mutations introduces a threonine into the transmembrane domain. Embodiment 188. The engineered cell of any of embodiments 183 to 187, wherein the one or more mutations introduces a trimer peptide of cysteine, proline, and another amino acid other than cysteine or proline into the transmembrane domain. Embodiment 189. The engineered cell of any of embodiments 183 to 188, wherein the one or more mutations introduces a trimer peptide of cysteine, proline, threonine (CPT or TCP) into the transmembrane domain. Embodiment 190. The engineered cell of any of embodiments 183 to 188, wherein the transmembrane domain is a variant IL-7R transmembrane domain and the one or more mutations is in the wild-type transmembrane sequence PILLTISILSFFSVALLVILACVLW (SEQ ID NO: 71). Embodiment 191. The engineered cell of any of embodiments 139 to 158 and 180 to 190, wherein the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 6, SEQ ID NO:7 or SEQ ID NO: 21. Embodiment 192. The engineered cell of any of embodiments 139 to 158 and 180 to 191, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:21. Embodiment 193. The engineered cell of any of embodiments 139 to 158, wherein the transmembrane domain comprises a transmembrane domain derived from Muc24.
30761-20002.40 Embodiment 194. The engineered cell of any of embodiments 139 to 158 and 193, wherein the transmembrane domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 23. Embodiment 195. The engineered cell of any of embodiments 139 to 158, 193 or 194, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 23. Embodiment 196. The engineered cell of any of embodiments 139 to 195, wherein the extracellular domain is between about 150 to 260 amino acids in length. Embodiment 197. The engineered cell of any of embodiments 139 to 196, wherein the extracellular domain is a dimerizing domain. Embodiment 198. The engineered cell of embodiment 197, wherein the dimerizing domain comprises a hinge region. Embodiment 199. The engineered cell of any of embodiments 139 to 198, wherein the extracellular domain promotes disulfide-linked dimerization. Embodiment 200. The engineered cell of any of embodiments 139 to 199, wherein the extracellular domain comprises 1 to 6 cysteine residues. Embodiment 201. The engineered cell of embodiment 199 or embodiment 200, wherein the disulfide-linked dimerization forms 1 to 4 disulfide bridges between polypeptide chains of the synthetic cytokine receptor. Embodiment 202. The engineered cell of any of embodiments 139 to 201, wherein the extracellular domain is derived from the extracellular domain of CD34, DAP12, Glycophorin A, CD8, or Muc24. Embodiment 203. The engineered cell of any of embodiments 139 to 202, wherein the extracellular domain comprises an extracellular domain derived from Thrombopoietin receptor. Embodiment 204. The engineered cell of any of embodiments 139 to 203, wherein the extracellular domain comprises an extracellular domain of CD8 or a truncated portion thereof comprising at least one cysteine residue. Embodiment 205. The engineered cell of any of embodiments 139 to 204, wherein the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 18 or SEQ ID NO: 19. Embodiment 206. The engineered cell of any of embodiments 139 to 205, wherein the extracellular domain comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19.
30761-20002.40 Embodiment 207. The engineered cell of any of embodiments 139 to 203, wherein the extracellular domain comprises an extracellular domain of CD34 or a truncated portion thereof comprising at least one cysteine residue. Embodiment 208. The engineered cell of any of embodiments 139 to 203 and 207, wherein the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 4. Embodiment 209. The engineered cell of any of embodiments 139 to 203, 207 and 208, wherein the extracellular domain comprises the amino acid sequence of SEQ ID NO: 4. Embodiment 210. The engineered cell of any of embodiments 139 to 203, wherein the extracellular domain is an extracellular domain of Muc24 or a truncated portion thereof comprising at least one cysteine residue. Embodiment 211. The engineered cell of any of embodiments 139 to 203 and 210, wherein the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 20. Embodiment 212. The engineered cell of any of embodiments 139 to 203, 210 and 211, wherein the extracellular domain comprises the amino acid sequence of SEQ ID NO: 20. Embodiment 213. The synthetic cytokine receptor of any of embodiments 139 to 203, wherein the extracellular domain is an extracellular domain of DAP12 or a truncated portion thereof comprising at least one cysteine residue. Embodiment 214. The engineered cell of any of embodiments 139 to 203 and 213, wherein the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 5. Embodiment 215. The engineered cell of any of embodiments 139 to 203, 213 and 214, wherein the extracellular domain comprises the amino acid sequence of SEQ ID NO: 5. Embodiment 216. The synthetic cytokine receptor of any of embodiments 139 to 203, wherein the extracellular domain is an extracellular domain of Glycophorin A (GpA) or a truncated portion thereof comprising at least one cysteine residue. Embodiment 217. The engineered cell of any of embodiments 139 to 203 and 216, wherein the extracellular domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 16 or SEQ ID NO: 17. Embodiment 218. The engineered cell of any of embodiments 139 to 203, 216 and 217, wherein the extracellular domain comprises the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17.
30761-20002.40 Embodiment 219. The engineered cell of any of embodiments 139 to 218, wherein the IL-9R intracellular domain or variant thereof is about 100 to 260 amino acids in length. Embodiment 220. The engineered cell of any of embodiments 139 to 219, wherein the IL-9R intracellular domain or variant thereof comprises a BOX1 motif and/or a BOX2 motif. Embodiment 221. The engineered cell of any of embodiments 139 to 220, wherein the IL-9R intracellular domain or variant thereof comprises a BOX2 motif. Embodiment 222. The engineered cell of any of embodiments 2 and 4 to 80, wherein the IL-9R intracellular domain or variant thereof is 230 amino acids in length. Embodiment 223. The engineered cell of any of embodiments 139 to 222, wherein the IL-9R intracellular domain or variant thereof is wild-type IL-9R intracellular domain or a variant thereof that comprises one or more mutations compared to the wild-type IL-9R intracellular domain set forth in SEQ ID NO: 8. Embodiment 224. The engineered cell of any of embodiments 139 to 223, wherein the one or more mutations comprises one or more amino acid insertions, deletions, and/or substitutions. Embodiment 225. The engineered cell of embodiment 223 or embodiment 224, wherein the one or more mutations promote signaling through STAT1, STAT3, and/or STAT5 pathways. Embodiment 226. The engineered cell of any of embodiments 139 to 225, wherein the IL-9R intracellular domain or variant thereof comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 8. Embodiment 227. The engineered cell of any of embodiments 139 to 226, wherein the IL-9R intracellular domain or variant thereof comprises an amino acid sequence of SEQ ID NO: 8. Embodiment 228. The engineered cell of any of embodiments 139 to 225, wherein the IL-9R intracellular domain or variant thereof comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56. Embodiment 229. The engineered cell of any of embodiments 139 to 225 and 228, wherein the IL-9R intracellular domain or variant thereof comprises an amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56. Embodiment 230. The engineered cell of any of embodiments 139 to 229, wherein the synthetic cytokine receptor comprises an amino acid sequence that is at least about 85%
30761-20002.40 identical to SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66 or SEQ ID NO: 67. Embodiment 231. The engineered cell of any of embodiments 139 to 230, wherein the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66 or SEQ ID NO: 67. Embodiment 232. The engineered cell of any of embodiments 140 to 231, wherein the IL-9R intracellular domain or variant thereof comprises one or more amino acid deletions with reference to wild-type IL-9R intracellular domain (SEQ ID NO: 8). Embodiment 233. The engineered cell of any of embodiments 140 to 232, wherein the IL-9R intracellular domain or variant thereof is a truncated IL-9R that lacks a contiguous sequence of amino acids at the C-terminus of wild-type IL-9R intracellular domain. Embodiment 234. The engineered cell of embodiment 233, wherein the truncated IL-9R intracellular domain or variant thereof is truncated by between 62 and 99 contiguous amino acids from the C-terminus of wild-type IL-9R intracellular domain. Embodiment 235. The engineered cell of any of embodiments 140 to 232, wherein the IL-9R intracellular domain or variant thereof is a truncated IL-9R that lacks amino acids 132 to 230 of SEQ ID NO:8 or lacks amino acids 134 to 230 of SEQ ID NO:8. Embodiment 236. The engineered cell of any of embodiments 140 to 235, wherein the IL-9R intracellular domain or variant thereof comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 54, SEQ ID NO: 103, or SEQ ID NO: 104. Embodiment 237. The engineered cell of any of embodiments 140 to 236, wherein the IL-9R intracellular domain or variant thereof comprises an amino acid sequence of SEQ ID NO: 54, SEQ ID NO: 103, or SEQ ID NO: 104. Embodiment 238. The engineered cell of any of embodiments 140 to 237, wherein the synthetic cytokine receptor comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63.
