Anti-mesothelin chimeric antigen receptors and methods of use

Abstract

Disclosed herein are polypeptides that bind to mesothelin and methods of use thereof.

Description

[0001] ANTI-MESOTHELIN CHIMERIC ANTIGEN RECEPTORS AND METHODS OF USE CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 582,826 filed on September 14, 2023, and U.S. Provisional Patent Application No. 63 / 670,688 filed on July 12, 2024, each of which is incorporated by reference in its entirety. BACKGROUND

[0002] There is an unmet need in the field for a safe and effective T cell therapy capable of targeting malignant cells that express mesothelin (MSLN). The disclosure provides a solution to this unmet need with MSLN-binding polypeptides that, when incorporated into a chimeric receptor expressed by a T-cell, optionally designed for an optimal immune synapse distance, demonstrate superior efficacy without compromising the safety profile. INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. SUMMARY

[0004] The disclosure provides a polypeptide comprising: (a) a complementarity determining region 1 (CDR1) comprising one or more of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, and 85; (b) a complementarity determining region 2 (CDR2) comprising one or more of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, and 86; and (c) a complementarity determining region 3 (CDR3) comprising one or more of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84 and 87. In some embodiments, a VHH comprises the polypeptide.

[0005] The disclosure provides a polypeptide of one or more of the following embodiments. In some embodiments of the disclosure, polypeptide (1) comprises (a) a CDR1 comprising SEQ ID NO: 1 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 2 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 3 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN). In some embodiments, polypeptide (1) comprises a sequence comprising SEQ ID NO: 88 or a sequence having at least 70% thereto.

[0006] In some embodiments of the disclosure, polypeptide (2) comprises (a) a CDR1 comprising SEQ ID NO: 4 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 5 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 6 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (2) comprises a sequence comprising SEQ ID NO: 89 or a sequence having at least 70% thereto.

[0007] In some embodiments of the disclosure, polypeptide (3) comprises (a) a CDR1 comprising SEQ ID NO: 7 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 8 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 9 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (3) comprises a sequence comprising SEQ ID NO: 90 or a sequence having at least 70% thereto.

[0008] In some embodiments of the disclosure, polypeptide (4) comprises (a) a CDR1 comprising SEQ ID NO: 10 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 11 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 12 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (4) comprises a sequence comprising SEQ ID NO: 91 or a sequence having at least 70% thereto.

[0009] In some embodiments of the disclosure, polypeptide (5) comprises (a) a CDR1 comprising SEQ ID NO: 13 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 14 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 15 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (5) comprises a sequence comprising SEQ ID NO: 92 or a sequence having at least 70% thereto.

[0010] In some embodiments of the disclosure, polypeptide (6) comprises (a) a CDR1 comprising SEQ ID NO: 16 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 17 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 18 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (6) comprises a sequence comprising SEQ ID NO: 93 or a sequence having at least 70% thereto.

[0011] In some embodiments of the disclosure, polypeptide (7) comprises (a) a CDR1 comprising SEQ ID NO: 19 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 20 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 21 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (7) comprises a sequence comprising SEQ ID NO: 94 or 117-128 or a sequence having at least 70% thereto.

[0012] In some embodiments of the disclosure, polypeptide (8) comprises (a) a CDR1 comprising SEQ ID NO: 22 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 23 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 24 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (8) comprises a sequence comprising SEQ ID NO: 95 or a sequence having at least 70% thereto.

[0013] In some embodiments of the disclosure, polypeptide (9) comprises (a) a CDR1 comprising SEQ ID NO: 25 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 26 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 27 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (9) comprises a sequence comprising SEQ ID NO: 96 or a sequence having at least 70% thereto.

[0014] In some embodiments of the disclosure, polypeptide (10) comprises (a) a CDR1 comprising SEQ ID NO: 28 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 29 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 30 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (10) comprises a sequence comprising SEQ ID NO: 97 or a sequence having at least 70% thereto.

[0015] In some embodiments of the disclosure, polypeptide (11) comprises (a) a CDR1 comprising SEQ ID NO: 31 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 32 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 33 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (11) comprises a sequence comprising SEQ ID NO: 98 or a sequence having at least 70% thereto.

[0016] In some embodiments of the disclosure, polypeptide (12) comprises (a) a CDR1 comprising SEQ ID NO: 34 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 35 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 36 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (12) comprises a sequence comprising SEQ ID NO: 99 or a sequence having at least 70% thereto.

[0017] In some embodiments of the disclosure, polypeptide (13) comprises (a) a CDR1 comprising SEQ ID NO: 37 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 38 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 39 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (13) comprises a sequence comprising SEQ ID NO: 100 or a sequence having at least 70% thereto.

[0018] In some embodiments of the disclosure, polypeptide (14) comprises (a) a CDR1 comprising SEQ ID NO: 40 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 41 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 42 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (14) comprises a sequence comprising SEQ ID NO: 101 or a sequence having at least 70% thereto.

[0019] In some embodiments of the disclosure, polypeptide (15) comprises (a) a CDR1 comprising SEQ ID NO: 43 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 44 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 45 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (15) comprises a sequence comprising SEQ ID NO: 102 or a sequence having at least 70% thereto.

[0020] In some embodiments of the disclosure, polypeptide (16) comprises (a) a CDR1 comprising SEQ ID NO: 46 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 47 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 48 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (16) comprises a sequence comprising SEQ ID NO: 103 or a sequence having at least 70% thereto.

[0021] In some embodiments of the disclosure, polypeptide (17) comprises (a) a CDR1 comprising SEQ ID NO: 49 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 50 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 51 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (17) comprises a sequence comprising SEQ ID NO: 104 or a sequence having at least 70% thereto.

[0022] In some embodiments of the disclosure, polypeptide (18) comprises (a) a CDR1 comprising SEQ ID NO: 52 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 53 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 54 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (18) comprises a sequence comprising SEQ ID NO: 105 or a sequence having at least 70% thereto.

[0023] In some embodiments of the disclosure, polypeptide (19) comprises (a) a CDR1 comprising SEQ ID NO: 55 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 56 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 57 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (19) comprises a sequence comprising SEQ ID NO: 106 or a sequence having at least 70% thereto.

[0024] In some embodiments of the disclosure, polypeptide (20) comprises (a) a CDR1 comprising SEQ ID NO: 58 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 59 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 60 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (20) comprises a sequence comprising SEQ ID NO: 107 or a sequence having at least 70% thereto.

[0025] In some embodiments of the disclosure, polypeptide (21) comprises (a) a CDR1 comprising SEQ ID NO: 61 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 62 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 63 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (21) comprises a sequence comprising SEQ ID NO: 108 or a sequence having at least 70% thereto.

[0026] In some embodiments of the disclosure, polypeptide (22) comprises (a) a CDR1 comprising SEQ ID NO: 64 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 65 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 66 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (22) comprises a sequence comprising SEQ ID NO: 109 or a sequence having at least 70% thereto.

[0027] In some embodiments of the disclosure, polypeptide (23) comprises (a) a CDR1 comprising SEQ ID NO: 67 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 68 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 69 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (23) comprises a sequence comprising SEQ ID NO: 110 or a sequence having at least 70% thereto.

[0028] In some embodiments of the disclosure, polypeptide (24) comprises (a) a CDR1 comprising SEQ ID NO: 70 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 71 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 72 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (24) comprises a sequence comprising one or more of SEQ ID NO: 111 or 129-134, or a sequence having at least 70% thereto.

[0029] In some embodiments of the disclosure, polypeptide (25) comprises (a) a CDR1 comprising SEQ ID NO: 73 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 74 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 75 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (25) comprises a sequence comprising SEQ ID NO: 112 or a sequence having at least 70% thereto.

[0030] In some embodiments of the disclosure, polypeptide (26) comprises (a) a CDR1 comprising SEQ ID NO: 76 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 77 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 78 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (26) comprises a sequence comprising SEQ ID NO: 113 or a sequence having at least 70% thereto.

[0031] In some embodiments of the disclosure, polypeptide (27) comprises (a) a CDR1 comprising SEQ ID NO: 79 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 80 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 81 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (27) comprises a sequence comprising SEQ ID NO: 114 or a sequence having at least 70% thereto.

[0032] In some embodiments of the disclosure, polypeptide (28) comprises (a) a CDR1 comprising SEQ ID NO: 82 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 83 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 84 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (28) comprises a sequence comprising SEQ ID NO: 115 or a sequence having at least 70% thereto.

[0033] In some embodiments of the disclosure, polypeptide (29) comprises (a) a CDR1 comprising SEQ ID NO: 85 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 86 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 87 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to MSLN. In some embodiments, polypeptide (29) comprises a sequence comprising SEQ ID NO: 116 or a sequence having at least 70% thereto.

[0034] In some embodiments of the disclosure, a VHH comprises one or more of polypeptides (1) through (29).

[0035] In some embodiments of the disclosure, a binding moiety comprises one or more of polypeptides (1) through (29). In some embodiments of the disclosure, a binding moiety comprises one or more VHH(s) of the disclosure.

[0036] In some embodiments of the disclosure, a polypeptide of the disclosure does not comprise one or more of polypeptide (4), polypeptide (10), polypeptide (11), polypeptide (12), polypeptide (15) and polypeptide (20).

[0037] The disclosure provides a polypeptide of one or more of the following embodiments. In some embodiments of the disclosure, the polypeptide comprises: a light chain variable region comprising: (a) a CDR1 comprising SASSSVSYMH (SEQ ID NO: 135); (b) a CDR2 comprising RWIYDTSKLAS (SEQ ID NO: 136); and (c) a CDR3 comprising QQWSGYPLT (SEQ ID NO: 137); and a heavy chain variable region comprising: (d) a CDR1 comprising GYSFTGYTMN (SEQ ID NO: 138); (e) a CDR2 comprising IGLITPYNGASS (SEQ ID NO: 139); and (f) a CDR3 comprising ARGGYDGRGFDY (SEQ ID NO: 140); wherein the light chain comprises a sequence having at least 70% identity to SEQ ID NO: 141 and wherein the heavy chain comprises a sequence having at least 70% identity to SEQ ID NO: 142. In some embodiments, a single chain variable fragment (scFv) comprises the polypeptide.

[0038] The disclosure provides a polypeptide of one or more of the following embodiments. In some embodiments of the disclosure, polypeptide (30) comprises: a light chain variable region comprising SEQ ID NO: 143 and a heavy chain variable region comprising SEQ ID NO: 144. In some embodiments of the disclosure, polypeptide (31) comprises: a light chain variable region comprising SEQ ID NO: 145 and a heavy chain variable region comprising SEQ ID NO: 146. In some embodiments of the disclosure, polypeptide (32) comprises: a light chain variable region comprising SEQ ID NO: 147 and a heavy chain variable region comprising SEQ ID NO: 148. In some embodiments of the disclosure, polypeptide (33) comprises: a light chain variable region comprising SEQ ID NO: 149 and a heavy chain variable region comprising SEQ ID NO: 150. In some embodiments of the disclosure, polypeptide (34) comprises: a light chain variable region comprising SEQ ID NO: 151 and a heavy chain variable region comprising SEQ ID NO: 152. In some embodiments of the disclosure, polypeptide (35) comprises: a light chain variable region comprising SEQ ID NO: 153 and a heavy chain variable region comprising SEQ ID NO: 154. In some embodiments of the disclosure, polypeptide (36) comprises: a light chain variable region comprising SEQ ID NO: 155 and a heavy chain variable region comprising SEQ ID NO: 156. In some embodiments of the disclosure, polypeptide (37) comprises: a light chain variable region comprising SEQ ID NO: 157 and a heavy chain variable region comprising SEQ ID NO: 158.

[0039] In some embodiments of the disclosure, a single chain variable fragment (scFv) comprises one or more of polypeptides (30) through (37).

[0040] In some embodiments of the disclosure, a binding moiety comprises one or more of polypeptides (30) through (37). In some embodiments of the disclosure, a binding moiety comprises one or more scFv(s) and VHH(s) of the disclosure.

[0041] In some embodiments of the disclosure, a polypeptide of the disclosure does not comprise polypeptide (30).

[0042] In some embodiments of the disclosure, a polypeptide of the disclosure has been (1) modified to decrease immunogenicity, (2) modified to increase stability, and / or (3) humanized.