30761-20002.40 Embodiment 239. The engineered cell of any of embodiments 140 to 238, wherein the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63. Embodiment 240. The engineered cell of any of embodiments 140 to 225, wherein the IL-9R intracellular domain or variant thereof comprises one or more amino acid substitutions with reference to wild-type IL-9R intracellular domain (SEQ ID NO: 8). Embodiment 241. The engineered cell of embodiment 240, wherein the IL-9R intracellular domain or variant thereof comprises a STAT binding motif or a variant thereof. Embodiment 242. The engineered cell of embodiment 241, wherein the STAT binding motif comprises a STAT1, STAT3, and/or STAT5 binding motif. Embodiment 243. The engineered cell of embodiment 241 or embodiment 242, wherein the STAT binding motif comprises YLPQ (SEQ ID NO: 171). Embodiment 244. The engineered cell of any of embodiments 241 to 243, wherein the STAT binding motif comprises a variant STAT binding motif. Embodiment 245. The engineered cell of any of embodiments 241 to 244, wherein the variant STAT binding motif comprises YRPQ (SEQ ID NO: 172). Embodiment 246. The engineered cell of any of embodiments 241 to 244, wherein the variant STAT binding motif comprises YLPL (SEQ ID NO: 173). Embodiment 247. The engineered cell of any of embodiments 241 to 244, wherein the variant STAT binding motif comprises YLKQ (SEQ ID NO: 174). Embodiment 248. The engineered cell of any of embodiments 140 to 247, wherein the variant IL-9R intracellular domain or variant thereof comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 107 or SEQ ID NO: 108, or SEQ ID NO: 109. Embodiment 249. The engineered cell of any of embodiments 140 to 248, wherein the variant IL-9R intracellular domain comprises an amino acid sequence of SEQ ID NO: 107 or SEQ ID NO: 108, or SEQ ID NO: 109. Embodiment 250. The engineered cell of embodiment 141, wherein the chimeric JAK/STAT fusion domain comprises a JAK binding domain from a type I cytokine receptor and a STAT binding domain from an IL-9R intracellular domain. Embodiment 251. The engineered cell of embodiment 250, wherein the IL-9R STAT binding domain comprises amino acid residues 73 to 230 of SEQ ID NO: 8. Embodiment 252. The engineered cell of embodiment 250 or embodiment 251, wherein the STAT binding domain is 59 to 158 amino acids in length and comprises an IL-9R STAT binding motif.
30761-20002.40 Embodiment 253. The engineered cell of embodiment 252, wherein the IL-9R STAT binding motif comprises YLPQ (SEQ ID NO: 171). Embodiment 254. The engineered cell of any of embodiments 250 to 253, wherein the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks a contiguous sequence of amino acids at the N-terminus of SEQ ID NO: 8. Embodiment 255. The engineered cell of any of embodiments 250 to 254, wherein the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks a contiguous sequence of amino acids at the C-terminus of SEQ ID NO: 8. Embodiment 256. The engineered cell of any of embodiments 250 to 255, wherein the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks amino acids at positions 1 to 72 and/or 132 to 230 of SEQ ID NO: 8. Embodiment 257. The engineered cell of any of embodiments 250 to 256, wherein the IL-9R STAT binding domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 122 or SEQ ID NO: 123. Embodiment 258. The engineered cell of any of embodiments 250 to 257, wherein the IL-9R STAT binding domain comprises an amino acid sequence of SEQ ID NO: 122 or SEQ ID NO: 123. Embodiment 259. The engineered cell of any of embodiments 250 to 258, wherein the type I cytokine receptor is selected from the group consisting of interleukin 2 receptor (IL- 2R), interleukin 4 receptor (IL-4R), interleukin 7 receptor (IL-7R), interleukin 13 receptor (IL-13R), interleukin 15 receptor (IL-15R), and interleukin 2 receptor (IL-21R). Embodiment 260. The engineered cell of any of embodiments 250 to 259, wherein the type I cytokine receptor is IL-7R. Embodiment 261. The engineered cell of any of embodiments 250 to 260, wherein the IL-7R JAK binding domain is 65 amino acids in length and comprises a box 1 motif. Embodiment 262. The engineered cell of any of embodiments 250 to 261, wherein the IL-7R JAK binding domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 121. Embodiment 263. The engineered cell of embodiment 250 to 262, wherein the IL-7R JAK binding domain comprises an amino acid sequence of SEQ ID NO: 121. Embodiment 264. The engineered cell of any of embodiments 250 to 263, wherein the chimeric JAK/STAT fusion domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 114 or SEQ ID NO: 116.
30761-20002.40 Embodiment 265 The engineered cell of any of embodiments 250 to 264, wherein the chimeric JAK/STAT fusion domain comprises an amino acid sequence of SEQ ID NO: 114 or SEQ ID NO: 116. Embodiment 266. The engineered cell of any of embodiments 139 to 265, wherein the synthetic cytokine receptor comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 113, SEQ ID NO: 115, or SEQ ID NO: 117. Embodiment 267. The engineered cell of any of embodiments 139 to 266, wherein the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 113, SEQ ID NO: 115, or SEQ ID NO: 117. Embodiment 268. The engineered cell of any of embodiments 139 to 141 and 152 to 267, wherein the synthetic cytokine receptor is a constitutively active cytokine receptor. Embodiment 269. The engineered cell of any of embodiments 139 to 268, wherein the synthetic cytokine receptor elicits signaling through STAT1, STAT3, and/or STAT5 pathways. Embodiment 270. The engineered cell of embodiment 269, wherein signaling through STAT1, STAT3, and/or STAT5 is increased compared to STAT1, STAT3, and/or STAT5 signaling via wild-type IL-9R. Embodiment 271. The engineered cell of embodiment 269 or embodiment 270, wherein signaling through STAT1, STAT3, and/or STAT5 is sustained for a longer period of time compared to STAT1, STAT3, and/or STAT5 signaling via wild-type IL-9R. Embodiment 272. The engineered cell of embodiment 271, wherein sustained STAT1, STAT3, and/or STAT5 signaling is determined by phosphorylation status of STAT1, STAT3, and/or STAT5. Embodiment 273. The engineered cell of any of embodiments 139 to 272, wherein the engineered cell further expresses at least one different type of engineered receptor. Embodiment 274. The engineered cell of embodiment 273, wherein the at least one different type of engineered receptor is a chimeric antigen receptor. Embodiment 275. The engineered cell of embodiment 274, wherein the extracellular domain of the chimeric antigen receptor binds to an antigen expressed on a cancer cell. Embodiment 276. The engineered cell of embodiment 275, wherein the extracellular domain of the chimeric antigen receptor binds to an idiotype of an antibody. Embodiment 277. The engineered cell of embodiment 276, wherein the antibody is against an antigen expressed on a cancer cell.