[0043] In some embodiments of the disclosure, a polypeptide of the disclosure further comprises a spacer sequence. In some embodiments, the spacer sequence comprises (a) one or more of SEQ ID NOs: 159-177, (b) a sequence having at least 90% identity to a sequence of (a), or (c) a sequence producing a protein having a length of between 25 and 250 angstroms, inclusive of the endpoints. In some embodiments, the spacer sequence comprises (a) SEQ ID NO: 164, (b) a sequence having at least 90% identity to a sequence of (a), or (c) a sequence producing a protein having a length of between 40 and 50 angstroms, inclusive of the endpoints. In some embodiments, the spacer sequence comprises (a) SEQ ID NO: 165, (b) a sequence having at least 90% identity to a sequence of (a), or (c) a sequence producing a protein having a length of between 50 and 60 angstroms, inclusive of the endpoints. In some embodiments, the spacer sequence comprises (a) SEQ ID NO: 166, (b) a sequence having at least 90% identity to a sequence of (a), or (c) a sequence producing a protein having a length of between 50 and 60 angstroms, inclusive of the endpoints. In some embodiments, the spacer sequence comprises (a) SEQ ID NO: 167, (b) a sequence having at least 90% identity to a sequence of (a), or (c) a sequence producing a protein having a length of between 60 and 70 angstroms, inclusive of the endpoints. In some embodiments, the spacer sequence comprises (a) SEQ ID NO: 168, (b) a sequence having at least 90% identity to a sequence of (a), or (c) a sequence producing a protein having a length of between 70 and 80 angstroms, inclusive of the endpoints.

[0044] In some embodiments of the disclosure, the spacer sequence further comprises a linker sequence. In some embodiments, the linker sequence comprises one or more of a small molecule, a DNA sequence, an RNA sequence, and an amino acid sequence. In some embodiments, the linker sequence comprises an amino acid sequence. In some embodiments, the linker comprises a Glycine (G) and / or a Serine (S). In some embodiments, the linker comprises GGGSG (SEQ ID NO: 178), GGSG (SEQ ID NO: 179) or GS. In some embodiments, the linker has a length of between 15 and 20 Angstroms. In some embodiments, the linker has a length of about 18 Angstroms.

[0045] The disclosure provides a chimeric receptor comprising a polypeptide of the disclosure, including, but not limited to, one or more of polypeptide (1) through (37). In some embodiments, the chimeric receptor is a Chimeric Antigen Receptor (CAR). In some embodiments, the CAR comprises a polypeptide of the disclosure, including, but not limited to, one or more of polypeptide (1) through (37).

[0046] In some embodiments of the disclosure, a CAR comprises (a) an extracellular domain comprising a polypeptide of the disclosure, including, but not limited to, one or more of polypeptide (1) through (37), (b) a transmembrane domain, and (c) an intracellular domain. In some embodiments, the transmembrane domain comprises a sequence isolated or derived from a human endogenous transmembrane protein. In some embodiments, the transmembrane domain comprises a sequence isolated or derived from a sequence encoding human CD3-zeta, human CD4, human CD8, human CD28 and / or human ICOS. In some embodiments, the intracellular domain comprises a sequence isolated or derived from a human CD3-zeta sequence. In some embodiments, the intracellular domain further comprises at least one co-stimulatory domain. In some embodiments, the at least one co-stimulatory domain comprises a sequence isolated or derived from a human CD28 sequence. In some embodiments, the at least one co-stimulatory domain comprises a sequence isolated or derived from a human 41BB sequence or an OX40 sequence. In some embodiments, the intracellular domain comprises a sequence isolated or derived from a cytokine inducer. In some embodiments, the cytokine inducer comprises an IL-12 cytokine inducer. In some embodiments, the cytokine inducer binds to a sequence isolated or derived from a Nuclear factor of activated T cells (NFAT) sequence. In some embodiments, the intracellular domain comprises a sequence isolated or derived from an IL-2 receptor (IL-2R). In some embodiments, the IL-2R comprises IL-2R beta.

[0047] The disclosure provides a nucleic acid sequence encoding the polypeptide of the disclosure or the chimeric receptor of the disclosure. In some embodiments, the nucleic acid sequence comprises a regulatory sequence capable of driving expression of the nucleic acid sequence in a mammalian cell. In some embodiments, the regulatory sequence comprises one or more of a promoter, a response element, an enhancer and any combination thereof. In some embodiments, the regulatory sequence comprises two or more response elements or repeated response elements, optionally, arranged in tandem. In some embodiments, the regulatory sequence comprises a constitutive promoter. In some embodiments, the regulatory sequence comprises an inducible promoter. In some embodiments, the regulatory sequence comprises a minimal promoter. In some embodiments, the regulatory sequence comprises a promoter comprising one or more transcription factor binding motifs. In some embodiments, the regulatory sequence comprises a promoter comprising one or more concatemerized sequences or motifs.

[0048] The disclosure provides a vector comprising a nucleic acid of the disclosure. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a delivery vector. In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a viral vector.

[0049] The disclosure provides a cell comprising one or more of (a) a polypeptide of the disclosure; (b) a chimeric receptor of the disclosure; (c) a nucleic acid sequence of the disclosure; and (d) a vector of the disclosure. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a stem cell or a T-cell precursor cell. In some embodiments, the cell is a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), a transdifferentiated cell, a dedifferentiated cell or a manufactured cell. In some embodiments, the cell is a somatic cell or a differentiated cell. In some embodiments, the cell is a T cell, a Natural Killer (NK) cell, or a macrophage. In some embodiments, the cell is isolated or derived from one or more of bone marrow, peripheral circulating blood, umbilical cord blood and placental blood. In some embodiments, the cell has been cryopreserved. In some embodiments, the cell has been modified to become an allogeneic cell. In some embodiments, the cell has been modified to produce an allogeneic cell.

[0050] In some embodiments of a cell of the disclosure, the cell is capable of expressing an exogenous protein capable of binding to or signaling through one or more cytokine receptor(s). In some embodiments, the exogenous protein comprises a cytokine. In some embodiments, the cytokine comprises one or more of a sequence isolated or derived from a human IL-2, a human IL-7, a human IL-15, a human IL-18, a human IL-21, a human IL-23, a human interferon (including but not limited to interferon alpha, beta, gamma and omega).

[0051] In some embodiments of a cell of the disclosure, the cell is capable of expressing a safety switch. In some embodiments, the safety switch comprises a sequence isolated or derived from a human caspase protein. In some embodiments, the safety switch comprises an inducible Caspase 9 (iCasp9). In some embodiments, the safety switch comprises a sequence isolated or derived from a human Epidermal growth factor receptor (EGFR) protein. In some embodiments, the safety switch comprises a truncated EGFR (EGFRt). In some embodiments, the EGFRt comprises a sequence of SEQ ID NO: 229 In some embodiments, the safety switch comprises an EGFRopt sequence. In some embodiments, the EGFRopt sequence comprises a sequence of any one of SEQ ID Nos: 230-244.

[0052] The disclosure provides a cell comprising a vector comprising a nucleic acid sequence encoding, from five-prime to three-prime, an inducible promoter that drives expression of the combination of an anti-MSLN CAR of the disclosure, a 2A sequence, an optimized EGFR sequence (also referred to as an EGFRopt sequence), a 2A sequence, and a CD8-targeted designed cytokine of the disclosure. In some embodiments, the inducible promoter comprises the sequence of GGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTG GGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGG GGACTTTCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATAATGGAAGCTCGAATTC CAG (SEQ ID NO: 477). In some embodiments, the inducible promoter further comprises a 5’ UTR sequence of ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC (SEQ ID NO: 478), optionally producing a combination of an inducible promoter + 5’UTR comprising the sequence of GGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTG GGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGG GGACTTTCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATAATGGAAGCTCGAATTC CAGACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC (SEQ ID NO: 479). In some embodiments, the anti-MSLN CAR comprises an scFv that binds MSLN and comprises the sequence of DIQMTQSPSTLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQWSGYPLTFGQGTKVEIKGGGGSGGGGSGGG GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMNWVRQAPGQGLEWMG LITPYNGASSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFD YWGQGTLVTVSS (SEQ ID NO: 480). In some embodiments, the anti-MSLN CAR comprises the sequence of DIQMTQSPSTLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQWSGYPLTFGQGTKVEIKGGGGSGGGGSGGG GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMNWVRQAPGQGLEWMG LITPYNGASSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFD YWGQGTLVTVSSVPCPVPPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQ PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 481) without a signal peptide or MVLQTQVFISLLLWISGAYGDIQMTQSPSTLSASVGDRVTITCSASSSVSYMHWYQQKP GKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQWSGYPLTFGQG TKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTS YTMNWVRQAPGQGLEWMGLITPYNGASSYAQKFQGRVTMTRDTSTSTVYMELSSLRS EDTAVYYCARGGYDGRGFDYWGQGTLVTVSSVPCPVPPFWVLVVVGGVLACYSLLVT VAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 482) with a signal peptide. In some embodiments, the optimized EGFR sequence comprises MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPV AFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQ FSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGEN SCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECI QCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADA GHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRR (SEQ ID NO: 483). In some embodiments, the designed cytokine comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK (SEQ ID NO: 459) and the anti-CD8 targeting moiety comprises the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGST YYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYY DMDYWGQGTQVTVSS (SEQ ID NO: 484). In some embodiments, the CD8-targeted designed cytokine comprises the sequence of MVLQTQVFISLLLWISGAYGAPTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEF LNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLE LKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQA PGKGREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAG SLYTCVQSIVVVPARPYYDMDYWGQGTQVTVSS (SEQ ID NO: 485). In some embodiments, the 2A sequence is a P2A sequence comprising SGATNFSLLKQAGDVEENPGP (SEQ ID NO: 486). In some embodiments, the 2A sequence is a T2A sequence comprising GSGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 487).

[0053] The disclosure provides a composition comprising one or more of: (a) a polypeptide of the disclosure; (b) a chimeric receptor of the disclosure; (c) a nucleic acid sequence of the disclosure; (d) a vector of the disclosure; and (e) a cell of the disclosure.

[0054] The disclosure provides a pharmaceutical composition comprising one or more of: (a) a polypeptide of the disclosure; (b) a chimeric receptor of the disclosure; (c) a nucleic acid sequence of the disclosure; (d) a vector of the disclosure; (e) a cell of the disclosure; and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the systemic administration comprises an intravenous injection or an intravenous infusion. In some embodiments, the pharmaceutical composition is formulated for local administration. In some embodiments, the local administration comprises an intratumoral injection or infusion, an intraspinal injection or infusion, an intracerebellar injection or infusion, or intracisternal injection or infusion.

[0055] The disclosure provides a use of one or more of a polypeptide of the disclosure, a chimeric receptor of the disclosure, a nucleic acid sequence of the disclosure, a vector of the disclosure, a cell of the disclosure, a composition of the disclosure or a pharmaceutical composition of the disclosure in the manufacture of a medicament for treating a disease or disorder. In some embodiments, the disease or disorder comprises a proliferative disorder. In some embodiments, the proliferative disorder is a metastatic disorder. In some embodiments, the metastatic disorder comprises a solid tumor.

[0056] The disclosure provides a use of one or more of a polypeptide of the disclosure, a chimeric receptor of the disclosure, a nucleic acid sequence of the disclosure, a vector of the disclosure, a cell of the disclosure, a composition of the disclosure or a pharmaceutical composition of the disclosure for treating a disease or disorder. In some embodiments, the disease or disorder comprises a proliferative disorder. In some embodiments, the proliferative disorder is a metastatic disorder. In some embodiments, the metastatic disorder comprises a solid tumor.

[0057] The disclosure provides a method of treating a disease or disorder comprising administering an effective amount of one or more of a polypeptide of the disclosure, a chimeric receptor of the disclosure, a nucleic acid sequence of the disclosure, a vector of the disclosure, a cell of the disclosure, a composition of the disclosure or a pharmaceutical composition of the disclosure to a subject in need thereof, thereby treating the disease or disorder. In some embodiments, the subject is at risk of developing the disease or disorder. In some embodiments, the subject has been diagnosed with the disease or disorder. In some embodiments, the disease or disorder comprises a proliferative disorder. In some embodiments, the proliferative disorder is a metastatic disorder. In some embodiments, the metastatic disorder comprises a solid tumor. In some embodiments, treating the disease or disorder comprises one or more of: (a) decreasing the severity of a sign or symptom of the disease or disorder; (b) improving a function negatively affected by the disease or disorder; (c) improving the subject’s prognosis; (d) increasing the subject’s duration of progression-free survival (PFS); (e) reducing a quantity of cells affected by the disease or disorder; (f) inducing a remission form the disease or disorder; (g) delaying or preventing a relapse of the disease or disorder; (h) delaying or preventing a second presentation of the disease or disorder; (i) increasing a population of memory cells capable of treating a relapse or second presentation of the disease or disorder. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG.1 is a schematic ribbon diagram depicting the folded configuration of the mesothelin (MSLN) polypeptide, including, from proximal to distal, the GPI-linker attached to the outer layer of a membrane, Region III, Region II and Region I.