30761-20002.40 Embodiment 278 The engineered cell of embodiment 277, wherein the cancer cell is a blood cancer cell or a solid tumor cancer cell. Embodiment 279. The engineered cell of any of embodiments 139 to 278, wherein the cell is an immune cell. Embodiment 280. The engineered cell of any of embodiments 139 to 279, wherein the cell is a lymphocyte. Embodiment 281. The engineered cell of any of embodiments 139 to 280, wherein the engineered cell is an immune effector cell. Embodiment 282. The engineered cell of any of embodiments 139 to 281, wherein the cell is a T cell or a Natural Killer (NK) cell. Embodiment 283. The engineered cell of embodiment 139 to 282, wherein the cell is a T cell and the T cell is a CD4+ T cell or a CD8+ T cell. Embodiment 284. The engineered cell of embodiment 283, wherein the immune effector cell is a cytotoxic T cell. Embodiment 285. The engineered cell of embodiment 283, wherein the immune effector cell is a natural killer cell. Embodiment 286. The engineered cell of any of embodiments 139 to 285, wherein the cell is a primary cell. Embodiment 287. The engineered cell of any of embodiments 139 to 286, wherein the cell is a human cell. Embodiment 288. A population of cells, comprising at least one engineered cell of any of embodiments 139 to 287. Embodiment 289. The population of cells of embodiment 288, wherein the at least one engineered cell comprises engineered CD4+ T cells and engineered CD8+ T cells. Embodiment 290. A pharmaceutical composition comprising the engineered cell of any of embodiments 90 to 191 or the population of cells of embodiment 288 or embodiment 289. Embodiment 291. The pharmaceutical composition of embodiment 290, wherein the pharmaceutical composition further comprises a pharmaceutical acceptable carrier. Embodiment 292. The pharmaceutical composition of embodiment 290 or embodiment 291, wherein the pharmaceutical composition further comprises a cryoprotectant Embodiment 293. The pharmaceutical composition of any of embodiments 290 to 292, for use in treating a cancer in a subject.
30761-20002.40 Embodiment 294. A method of treating a disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any of embodiments 290 to 293. Embodiment 295. The method of embodiments 294, wherein the disease or condition is a cancer. Embodiment 296. The method of embodiment 294 or embodiment 295, wherein the engineered cell of the pharmaceutical composition expresses an engineered antigen receptor that binds to an antigen expressed on a cell of the cancer. Embodiment 297. The method of embodiment 296, wherein the engineered antigen receptor is a chimeric antigen receptor. Embodiment 298. The method of embodiment 296, wherein the engineered antigen receptor is a T cell receptor. Embodiment 299. A method of improving function of an immune cell, comprising introducing to the immune cell the polynucleotide of embodiment 135 or the vector of embodiment 136, wherein improving immune cell function comprises increased STAT1, STAT3, and/or STAT5 signaling compared to an immune cell expressing wild-type IL-9R. Embodiment 300. A method of improving cytotoxicity of an immune cell, comprising introducing to the immune cell the polynucleotide of embodiment 135 or the vector of embodiment 136, wherein improving immune cell cytotoxicity comprises increased target cell killing compared to an immune cell expressing wild-type IL-9R. Embodiment 301. A method of improving viability of an immune cell, comprising introducing to the immune cell the polynucleotide of embodiment 135 or the vector of embodiment 136, wherein improving viability of the immune cell comprises decreased immune cell death compared to an immune cell expressing wild-type IL-9R. Embodiment 302. A method of improving cytokine secretion by an immune cell, comprising introducing to the immune cell the polynucleotide of embodiment 135 or the vector of embodiment 136, wherein improving cytokine secretion comprises increased secretion of interferon compared to an immune cell expressing wild-type IL-9R. Embodiment 303. The method of embodiment 302, wherein the interferon comprises IFNȖ. Embodiment 304. The method of any of embodiments 299 to 303, wherein the immune cell further comprises an engineered antigen receptor. Embodiment 305. The method of embodiment 304, wherein the one different type of engineered receptor is a chimeric antigen receptor (CAR).
30761-20002.40 Embodiment 306. The method of embodiment 305, wherein the extracellular domain of the CAR binds to an antigen expressed on a cancer cell. Embodiment 307. The method of embodiment 306, wherein the cancer cell is a blood cancer cell or a solid tumor cancer cell. Embodiment 308. The method of any of embodiments 299 to 307, wherein the immune cell is a lymphocyte. Embodiment 309. The method of any of embodiments 299 to 308, wherein the immune cell is an effector cell. Embodiment 310. The method of any of embodiments 299 to 309, wherein the immune cell is a T cell or NK cell. Embodiment 311. The method of any of embodiments 299 to 310, wherein the immune cell is a T cell. Embodiment 312. The method of any of embodiments 299 to 310, wherein the immune cell is a T cell and the T cell is a CD4+ T cell or a CD8+ T cell. Embodiment 313. The method of any of embodiments 299 to 310, wherein the immune cell is a cytotoxic T cell. Embodiment 314. The method of any of embodiments 299 to 311, wherein the immune cell is a NK cell. Embodiment 315. The method of any of embodiments 299 to 313, wherein the immune cell is a primary cell. Embodiment 316. A method of improving function of an immune cell, comprising introducing to the immune cell the polynucleotide of claim 135 or the vector of claim 136, thereby improving function of the immune cell. Embodiment 317. The method of embodiment 316, wherein improving immune cell function comprises one or more of increased STAT signaling, increased cytotoxicity, increased proliferation, increased viability, increased cytokine secretion, and increased cytotoxic protein secretion compared to a reference cell. Embodiment 318. The method of embodiment 317, wherein the reference cell comprises a non-engineered immune cell or an engineered immune cell. Embodiment 319. The method of embodiment 318, wherein the engineered immune cell expresses a engineered antigen receptor. Embodiment 320. The method of embodiment 319, wherein the engineered antigen receptor is a wild-type IL-9R, a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
30761-20002.40 Embodiment 321. The method of any of embodiments 317-320, wherein increased STAT signaling comprises increased STAT1, STAT3 and/or STAT5 signaling. Embodiment 322. The method of any of embodiments 317-321, wherein increased cytotoxicity comprises increased killing of a target cell. Embodiment 323. The method of any of embodiments 317-322, wherein increased cytokine secretion comprises increased secretion of one or more of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-10 (IL-10), and TNFĮ. Embodiment 324. The method of any of embodiments 317-323, wherein increased cytotoxic protein secretion comprises increased secretion of one or more of granzyme A, granzyme B, granulysin and perforin. Embodiment 325. The method of any of embodiments 317-324, wherein increased viability comprises decreased cell death. Embodiment 326. The method of any of embodiments 316-325, wherein the immune cell is a lymphocyte. Embodiment 327. The method of any of embodiments 316-326, wherein the immune cell is an effector cell. Embodiment 328. The method of any of embodiments 316-327, wherein the immune cell is a T cell or NK cell. Embodiment 329. The method of any of embodiments 316-327, wherein the immune cell is a T cell and the T cell is a CD4+ T cell or a CD8+ T cell. Embodiment 330. The method of any of embodiments 316-329, wherein the immune cell is a cytotoxic T cell. Embodiment 331. The method of any of embodiments 316-328, wherein the immune cell is a NK cell. Embodiment 332. The method of any of embodiments 316-331, wherein the immune cell is a primary cell.. IX. EXAMPLES [0453] The following is a description of various methods and materials used in the studies. They are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below were performed and are all of the experiments that may be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that
30761-20002.40 the descriptions can be performed to generate the data and the like associated with the teachings of the present invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental errors and deviations should be accounted for. EXAMPLE 1—Design of Synthetic Cytokine Receptors [0454] This example provides exemplary synthetic cytokine receptors capable of driving interleukin-9 (IL-9) signaling in the absence of their cognate cytokine ligand (i.e., constitutively active). [0455] As shown in FIG.1, each of the exemplary synthetic cytokine receptors comprises an extracellular domain (ECD), a transmembrane domain (TMD), and an interleukin 9 receptor (IL-9R) intracellular domain (ICD). Moreover, in the encoded protein, a Flag tag (set forth in SEQ ID NO: 3) was inserted between the signal peptide (e.g., set forth in SEQ ID NO: 1 or SEQ ID NO:2) and the ECD. The signal peptide is expected to be cleaved when the cytokine receptor is expressed on the surface of a cell. The Flag tag was included to facilitate detection of the receptors; however the constructs can be generated with an alternative tag, or without a Flag tag such as depicted in Table E1. IL7R* denotes a mutant IL-7R TMD; IL7R*2 denotes another mutant IL-7R TMD. [0456] The ECD and TMD were designed to promote the oligomerization (e.g., dimerization) of synthetic cytokine receptors so that the synthetic cytokine receptors are constitutively active. The ICD domain was designed to trigger a strong JAK/STAT signal cascade where STAT1, STAT3, and STAT5 could all be activated. The exemplary synthetic cytokine receptors are shown in Table E1 below. Table E1 Design of Synthetic Cytokine Receptors
[0457] Polynucleotides encoding the exemplary synthetic cytokine receptors were cloned into lentiviral vectors for efficient transduction of primary human CD3-positive T cells. The cells were activated on Day 0 and transduced on Day 1. The transduced cells were sorted for Flag-positive cells 5 days after activation with an anti-CD3/anti-CD28 activation reagent (Dynabead activation). The sorted cells were expanded for an additional 7 days, stained with an anti-Flag antibody, and measured by flow cytometry to assess surface expression level of the exemplary synthetic cytokine receptors.