[0059] FIG.2 is a schematic diagram depicting folded configuration of the mesothelin (MSLN) polypeptide, highlighting the GPI-linker attached to the outer layer of a membrane in red, and the distance occupied from the cell surface of epitopes located in Region I, which is approximately 90 Angstroms.

[0060] FIG.3 is a schematic diagram depicting an exemplary immune-synapse, with a distance from the surface of a CAR-expressing cell to a target cell surface of about 200 Angstroms. Spacers and / or linkers of the disclosure may be adjusted to reach this optimal distance. For example, a spacer used in combination with a binder raised against an epitope located in Region III of MSLN will be longer than a spacer used in combination with a binder raised against an epitope located in Region I of MSLN.

[0061] FIG.4A is a plot showing Area Under the Curve (AUC) as a function of Interferon gamma production for all candidate MSLN binding polypeptides.

[0062] FIG.4B is a plot showing Area Under the Curve (AUC) as a function of Interferon gamma production for selected MSLN binding polypeptides.

[0063] FIG.5A is a plot showing Area Under the Curve (AUC) as a function of IL-2 production for all candidate MSLN binding polypeptides.

[0064] FIG.5B is a plot showing Area Under the Curve (AUC) as a function of IL-2 production for selected MSLN binding polypeptides.

[0065] FIG.6A is a schematic diagram depicting is a plot showing Area Under the Curve (AUC) as a function of IL-2 production for selected MSLN binding polypeptides in ovarian cancer cells.

[0066] FIG.6B is a schematic diagram depicting is a plot showing Area Under the Curve (AUC) as a function of IL-2 production for selected MSLN binding polypeptides in non-small cell lung cancer cells.

[0067] FIG.7 is a series of schematic diagrams representing the constructs used for generating the data depicted in graphs below them. Collectively, the data demonstrate that a combination of technologies, including anti-MSLN CAR-expressing T-cells that can secrete a designed cytokine of the disclosure, progressively improve anti-tumor efficacy and expansion. In a first, “CAR- only” construct, the expression of an anti-MSLN chimeric antigen receptor of the disclosure is under the control of a constitutive MND promoter (and combined by a 2A sequence with an EGFR sequence that serves as both a cell marker and a safety switch (see WO 2021 / 189008)). In a second construct, the expression of the anti-MSLN chimeric antigen receptor of the disclosure is under the control of an inducible promoter (and combined by a 2A sequence with an EGFR sequence that serves as both a cell marker and a safety switch (see WO 2021 / 189008)). The inducible promoter comprises the sequence GGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTG GGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGG GGACTTTCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATAATGGAAGCTCGAATTC CAG (SEQ ID NO: 477). In a preferred embodiment of the disclosure, the inducible promoter depicted in this figure may be used in combination with a 5’ UTR sequence comprising ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC (SEQ ID NO: 478). When used in combination, the resultant inducible promoter + 5’UTR comprises the sequence GGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTG GGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGG GGACTTTCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATAATGGAAGCTCGAATTC CAGACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC (SEQ ID NO: 479). In a third construct, the expression of the anti-MSLN chimeric antigen receptor of the disclosure is under the control of the inducible promoter (and combined by a 2A sequence with an EGFR sequence that serves as both a cell marker and a safety switch (see WO 2021 / 189008)), and further combined by a 2A sequence to a designed cytokine of the disclosure. The designed cytokine depicted in this figure comprises the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK (SEQ ID NO: 459). When combined with a preferred anti-CD8 VHH of the disclosure comprising a sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGST YYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYY DMDYWGQGTQVTVSS (SEQ ID NO: 484), the resulting polypeptide may contain a linker and may comprise the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELKGGGGSGGGGSGGGGSE VQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGSTY YADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYD MDYWGQGTQVTVSS (SEQ ID NO: 488). In each of these constructs, the anti-MSLN CAR of the disclosure comprises an scFv that binds MSLN and comprises the sequence of DIQMTQSPSTLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQWSGYPLTFGQGTKVEIKGGGGSGGGGSGGG GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMNWVRQAPGQGLEWMG LITPYNGASSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFD YWGQGTLVTVSS (SEQ ID NO; 480). In some embodiments of the disclosure, the anti-MSLN CAR of the disclosure comprises a sequence of DIQMTQSPSTLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQWSGYPLTFGQGTKVEIKGGGGSGGGGSGGG GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMNWVRQAPGQGLEWMG LITPYNGASSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFD YWGQGTLVTVSSVPCPVPPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQ PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 481). Using these constructs, the anti- tumor efficacy was assessed by measuring tumor volume (mm3) as a function of days post-tumor engraftment (see FIG.8 for a description of the xenograft mouse model used). Using these constructs, the CAR T cell expansion was assessed by measuring hCD45+ cell number (cells / milliliter) as a function of days post-T cell injection.

[0068] FIG.8 is a series of schematic diagrams and graphs depicting elimination of intraperitoneal (IP) engrafted SKVO3 ovarian cancer cells following intravenous (IV) treatment with a CAR-T composition containing a designed cytokine of the disclosure. The schematic diagram demonstrates the generation of the mouse xenograft model used for this study. Briefly, fluorescently-labeled (FFluc) SKVO3 ovarian cancer cells from one of two donors were introduced into an immune deficient mouse to produce an intraperitoneal (IP) engraftment. Twenty days post-injection of the SKOV3 cells, the CAR-T composition was introduced intravenously via a tail vein. T cell compositions were provided at varying dosages of 50 thousand cells per animal, 200 thousand cells per animal, and 500 thousand cells per animal. Each CAR-T provided for treatment contained a construct in which the expression of the anti- MSLN chimeric antigen receptor, an EGFRopt sequence (see WO 2021 / 189008), and a designed cytokine of the disclosure were under the control of an inducible promoter. The designed cytokine depicted in this figure comprises the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK (SEQ ID NO: 459). When combined with a preferred anti-CD8 VHH of the disclosure comprising a sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGST YYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYY DMDYWGQGTQVTVSS (SEQ ID NO: 484), the resulting polypeptide may contain a linker and may comprise the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELKGGGGSGGGGSGGGGSE VQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGSTY YADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYD MDYWGQGTQVTVSS (SEQ ID NO: 488). The inducible promoter comprises the sequence GGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTG GGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGG GGACTTTCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATAATGGAAGCTCGAATTC CAG (SEQ ID NO: 477). In a preferred embodiment of the disclosure, the inducible promoter depicted in this figure may be used in combination with a 5’ UTR sequence comprising ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC (SEQ ID NO: 478). When used in combination, the resultant inducible promoter + 5’UTR comprises the sequence GGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTG GGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGG GGACTTTCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATAATGGAAGCTCGAATTC CAGACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC (SEQ ID NO: 479). In the construct used to generate the data in this study, the anti-MSLN CAR of the disclosure comprises an scFv that binds MSLN and comprises the sequence of DIQMTQSPSTLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQWSGYPLTFGQGTKVEIKGGGGSGGGGSGGG GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMNWVRQAPGQGLEWMG LITPYNGASSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFD YWGQGTLVTVSS (SEQ ID NO: 480). In some embodiments of the disclosure, the anti-MSLN CAR of the disclosure comprises a sequence of DIQMTQSPSTLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQWSGYPLTFGQGTKVEIKGGGGSGGGGSGGG GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMNWVRQAPGQGLEWMG LITPYNGASSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFD YWGQGTLVTVSSVPCPVPPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQ PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 481). Elimination of the SKOV3 tumor cells was determined by measuring radiance as a function of days post-engraftment. The data show that in both donors and at least at the doses of 200K and 500K cells per animal, the fluorescent cells are eliminated as the radiance decreases precipitously.

[0069] FIG.9 is a series of photographs providing a qualitative analysis of the reduction of fluorescent label based on the study performed in FIG.8.

[0070] FIG.10 is a schematic diagram and graphs extending the study performed in FIG.8 using two additional T-cell donors and a broader range of doses for the T-cell composition (20 thousand cells per animal, 50 thousand cells per animal, 200 thousand cells per animal, 500 thousand cells per animal). The results of this study confirm the results of the study shown in FIG.8. The elimination of SKOV3 tumor cells occurs following treatment with the T cell composition in a dose-dependent manner.

[0071] FIG.11 is a graph showing that binding of a targeted designed cytokine (with an anti- CD8 targeting moiety) does not impair endogenous coreceptor activity required for proper T-cell activation. Specifically, this graph depicts activation of a T-cell expressing an exogenous TCR construct (TCR-T), measured as a percent (%) of interferon gamma (IFNJ) secreting TCR-T cells as a proportion of CD8 positive (CD8+) and EGFR-marker expressing (EGFR+) TCR-T cells, as a function of a nanomolar (nM) concentration of peptide to which the TCR-T binds. Three conditions were tested: the “no cytokine” (negative control) condition measures TCR-T activation in the absence of a designed cytokine of the disclosure; the “designed cytokine” condition measures TCR-T activation in the presence of an exemplary designed cytokine of the disclosure; the “targeted designed cytokine” condition measures TCR-T activation in the presence of an exemplary CD8-targeted designed cytokine of the disclosure. The data demonstrate that the addition of the anti-CD8 targeting moiety to an exemplary designed cytokine of the disclosure does not negatively impact TCR-T activation. The exemplary designed cytokine used for the two test conditions of this study comprises the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK (SEQ ID NO: 459). The exemplary anti-CD8 targeting moiety used for the targeted designed cytokine of this study comprises the sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGST YYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYY DMDYWGQGTQVTVSS (SEQ ID NO: 484). The designed cytokine may contain a linker sequence and may comprise the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELKGGGGSGGGGSGGGGSE VQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGSTY YADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYD MDYWGQGTQVTVSS (SEQ ID NO: 488). The exemplary EGFR-marker used in this study comprises the sequence of MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPV AFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQ FSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGEN SCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECI QCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADA GHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRR (SEQ ID NO: 483).

[0072] FIG.12A-12B is a series of schematic diagram depicting an experimental design for a study to assess T-cell homeostasis and proliferation in T-cells of the disclosure expressing an anti-MSLN CAR of the disclosure, alone or in combination with an inducible cytokine of the disclosure (with and without an anti-CD8 targeting moiety) when compared to T-cells expressing the same anti-MSLN CAR in combination with exogenous wildtype IL-2 or IL-15. According to this experimental design, T cells transduced with lentiviral vectors (comprising the constructs described in FIG.13) were rested overnight in the absence of exogenous cytokines. EGFR- marker expressing cells (EGFR+ cells) were replated at 1.6E6 cells / ml in the presence or absence of 0.65 nM test articles (see FIG.13). Every 3 to 4 days over the course of 4 weeks, a subset of cells was analyzed by flow cytometry, and the remaining cells washed and replated in fresh cytokine media. Transduced (EGFRopt+) and bystander (EGFRopt-) cells within the same well were analyzed separately. (A) Stimulation was performed by contacting each test article with a cell media containing a combination of MSLN, CD58 and ICAM1. (B) Resting condition performed by contacting each test article with cell media lacking exogenous cytokines.