30761-20002.40 [0458] As shown in FIG.2, 9RC, 9RC2, and 9RD2 were stained positive for Flag, indicating that these three exemplary synthetic cytokine receptors could express on the surface of primary human CD3-positive T cells. Surface expression of 9RD was not observed (data not shown). EXAMPLE 2—T Cells Expressing Synthetic Cytokine Receptors [0459] This example shows features of primary human CD3-positive T cells expressing the exemplary synthetic cytokine receptors disclosed in Example 1 above. Primary human CD3-positive T cells were transduced with a lentiviral vector as described in Example 1 and were expanded as described. In some experiments, cells were co-transduced with a chimeric antigen receptor (CAR) by transduction with a lentiviral vector containing a polynucleotide comprising a nucleic acid sequence encoding the CAR separated from the nucleic acid sequence encoding the synthetic cytokine receptor by a IRES or 2a sequence. In these experiments, the exemplary CAR contained an antigen-binding domain directed against a cell surface antigen, a transmembrane domain and a intracellular signaling domain composed of a 4-1BB costimulatory signaling domain and a CD3zeta signaling domain (exemplary CAR designated “40bbz”). [0460] The differentiation state of the transduced cells sorted for Flag-positive cells 5 days after anti-CD3/anti-CD28 (e.g., Dynabead) activation was evaluated by staining the cells with anti-CD27 and anti-CD45RA antibodies followed by flow cytometric analysis. As shown in FIGS.3A and 3B, cells expressing the 9RC synthetic cytokine receptor had a higher percentage of CD27-positive and CD45RA-positive cells when compared with un-transduced cells. Such results indicate that the 9RC synthetic cytokine receptor polarized primary human CD3-positive T cells toward a naiver phenotype (i.e., assuming characteristics of stem memory T cells). [0461] The differentiation state of the sorted cells expanded in vitro for an additional 7 days was also evaluated. As shown in FIG.4, cells expressing an exemplary synthetic cytokine receptor (9RC, 9RC2, or 9RD2) had a higher percentage of CD27-positive and CD45RA-positive cells when compared with un-transduced cells. Such results indicate that the synthetic cytokine receptors (9RC, 9RC2, or 9RD2) polarized primary human CD3-positive T cells toward a naiver phenotype (i.e., assuming characteristics of stem memory T cells). [0462] The differentiation state of the sorted cells expanded in vitro for an additional 11 days was also evaluated. As shown in FIG.5, cells expressing an exemplary synthetic cytokine receptor (9RC or 9RC2) had a higher percentage of CD27-positive and Fas-positive cells when compared with un-transduced cells. Such results provide further evidence that the synthetic cytokine receptors (9RC or 9RC2) polarized primary human CD3-positive T cells toward a naiver phenotype (i.e., assuming characteristics of stem memory T cells). [0463] The results shown in FIG.5 were confirmed by a repeated experiment. Specifically, FIG. 6A shows the percentage of CD27-positive and Fas-positive cells among all the transduced cells before being sorted for Flag-positive cells; FIG.6B shows the percentage of CD27-positive and Fas- positive cells among the transduced cells after being sorted for Flag-positive cells.
30761-20002.40 [0464] The surface expression of the chemokine receptor CCR7 in combination with CD45RA was evaluated. FIG.7A shows the percentage of CCR7-positive and CD45RA-positive cells among all the transduced cells before being sorted for Flag-positive cells; FIG.7B shows the percentage of CCR7-positive and CD45RA-positive cells among the transduced cells after being sorted for Flag- positive cells. Consistent with the data shown above, cells expressing an exemplary synthetic cytokine receptor (9RC and 9RC2) had a higher percentage of CCR7-positive and CD45RA-positive cells when compared with un-transduced cells. Such results provide further evidence that the synthetic cytokine receptors (9RC or 9RC2) polarized primary human CD3-positive T cells toward a naiver phenotype (i.e., assuming characteristics of stem memory T cells). [0465] It is noteworthy that FIGS.6A-7B show that the addition of CAR molecules in primary human CD3-positive T cells did not impede 9RC polarizing the cells toward a naiver phenotype (i.e., assuming characteristics of stem memory T cells). [0466] As shown in FIG.8, upregulated CD27 surface expression level was observed in 9RC expressing primary human CD3-positive T cells. Moreover, the expression of cellular senescence marker CD57 (FIGS.9A and 9B) and inhibitory receptors TIM3 and LAG3 (FIG.10) was markedly reduced in 9RC expressing primary human CD3-positive T cells. The quantitative measurement of the expression of TIM3 and LAG3 is shown in Table E2 below. The expression level was determined by mean fluorescence intensity (gMFI). Table E2 Expression of TIM-3 and LAG-3
[0467] Another defining feature of IL-9 signaling is the skewing of the CD4/CD8 ratio in favor of CD4. To test if the exemplary synthetic cytokine receptors can trigger such feature, primary human CD3-positive T cells were transduced with the lentiviral vectors expressing an exemplary synthetic cytokine receptor (9RC, 9RC2, or 9RD2). The transduced cells were sorted for Flag-positive cells 5 days after anti-CD3/anti-CD28 (e.g., Dynabead) activation. The sorted cells were expanded for an additional 11 days and stained with anti-CD4 and anti-CD8 antibodies. As shown in FIG.11, the ratio of CD4-positive and CD8-positive T cells in cells expressing synthetic cytokine receptor 9RC or 9RC2 was around 2:1. In contrast, the ratio of CD4-positive and CD8-positive T cells in un- transduced cells was around 1:2. Such results indicate that the synthetic cytokine receptors skewed the CD4/CD8 ratio in favor of CD4. [0468] To test if the exemplary synthetic cytokine receptors can trigger malignant transformation, primary human CD3-positive T cells transduced with the lentiviral vectors expressing
30761-20002.40 an exemplary synthetic cytokine receptor (9RC or 9RC2) were kept in culture for 30 days and cell counts were taken at regular intervals. As shown in FIG.12, none of the un-transduced or transduced cells exhibited signs of uncontrolled expansion, indicating there is no malignant transformation in primary human CD3-positive T cells expressing the synthetic cytokine receptors. EXAMPLE 3—Additional Design of Synthetic Cytokine Receptors [0469] This example provides additional exemplary synthetic cytokine receptors capable of driving interleukin-9 (IL-9) signaling in the absence of their cognate cytokine ligand (i.e., constitutively active). A. Extracellular Domain (ECD) [0470] The first sub-group of additional exemplary synthetic cytokine receptors is shown in Table E3 below. They were designed to comprise (1) a full-length or truncated ECD from Glycophorin A (GpA), CD8 or Muc24, and (2) an IL7R TMD and an IL9R ICD. A Flag-tag was inserted between the signal peptide and the extracellular domain for detection, however, constructs also are generated without the Flag tag. Table E3 Design of Synthetic Receptors with Varied ECDs
[0471] Polynucleotides encoding the exemplary synthetic cytokine receptors were cloned into lentiviral vectors for efficient transduction of primary human CD3-positive T cells. The cells were activated on Day 0 by anti-CD3/anti-CD28 activation (Dynabead) and transduced on Day 1. The transduced cells were sorted for Flag-positive cells 5 days after Dynabead activation. The sorted cells were stained with an anti-Flag antibody and measured by flow cytometry to assess surface expression level of the exemplary synthetic cytokine receptors. [0472] FIGS.13A-13E show the design and expression of additional exemplary synthetic cytokine receptors comprising an IL7R TMD and an IL9R ICD. B. Transmembrane Domain (TMD) [0473] The second sub-group of additional exemplary synthetic cytokine receptors [0474] were designed to comprise (1) a full-length ECD and a TMD from GpA or Muc24, and (2) an IL9R ICD. A Flag-tag was inserted between the signal peptide and the extracellular domain for
30761-20002.40 detection, however, constructs also are generated without the Flag tag. The second sub-group of additional exemplary synthetic cytokine receptors are shown in Table E4 below. Table E4 Design of Synthetic Cytokine Receptors with Varied TMDs
[0475] Polynucleotides encoding the exemplary synthetic cytokine receptors were cloned into lentiviral vectors for efficient transduction of primary human CD3-positive T cells. The cells were activated on Day 0 with anti-CD3/anti-CD28 (Dynabeads) and transduced on Day 1. The transduced cells were sorted for Flag-positive cells 5 days after Dynabead activation. The sorted cells were stained with an anti-Flag antibody and measured by flow cytometry to assess surface expression level of the exemplary synthetic cytokine receptors. [0476] FIGS.14A-14B show the design and expression of additional exemplary synthetic cytokine receptors comprising an IL9R ICD and a transmembrane domain other than an IL7R TMD, such as Muc24 or GpA transmembrane domain. C. Extracellular Domain (ECD)-Transmembrane Domain (TMD) [0477] The third sub-group of additional exemplary synthetic cytokine receptors were designed to comprise (1) a full-length or truncated ECD from GpA, CD8 or Muc24, (2) a TMD from GpA, truncated GpA (designated GpA*), TACI, CPT1 or DR5, and (3) an IL9R ICD. A Flag-tag was inserted between the signal peptide and the extracellular domain for detection, however, constructs also are generated without the Flag tag. The third sub-group of additional exemplary synthetic cytokine receptors are shown in Table E5 below. Table E5 Design of Synthetic Cytokine Receptors with Varied ECDs and TMDs
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[0478] Polynucleotides encoding the exemplary synthetic cytokine receptors were cloned into lentiviral vectors for efficient transduction of primary human CD3-positive T cells. The cells were activated on Day 0 with anti-CD3/anti-CD28 (Dynabeads) and transduced on Day 1. The transduced cells were sorted for Flag-positive cells 5 days after Dynabead activation. The sorted cells were stained with an anti-Flag antibody and measured by flow cytometry to assess surface expression level of the exemplary synthetic cytokine receptors. FIGS.15A-15H show the design and expression of additional exemplary synthetic cytokine receptors. EXAMPLE 4—T Cells Expressing Additional Designs of Synthetic Cytokine Receptors [0479] This example provides features of primary human CD3-positive T cells expressing the additional designs of the synthetic cytokine receptors described in Examples 2 and 3. [0480] The effect of ECD minimization (i.e., truncation) on transduction efficiency was assessed by comparing the degree of expression of various cytokine receptor constructs that differ in the size of the ECD sequence. For instance, 46 amino acids were cleaved from the N-terminus of GpA ECD of the synthetic receptor G-G-9R, resulting in a GpA ECD length of 45 amino acids. [0481] Briefly, and as described above, lentiviral vectors containing polynucleotides encoding the synthetic cytokine receptors were transduced into primary human CD3-positive T cells. The cells were activated on Day 0 with anti-CD3/anti-CD28 (Dynabeads) and transduced on Day 1. The transduced cells were sorted for Flag-positive cells 5 days after activation. The sorted cells were stained with an anti-Flag antibody and measured by flow cytometry to assess surface expression level of the exemplary synthetic cytokine receptors. [0482] Overall, ECD minimization improved transduction efficiency for certain synthetic cytokine receptors. In some instances, transduction efficiency of truncated receptors was comparable to full-length receptors (see, e.g., CD8-7-9R and tCD8-T-9R). [0483] The differentiation state of cells following expression of additional exemplary synthetic cytokine receptors was also evaluated. Briefly, CD3+ T cells were activated on Day 0 and transduced
30761-20002.40 on Day 1. On Day 5 cells were de-beaded, expanded for 8 more days to allow T cell differentiation, stained for the Flag-tag, and sorted by flow cytometry. As shown in FIGS.17A-17C, cells expressing exemplary synthetic cytokine receptor (e.g., 9RC, G-G-9R, G-7-9R, tG-7-9R, tCD8-G-9R, tCD8-G*- 9R, tCD8-7-9R, tCD8-T-9R, tCD8-D-9R, CD8-7-9R, CD8-G-9R, M-M-9R, M-7-9R, or M-G-9R) had a higher percentage of CD27-positive and CD45RA-positive cells when compared with un- transduced cells. Such results indicate that the synthetic cytokine receptors polarized primary human CD3-positive T cells toward a naiver phenotype (i.e., assuming characteristics of stem memory T cells). Expression of certain synthetic cytokine receptor constructs in T cells induced an increase in the percentage of CD27+CD45RA+ cells to greater than 20% of cells (compared to less than 3% in the untransduced control lacking a constitutive synthetic cytokine receptor). EXAMPLE 5—Design of Synthetic Cytokine Receptor Variants with Varied Intracellular Domains (ICDs) [0484] This example provides designs for variants of the exemplary 9RC synthetic cytokine receptors described in Example 1. [0485] The wild-type IL9R signaling domain is negatively regulated on a temporal scale not seen in other common-gamma chain cytokine receptors (e.g., IL7R). Thus, the ICD variants in Table E6 were designed by removing segments of the ICD (SEQ ID NOS: 51-56, 84-106), by mutating STAT binding domains to improve STAT binding (SEQ ID NOS: 107-109), or by inserting STAT5 binding domains (SEQ ID NOS: 110-112). Table E6 ICD Variants
30761-20002.40 [0486] The 9RC synthetic cytokine receptor variants are shown in Table E7 below. The 9RC synthetic cytokine receptors were designed to containing: (1) a CD34 ECD; (2)an IL7R* TMD; and (3) one of the variant ICD from Table E6. In addition, also generated were receptors containing: (1) an IL9R ECD; (2) an IL9R TMD; and (3) one of the variant ICD from Table E6. Table E7 Design of 9RC Variants
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EXAMPLE 6—In Vivo Survival of Mice Administered T Cells Expressing CAR Molecules and Synthetic Cytokine Receptors [0487] The ability of mice that received in vivo administration of T cells expressing exemplary synthetic cytokine receptors to survive was tested. [0488] Primary human CD3-positive T cells were transduced with lentiviral vectors co- expressing an exemplary CAR molecule (CAR-40BBz) and one of five exemplary synthetic cytokine receptors (9RC, tCD8-G-9R, tCD8-T-9R, tCD8-D-9R, and M-M-9R). Following transduction, the administered cells were selected based on high CD45RA and CD27 positivity. Human colorectal carcinoma cells (CRC), HCT116, were engineered to express the antigen recognized by the CAR, and tumor-bearing mice were generated by injecting 1 x 106 antigen-expressing HCT116 cells into the flanks of mice. Tumors were allowed to grow for 9 days and then 1 x 105 of indicated CAR-T cells or UTD T cells were administered to each group of mice. Tumor growth was measured with calipers over the course of the experiment. [0489] As shown in FIG.18, in vivo survival rates of mice increased with either expression of CAR molecules alone or co-expression of CAR molecules and synthetic cytokine receptors (9RC, tCD8-G-9R, tCD8-T-9R, tCD8-D-9R, and M-M-9R), when compared with an un-transduced (UTD) control. The results indicate that the co-expression of synthetic cytokine receptors on T cells along with the CAR did not impede CAR T cell-directed anti-tumor activity.