[0073] FIG.13 is a pair of graphs depicting the results of the study described in FIG.12A-B. the long-term proliferation of T cells expressing a test article and an EGFR-marker of the disclosure was measured following acute stimulation for 24 hours with a MSLN antigen (FIG. 12A) or a negative control (rest for 24 hours, see FIG.12B). The results are depicted, in the left- hand plot, by calculating the area under the curve (AUC) of proliferation (Fold proliferation) as a function number of days. Thus, larger values indicate greater proliferation of the T-cells. The results are depicted, in the right-hand plot, by calculating the AUC of CD8-expressing (CD8+) cells as a proportion of all cells expressing an EGFR-marker of the disclosure as a function of number of days. With respect to each Test Article of this study, the exemplary anti-MSLN CAR comprises a MSLN binder having the sequence of DIQMTQSPSTLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQWSGYPLTFGQGTKVEIKGGGGSGGGGSGGG GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMNWVRQAPGQGLEWMG LITPYNGASSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFD YWGQGTLVTVSS (SEQ ID NO: 480), and the exemplary anti-MSLN CAR comprises the sequence of DIQMTQSPSTLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQWSGYPLTFGQGTKVEIKGGGGSGGGGSGGG GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMNWVRQAPGQGLEWMG LITPYNGASSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFD YWGQGTLVTVSSVPCPVPPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQ PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 481). Each Test Article of this study also contains an EGFR-marker of the disclosure having the sequence of MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPV AFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQ FSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGEN SCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECI QCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADA GHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRR (SEQ ID NO: 483). Each Test Article of this study contains an inducible promoter of the disclosure, driving expression of the exemplary anti-MSLN CAR, the exemplary EGFR-marker and, when present, a cytokine, the inducible promoter having the sequence of GGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTG GGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGG GGACTTTCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATAATGGAAGCTCGAATTC CAGACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC (SEQ ID NO: 479). Test Article 1 comprises T-cells transduced with a construct containing an inducible promoter of the disclosure, the exemplary anti-MSLN CAR, the exemplary EGFR- marker, and an exemplary CD8-targeted designed cytokine of the disclosure. The exemplary CD8-targeted designed cytokine of Test Article 1 comprises the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELKGGGGSGGGGSGGGGSE VQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGSTY YADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYD MDYWGQGTQVTVSS (SEQ ID NO: 488). Test Article 1 was not contacted with exogenous cytokines. Test Article 2 comprises T-cells transduced with a construct containing an inducible promoter of the disclosure, the exemplary anti-MSLN CAR and the exemplary EGFR-marker. Test Article 2 was not contacted with exogenous cytokines. Test Articles 3-6 comprise T-cells transduced with a construct containing an inducible promoter of the disclosure, the exemplary anti-MSLN CAR and the exemplary EGFR-marker. Test Article 3 was contacted with exogenous designed cytokine, having a sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK (SEQ ID NO: 459). Test Article 4 was contacted with exogenous CD8-targeted designed cytokine, having a sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELKGGGGSGGGGSGGGGSE VQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGSTY YADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYD MDYWGQGTQVTVSS (SEQ ID NO: 488). Test Article 5 was contacted with exogenous wild type IL-2. Test Article 6 was contacted with exogenous wild type IL-15. The results of this study demonstrate that, in the first 14 days, Test Article 1 cultures increased significantly more than Test Article 2, cultures), but less than the cultures of test articles 3-6, which were supplemented with exogenous cytokine. Growth of the Test Article 1 and Test Article 2 EGFR+ populations was limited and declined starting at day 14 such that by day 28, fewer cells were present than the initial seeding number on day 0. By day 28, the proportion of EGFR+ population was 10-fold greater in Test Article 1 than in Test Article 2, but 100-fold less than in Test Articles 3-6, which maintained similar cell numbers after day 14. Test articles with a targeted designed cytokine of the disclosure incorporated into the lentiviral transgene (e.g., Test Article 1), or supplemented as a recombinant protein (Test Article 4) had greater %CD8 AUC, indicating a strong bias towards CD8+ T cells. Taken together, the results of this study demonstrate that anti-MSLN CAR-T cells of the disclosure proliferate effectively, but not constitutively, which preserves homeostasis. Maintaining homeostasis is an important characteristic of T-cell therapies that, in part, ensures safety of the use of these cells in a cellular therapy. ^

[0074] FIG.14 is a pair of graphs demonstrating in vivo efficacy of anti-MSLN CAR-T cells of the disclosure, against, for example, a subcutaneous graft of human MSLN+ H1650 lung cells forming a tumor in this xenograft study. For two T-cell donors, the tumor volume (measured incubic millimeters (mm3) was examined as a function of time (measured as days post-engraftment). In each treatment condition, 5 mice were used as hosts for the human MSLN+ H1650 lung cells. The “Mock T-Cells (2M cells)” condition is composed of two (2) million (2M) mock-transfected T-cells that receive and empty lentivector and serve as a negative control. The “CAR-only (2M cells)” condition is composed of two (2) million (2M) T cells modified to express an exemplary anti-MSLN CAR of the disclosure and an exemplary EGFR- marker of the disclosure. In each condition the exemplary anti-MSLN CAR comprises a MSLN binder comprising the sequence of DIQMTQSPSTLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQWSGYPLTFGQGTKVEIKGGGGSGGGGSGGG GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMNWVRQAPGQGLEWMG LITPYNGASSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFD YWGQGTLVTVSS (SEQ ID NO: 480), and the exemplary anti-MSLN CAR comprises the sequence of DIQMTQSPSTLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQWSGYPLTFGQGTKVEIKGGGGSGGGGSGGG GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMNWVRQAPGQGLEWMG LITPYNGASSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFD YWGQGTLVTVSSVPCPVPPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQ PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 481). Each condition of this study also contains an EGFR-marker of the disclosure having the sequence of MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPV AFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQ FSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGEN SCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECI QCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADA GHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRR (SEQ ID NO: 483). The “CAR-only (500K cells)” condition is composed of five hundred thousand (500K) T cells modified to express an exemplary anti-MSLN CAR of the disclosure and an exemplary EGFR-marker of the disclosure. The “MSLN CAR T (500K cells)” condition is composed of five hundred thousand (500K) T cells modified to express an exemplary inducible promoter of the disclosure controlling expression of an exemplary anti-MSLN CAR of the disclosure, an exemplary EGFR-marker of the disclosure and an exemplary targeted designed cytokine of the disclosure. In this study, the exemplary inducible promoter comprises the sequence of GGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTG GGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGG GGACTTTCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATAATGGAAGCTCGAATTC CAGACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC (SEQ ID NO: 479). In this study, the exemplary targeted designed cytokine comprises the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELKGGGGSGGGGSGGGGSE VQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGSTY YADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYD MDYWGQGTQVTVSS (SEQ ID NO: 488). The “MSLN CAR T (200K cells)” condition is composed of two hundred thousand (200K) T cells modified to express an exemplary inducible promoter of the disclosure controlling expression of an exemplary anti-MSLN CAR of the disclosure, an exemplary EGFR-marker of the disclosure and an exemplary targeted designed cytokine of the disclosure. The “MSLN CAR T (50K cells)” condition is composed of fifty thousand (50K) T cells modified to express an exemplary inducible promoter of the disclosure controlling expression of an exemplary anti-MSLN CAR of the disclosure, an exemplary EGFR- marker of the disclosure and an exemplary targeted designed cytokine of the disclosure. The data presented in this figure demonstrate that anti-MSLN CARs of the disclosure, either alone, or in combination with a designed cytokine of the disclosure, effectively reduce tumor volume in vivo. The data presented also demonstrate that the combination of an anti-MSLN CAR of the disclosure and a designed cytokine of the disclosure are a synergistic combination that increase efficacy of the combination to a supra-additive level.