30761-20002.40 EXAMPLE 7 — T Cells Expressing Synthetic Cytokine Receptor Variants with Varied Intracellular Domains (ICDs) [0490] This example describes assessment of primary human CD3-positive T cells expressing synthetic cytokine receptors with an ICD variant described in Table E6 of Example 5. Specifically, in this Example, the ICD variants in Table E6 (See SEQ ID NOS: 51, 53-57, 85-90, 92, 96-106, and 108-112) were tested in synthetic receptors comprising ECD and TMD derived from wild-type IL-9R. See SEQ ID NOS: 127, 128, 130-135, 137, 140, 142-149, 150-154, and 156-161 in Table E7. [0491] Briefly and as described in Example 1, lentiviral vectors containing polynucleotides encoding the synthetic cytokine receptors were transduced into primary human CD3-positive T cells. The cells were activated on Day 0 with anti-CD3/anti-CD28 (Dynabeads) and transduced on Day 1. The transduced cells were assessed for pSTAT3 signaling 5 days after activation at 0 hours, 20 minutes, 2 hours and 24 hours either in the presence or absence of IL-9. Following activation, cells were washed, fixed in 4% paraformaldehyde for 15 minutes with agitation, and permeabilized with methanol for at least 1 hour at -80°C. Cells were then washed with FACS buffer before staining with pSTAT3 antibody for 2 hours at 4°C in the dark. Cells were washed and analyzed, and pSTAT3 signaling was represented as mean fluorescence intensity (MFI). pSTAT3 was measured in a multiplex stain. Results are shown in FIG.19.40BBz = CAR-40BBz as described in Example 6. [0492] As shown in FIG.19, T cell activation as measured by pSTAT3 signaling was enhanced in T cells expressing 9RC Large D2 (i.e., IL9R(LargeD4)) and 9RC Large D2’ (i.e., IL9R(LargeD4’)) compared to T cells expressing wild-type IL9R. T cells expressing wild-type IL9R were activated at 20 minutes post-IL9 stimulation and downregulated by 24 hours post-IL9 stimulation. [0493] These results indicate that truncations to the IL9R signaling domain reduces intrinsic IL9R downregulation. These results also indicate that these ICD variants are suitable for combination with ECDs and TMDs that are not derived from wild-type IL9R. EXAMPLE 8—Design of Synthetic Cytokine Receptors with IL7R/IL9R Fusion Intracellular Domains (ICDs) and Expression in T cells [0494] This example provides designs for a variant ICD comprising a JAK binding domain of an IL7R ICD fused to a STAT binding domain of an IL9R ICD (hereinafter 7R/9R JAK/STAT fusion). [0495] The 7R/9R JAK/STAT fusion is shown in Table E8 below and was designed to comprise: (1) a CD34 ECD; (2) an IL7R* TMD; and (3) the 7R/9R JAK/STAT. The goal of the ICD 7R/9R JAK/STAT fusion was to reduce intrinsic downregulation of the IL9R signaling domain. [0496] Additional synthetic cytokine receptors used as positive controls in the experiments described herein included C7R, CAR-IL15 turbo or turbo 15.3, constitutively active GP130 receptor, and Thrombopoietin Receptor (TpoR). The synthetic cytokine receptor designated C7R (SEQ ID NO: 118) represents a synthetic receptor comprising a CD34 ECD (SEQ ID NO: 4), an IL7R* TMD (SEQ ID NO: 6) and an IL7R ICD (SEQ ID NO: 119), and was included as a control since IL7R is not
30761-20002.40 subject to intrinsic downregulation to the same extent as IL9R. The synthetic cytokine receptor designated TpoR-IL15R (SEQ ID NO: 167) represents a synthetic receptor comprising a thrombopoietin ECD (SEQ ID NO: 162), TMD (SEQ ID NO: 163), and an ICD (SEQ ID NO: 166) comprising a portion of the TpoR ICD (SEQ ID NO: 164) fused to IL-15RB turbodomains (SEQ ID NO: 165). Table E8 Design of 9RC Variants
[0497] Primary human CD3-positive T cells expressing C-7-9R(a) was assessed. Briefly and as described in Example 1, lentiviral vectors containing polynucleotides encoding the synthetic cytokine receptors were transduced into primary human CD3-positive T cells obtained from healthy donors. The cells were activated on Day 0 with anti-CD3/anti-CD28 (Dynabeads) and transduced on Day 1. The transduced cells were sorted for Flag-positive cells 5 days after activation. [0498] In some experiments, cells were co-transduced with a chimeric antigen receptor (CAR) by transduction with a lentiviral vector containing a polynucleotide comprising a nucleic acid sequence encoding the CAR separated from the nucleic acid sequence encoding the synthetic cytokine receptor by a IRES or 2a sequence. In these experiments, the exemplary CAR contained an antigen- binding domain directed against a cell surface antigen, a transmembrane domain and a intracellular signaling domain composed of a 4-1BB costimulatory signaling domain and a CD3zeta signaling domain (exemplary CAR designated “40bbz”). [0499] The sorted cells were stained with an anti-Flag antibody and measured by flow cytometry to assess surface expression level of the exemplary synthetic cytokine receptors. The cells were also assessed for pSTAT signaling, killing activity via a T cell-mediated killing assay, phenotype post- killing assay, total cell count post-killing assay, cytokine production in a stimulation assay and activation-induced cell death (AICD) in a stimulation assay. A. Synthetic Cytokine Receptor Expression [0500] Overall, 7R/9R JAK/STAT fusion in the ICD improved transduction efficiency of C-7- 9R(a), which was comparable to synthetic cytokine receptor C7R comprising a full-length IL7R ICD (see FIG.20). B. pSTAT Signaling
30761-20002.40 [0501] Following transduction, cells were washed and permeabilized with ice-cold 100% methanol for 60 min at on ice or stored at -80°C overnight. Cells were washed with FACS buffer before staining with pSTAT1, pSTAT3, or pSTAT5 antibodies for 1 hour at 4°C in the dark. Cells were washed and analyzed on FACSymphony A3 flow cytometer. Data represent mean fluorescence intensity (MFI). [0502] As shown in FIG.21, pSTAT1 and pSTAT3 signaling was enhanced in cells expressing C-7-9R(a) compared to cells expressing IL-15 Turbo or C7R. pSTAT5 signaling was enhanced in cells expressing C-7-9R(a) compared to cells expressing IL-15 Turbo but not cells expressing C7R. C. T Cell-Mediated Killing Assay [0503] T cell killing activity was assessed in a T cell-mediated killing assay. Briefly, killing assays were performed against HCT116-mCherry CRC target cells expressing an antigen recognized by the CAR-40BBz on the CellCyteX Live Cell Analyzer (Discover Echo). [0504] HCT116 cells were seeded in a 96-well plate at 10,000 cells per well to track cell growth overnight. To allow cells to settle, fluorescence measurements were initiated 120 minutes after plating. On the next day, T cells were added at various effector:target ratios (1:2 and 1:4). Measurements were acquired every 4 hours for the duration of the assay assessing the detection of mCherry as a measure of tumor viability. For subsequent rounds of killing, T cells were added to the co-culture after 50 and 100 hrs. Killing activity was monitored for approximately 170 hours or 7 days. [0505] As shown in FIG.22, T cells co-expressing C-7-9R(a) and CAR-40BBz demonstrated improved T cell-mediated killing after three rounds compared to T cells expressing CAR-40BBz only. As shown in FIG.23, T cells co-expressing C-7-9R(a) and CAR-40BBz demonstrated improved T cell-mediated killing after two rounds of killing compared to T cells expressing CAR-40BBz only, IL- 15 Turbo, or C7R. D. Post-Killing T Cell Phenotype and Quantification [0506] The differentiation state of the transduced T cells was evaluated by staining the cells with anti-CD27 and anti-CD45RA antibodies followed by flow cytometric analysis. CD27+/CD45RA- cells represent central memory (CM) T cells; CD27+/CD45RA+ cells represent naïve-like T cells or T memory stem-like cells; CD27-/CD45RA- cells represent effector memory (EM) T cells; CD27- /CD45RA+ cells represent effector memory cells re-expressing CD45RA (TEMRA). As shown in FIG.24, expression of C-7-9R(a) significantly impacted T cell phenotype. The majority of T cells expressing C-7-9R(a) were CD27+/CD45RA+, which represent naïve-like T cells. [0507] Total cell count was performed using standard techniques known in the art. Briefly, cells were collected from the 96-well plate after two rounds of killing. Cell count was normalized based on the amount of cells expressing CAR-40BBz only. As shown in FIG.25, after 2 rounds of killing there were more T cells expressing C-7-9R(a) compared to T cells expressing C7R or IL-15 Turbo. E. Cytokine Production and Activation-Induced Cell Death Post-Stimulation Assay
30761-20002.40 [0508] Cytokine production was measured by intracellular cytokine staining after coculture of transduced T cells and the lymphoblast cell line K562 expressing cognate CAR-T antigen. An antigen recognized by the CAR expressed on the transduced T cells was overexpressed in K562 cells to stimulate the T cells. Transduced T cells and K562 cells were cocultured for 1 week. At 1 week, T cells were rechallenged with fresh target cells in 1x eBioscience™ Protein Transport Inhibitor Cocktail for 4 hours, and fixed/permeabilized using the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set according to the manufacturer’s protocol. Cells were stained overnight at 4°C in 1x permeabilization buffer for IL-2, IFN-Ȗ, and TNF-Į. [0509] [0510] As shown in FIG.26, IFN-Ȗ expression was increased in T cells expressing C-7-9R(a) compared to T cells expressing CAR-40BBz only. [0511] The percentage of live CAR T cells was measured 1 and 2 weeks post-stimulation with K562 cells. As shown in FIG.27, T cells expressing C-7-9R(a) persisted through 2 rounds of stimulation compared T cells expressing CAR-40BBz only or wild-type IL9R. [0512] These results show that 7R/9R JAK/STAT fusion can improve CAR T cell function and persistence. EXAMPLE 9—Design of Synthetic Cytokine Receptors with IL7R/IL9R Fusion Intracellular Domains (ICDs) Comprising Deletions and/or Mutations [0513] This example provides designs for combined features of the exemplary 9RC synthetic cytokine receptors described in Example 3 and Example 8. [0514] The 9RC variants are shown in Table E9 below. The 9RC variants were designed to comprise: (1) a CD8 or truncated CD8 (tCD8) ECD; (2) an IL7R* TMD; and (3) an IL7R ICD fused to a STAT binding domain of an IL9R ICD (hereinafter 7R/9R JAK/STAT fusion). Unlike the 7R/9R JAK/STAT fusion ICD in Example 8, the 7R/9R JAK/STAT in the present Example is truncated, comprising 99 fewer amino acid residues in the 9R STAT portion of the fusion as shown in SEQ ID NO: 116. Table E9 Design of 9RC Variants
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[0515] Overall, a combination of the 7R/9R JAK/STAT fusion with CD8 or tCD8 ECD resulted in improved synthetic cytokine receptor expression in T cells compared to T cells expressing CAR- 40BBz only (see FIGS.28A-28B). EXAMPLE 10 — T Cells Expressing Synthetic Cytokine Receptor Variants with Truncated Intracellular Domains (ICDs) [0516] This example describes assessment of primary human CD3-positive T cells expressing synthetic cytokine receptors described in Table E7 of Example 5. Specifically in this Example, the synthetic cytokine receptors tested included SEQ ID NOS: 113, 179, 180, 182-184 and 188, which comprise an ECD derived from CD34, CD8 or truncated CD8 (SEQ ID NOS: 4, 185 and 19), a TMD derived from IL-7R (SEQ ID NO: 6) and a chimeric JAK/STAT fusion intracellular domain (SEQ ID NOS: 114, 116 and 181). See Table E10. Table E10 Design of 9RC Variants
[0517] Additional synthetic cytokine receptors used as a reference or control in the experiments described herein included 40BBz (as described in Example 6), GP130 (as described in Example 8),
30761-20002.40 C7R (as described in Example 8), CAR-IL18 (a CAR that specifically binds IL-18), Turbo_IL15 (as described in Example 8), wildtype IL-9R, wt9R + IL-9 (wild-type IL-9R and IL-9). [0518] As described in Example 1, lentiviral vectors containing polynucleotides encoding the synthetic cytokine receptors were transduced into primary human CD3-positive T cells obtained from healthy donors. The cells were activated on Day 0 with anti-CD3/anti-CD28 (Dynabeads) and transduced on Day 1. The transduced cells were sorted for Flag-positive cells 5 days after activation. [0519] Additionally, in some experiments, T cells were co-transduced with a chimeric antigen receptor (CAR) by transduction with a lentiviral vector containing a polynucleotide comprising a nucleic acid sequence encoding the CAR separated from the nucleic acid sequence encoding the synthetic cytokine receptor by a IRES or 2a sequence. In these experiments, the exemplary CAR contained an antigen-binding domain directed against a cell surface antigen, a transmembrane domain and a intracellular signaling domain composed of a 4-1BB costimulatory signaling domain and a CD3zeta signaling domain (exemplary CAR designated “40bbz”). [0520] The cells were assessed for pSTAT signaling, cytokine production and secretion, and T cell phenotype, quantification and expansion. A. Transduction Efficiency [0521] Transduction efficiency was assessed for the different synthetic cytokine receptors described above. Cells were stained with an idiotypic antibody directed against the CAR. As the CAR was on the same construct as the synthetic cytokine receptors, measuring the CAR expression is a surrogate for assessing transduction efficiency. Flow cytometry was performed on the transduced cells to quantify the percentage of cells that stained positive for the CAR, indicating the cells were transduced. [0522] As another method of assessing transduction efficiency, the construct that contained the CAR and the synthetic cytokine receptors also included a FLAG peptide. The cells were stained with an antibody that was specific to the FLAG peptide. Flow cytometry was performed on the transduced cells to quantify the percentage of cells that stained positive for the FLAG peptide, indicating they were transduced. Many of the cells that were transduced with synthetic cytokine receptors had increased transduction efficiency. Table E11 below summarizes the different synthetic cytokine receptors and their transduction efficiency as measured by both the percent of cells CAR+ and Flag+. Without wishing to be bound by theory, the ICD variants may increase the transduction efficiency as they are smaller. Table E11: Percent of Cells positive for CAR or FLAG
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B. pSTAT Signaling [0523] Following transduction, cells were washed and permeabilized with ice-cold 100% methanol for 60 min on ice or stored at -80°C overnight. Cells were washed with FACS buffer before staining with pSTAT1, pSTAT3, or pSTAT5 antibodies for 1 hour at 4°C in the dark. Cells were washed and analyzed on FACSymphony A3 flow cytometer. Data represent the percentage of the highest signal observed for each of the individual STATs. As shown in FIG.29, pSTAT1, -3, and -5 signaling was enhanced in T cells expressing C-7-9R(c). C. Cytokine Production and Secretion [0524] Cytokine production and secretion was tested in CD3-positive T cells co-expressing an exemplary synthetic cytokine receptor and a CAR or only a CAR. As described in Example 8, cytokine production was measured by intracellular cytokine staining after coculture of transduced T cells and the lymphoblast cell line K562 expressing cognate CAR-T antigen. An antigen recognized by the CAR expressed on the transduced T cells was overexpressed in K562 cells to stimulate the T cells. Transduced T cells and K562 cells were cocultured 1x eBioscience™ Protein Transport Inhibitor Cocktail for 4 hours, and fixed/permeabilized using the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set according to the manufacturer’s protocol. Cells were stained overnight at 4°C in 1x permeabilization buffer for IFN-Ȗ, IL-2, IL-10 and TNF-Į. Results are shown in FIG.30. [0525] As shown in FIG.30, IFN-Ȗ expression was increased in T cells co-expressing various synthetic cytokine receptors and the exemplary CAR-40BBz compared to T cells expressing control receptors (CAR-IL18, Turbo_IL15, wt9R + IL-9) or CAR-40BBz only. IL-2 expression in T cells co- expressing C-7-9R(c) or C-7-9R(b) and CAR-40BBz was comparable to T cells expressing only CAR-40BBZ only. IL-10 expression was highest in T cells co-expressing C-7-9R(c) or C-7-9R(b) and CAR-40BBz compared to T cells expressing control receptors or CAR-40BBz only. TNFĮ
30761-20002.40 expression was highest in T cells co-expressing C-7-9R(c), C-7-9R(b), or C-7-9R(d) and CAR- 40BBZ and was comparable to TNFĮ expression in T cells expressing only a CAR. [0526] CAR-T cell efficacy is determined by various factors including persistence, proliferation and potency (i.e., cell killing ability). Cytokine production by CAR-T cells is highly linked to CAR-T cell efficacy. For example, IL-2 has been shown to promote CAR-T cell proliferation, IL-10 and TNFĮ have been shown to improve persistence and reduce T cell exhaustion, and IFNȖ has been shown to promote CAR-T cell potency and target cell killing. Thus, these data indicate that T cells expressing the synthetic cytokine receptors may have improved persistence and/or reduced exhaustion as demonstrated by increased IL-10 and TNFĮ expression, increased proliferation as demonstrated by increased IL-2 expression, and increased potency as demonstrated by increased IFNȖ expression. [0527] A cytokine bead array was used to individually measure IL-2, IL-10, IL-6, TNF-Į, Granzyme A, Granzyme B, Perforin and Granulysin secretion in the supernatant of a coculture of transduced T cells and the lymphoblast cell line K562 expressing cognate CAR-T antigen. An antigen recognized by the CAR expressed on the transduced T cells was overexpressed in K562 cells to stimulate the T cells. Transduced T cells and K562 cells were cocultured for 24 hours. Supernatant was collected and analyzed for cytokines using the Legendplex Human CD8/NK panel kit (BioLegend) per the manufacturer’s protocol. Results are shown in FIG.31. [0528] As shown in FIG.31, on average, cytokine secretion was increased in K562 cocultures containing T cells co-expressing C-7-9R(a), C-7-9R(c), tCD8-7-9R(f), or tCD8-7-9R(e) compared to cocultures containing T cells expressing CAR-40BBz only, GP130 or C7R. D. T Cell Phenotype, Quantification and Expansion [0529] The differentiation state of the transduced T cells was evaluated by staining the cells with anti-CD27 and anti-CD45RA antibodies followed by flow cytometric analysis. CD27+/CD45RA- cells represent central memory (CM) T cells; CD27+/CD45RA+ cells represent naïve-like T cells or T memory stem-like cells; CD27-/CD45RA- cells represent effector memory (EM) T cells; CD27- /CD45RA+ cells represent effector memory cells re-expressing CD45RA (TEMRA). [0530] As shown in FIG.32A, expression of C-7-9R(a) and C-7-9R(c) impacted T cell phenotype compared to expression of CAR-40BBz only. CD27+/CD45RA+ cells increased from week 1 to week 2. At week 3, there were more CD27+/CD45RA+ T cells in T cells expressing C-7- 9R(a) and C-7-9R(c) compared to T cells expressing CAR-40BBz only (FIG.32B). [0531] Total cell count was performed using standard techniques known in the art. Briefly, T cells expressing synthetic cytokine receptors or various controls were continuously co-cultured with target cells across three weeks in a 96-well plate. After 3 weeks of continuous co-culture, T cells were collected and counted. Cell count was normalized based on the fraction of input T cells. [0532] As shown in FIG.33A, total T cell count peaked at week 1 for T cells expressing C-7- 9R(a) and C-7-9R(c). At week 3, total T cell count was higher for T cells expressing C-7-9R(a) and C-7-9R(c) compared to T cells expressing C7R or CAR-40BBz only (FIGS.33A and 33B). In order
30761-20002.40 to determine whether the observed decreases in cell counts were due to T cell exhaustion, CD39 (a marker of T cell exhaustion) was measured at week three. As shown in FIG.33C, CD39 expression in T cells expressing C-7-9R(a) or C-7-9R(c) was comparable to cells expressing wild-type IL-9R and cells that were untransduced. Notably, CD39 expression was decreased in cells expressing C-7-9R(a) or C-7-9R(c) compared to cells expressing CAR only. [0533] T cell expansion was measured across 4 weeks by culturing T cells expressing various synthetic cytokine receptors or controls in the presence or absence of IL-2. Briefly, lentiviral vectors containing polynucleotides encoding the synthetic cytokine receptors were transduced into primary human CD3-positive T cells obtained from healthy donors. The cells were activated on Day 0 with anti-CD3/anti-CD28 (Dynabeads) and transduced on Day 1. The transduced cells were sorted for Flag-positive cells 5 days after activation. T cells were then seeded in a 96-well cell culture plate at a standard density of 2.5e5/mL and cultured in the presence of IL-2 at a concentration of 100IU/mL or in the absence of IL-2. IL-2 was replenished in cell culture medium every 2-3 days. [0534] As shown in FIG.34, T cell expansion was highest in T cells expressing C-7-9R(a), C-7- 9R(c), tCD8-7-9R(e), and tCD8-7-9R(f) in the presence of IL-2, which was comparable to T cell expansion in T cells expressing CAR-40BBz only in the presence of IL-2. Notably, no T cells expressing synthetic cytokine receptors expanded in the absence of IL-2. [0535] Taken together, these results demonstrate that IL-9R based synthetic cytokine receptors confer enhanced cytokine production, persistence, and stem-like phenotypic properties relative to control CAR-expressing T cells without causing aberrant proliferation. EXAMPLE 11—Tumor Growth in Mice Administered T Cells Expressing CAR Molecules and Synthetic Cytokine Receptors [0536] Tumor growth in mice administered T cells expressing exemplary synthetic cytokine receptors was tested. [0537] Primary human CD3-positive T cells were transduced with lentiviral vectors co- expressing an exemplary CAR molecule (CAR-40BBz) and one of four exemplary synthetic cytokine receptors described in Example 10 (C-7-9R(a), C-7-9R(c), tCD8-7-9R(f), tCD8-7-9R(e)). Following transduction, the administered cells were selected based on high CD45RA and CD27 positivity. [0538] Human adenocarcinoma human alveolar basal epithelial cells, A549, were engineered to express the antigen recognized by the CAR, and tumor-bearing mice were generated by injecting 5 x 106 antigen-expressing A549 cells into the flanks of mice on day 0. Tumors were allowed to grow for 18 days and then 1 x 106 (FIG.35) or 3 x 105 (FIG.36) of the indicated CAR-T cells or UTD T cells were administered to the mice. Tumor growth was measured twice weekly with calipers over the course of the experiment.
30761-20002.40 [0539] As shown in FIG.35, tumor volume was highest in mice that received UTD cells and tumor volume was lowest in mice that received cells expressing CAR only or CAR and a synthetic cytokine receptor. As shown in FIG.36, tumor volume was highest in mice that received UTD cells and cells expressing CAR only. Tumor volume was lowest in mice that received cells expressing CAR and a synthetic receptor. [0540] The results indicate that the ability of T cells co-expressing a synthetic cytokine receptor and a CAR to reduce tumor growth was improved compared to T cells expressing CAR only, particularly at the low dose tested in these experiments. Without wishing to be bound by theory, these results support the utility of CAR-T cells co-expressing such synthetic cytokine receptors to improve CAR-directed anti-tumor responses in vivo where a lower ratio of CAR-expressing T cells to tumor- expressing cells would be present. * * * * * [0541] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the present description. [0542] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.
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