[0075] FIG.15 is a pair of graphs depicting the results of a replication of the study described in FIG.14 to further validate the consistency of the results. All labels in this figure have the same meaning as in FIG.14.

[0076] FIG.16 is a series of graphs demonstrating in vivo efficacy of anti-MSLN CAR-T cells of the disclosure (derived from a single donor), against, for example, a subcutaneous graft of human MSLN+ H1650 lung cells forming a tumor in this xenograft study. For each of the 6 mice used (each plot representing a single mouse), the tumor volume (measured in cubic millimeters (mm3) was examined as a function of time (measured as days post-engraftment). All labels in this figure have the same meaning as in FIG.14.

[0077] FIG.17 is a graph demonstrating that expansion of anti-MSLN CAR-T cells of the disclosure is tumor-dependent. Expansion of CAR-T cells of the disclosure (in four (4) distinct conditions) is measured (as EGFR-marker expressing (EGFR+) cells / milliliter) as a function of days post-injection, using the xenograft model described in FIGs.14-16. “No Tumor” conditions represent a mouse lacking the subcutaneous graft of human MSLN+ H1650 lung cells to form a tumor (negative control). “Tumor bearing” conditions represent a mouse having the subcutaneous graft of human MSLN+ H1650 lung cells to form a tumor. The “CAR-only” condition describes a population of T cells modified to express an exemplary anti-MSLN CAR of the disclosure and an exemplary EGFR-marker of the disclosure. The “MSLN CAR-T” condition describes a population of T cells modified to express an exemplary inducible promoter of the disclosure controlling expression of an exemplary anti-MSLN CAR of the disclosure, an exemplary EGFR-marker of the disclosure and an exemplary targeted designed cytokine of the disclosure. As used in this study: the exemplary anti-MSLN CAR comprises a MSLN binder comprising the sequence of DIQMTQSPSTLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQWSGYPLTFGQGTKVEIKGGGGSGGGGSGGG GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMNWVRQAPGQGLEWMG LITPYNGASSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFD YWGQGTLVTVSS (SEQ ID NO: 480), and the exemplary anti-MSLN CAR comprises the sequence of DIQMTQSPSTLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQWSGYPLTFGQGTKVEIKGGGGSGGGGSGGG GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMNWVRQAPGQGLEWMG LITPYNGASSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFD YWGQGTLVTVSSVPCPVPPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQ PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 481); the EGFR-marker of the disclosure comprises the sequence of MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPV AFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQ FSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGEN SCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECI QCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADA GHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRR (SEQ ID NO: 483); the exemplary inducible promoter comprises the sequence of GGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTG GGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGG GGACTTTCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATAATGGAAGCTCGAATTC CAGACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC (SEQ ID NO: 479); and the exemplary targeted designed cytokine comprises the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELKGGGGSGGGGSGGGGSE VQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGSTY YADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYD MDYWGQGTQVTVSS (SEQ ID NO: 488). The results of this study demonstrate that expansion of the T-cell population over time occurs at statistically significant levels only in those conditions in which the mouse recipient contains the xenograft tumor.

[0078] FIG.18 is a series of graphs demonstrating that serum levels of exemplary targeted designed cytokines of the disclosure measured following treatment of xenografted mice with a population (composition) of T cells of the disclosure (derived from one of two donors) expressing both an anti-MSLN CAR-T of the disclosure and an inducible targeted designed cytokine of the disclosure decrease after tumor elimination even with high levels of engraftment of the T-cell composition. In this study and for each T-cell donor, tumor growth was determined by measuring tumor volume (in cubic millimeters) as a function of days following T-cell treatment. The data demonstrate that with tumor volume begins to decrease sharply after about 10 days post T-cell treatment. Upon activation of the T-cells by contact with the tumor, the T- cells derived from each donor and modified to express an anti-MSLN CAR-T of the disclosure and an inducible targeted designed cytokine of the disclosure, secrete the targeted designed cytokine. By measuring the serum concentration (picograms / milliliter) from the xenografted and treated mouse, the data demonstrate that not only can secretion be initiated by contact of the T- cell treatment to the tumor, but also, that secretion no longer occurs in the absence of the tumor. These data are important for showing a robust but tumor-dependent response, which improves the safety profile of anti-MSLN CAR-T cells of the disclosure that further comprises an inducible targeted designed cytokine of the disclosure. In the right column of plots, the data show that the CAR-T expansion in each condition is a result of the abundance (count in cells / milliliter) of cells expressing both the anti-MSLN CAR-T of the disclosure and the inducible targeted designed cytokine of the disclosure as a function of time. Thus, even while the T-cell abundance increases due to successful engraftment, those cells are no longer secreting the targeted designed cytokine once the tumor has been eliminated. As used in this study: the exemplary anti-MSLN CAR comprises a MSLN binder comprising the sequence of DIQMTQSPSTLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQWSGYPLTFGQGTKVEIKGGGGSGGGGSGGG GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMNWVRQAPGQGLEWMG LITPYNGASSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFD YWGQGTLVTVSS (SEQ ID NO: 480), and the exemplary anti-MSLN CAR comprises the sequence of DIQMTQSPSTLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQWSGYPLTFGQGTKVEIKGGGGSGGGGSGGG GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMNWVRQAPGQGLEWMG LITPYNGASSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFD YWGQGTLVTVSSVPCPVPPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQ PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 481); the EGFR-marker of the disclosure comprises the sequence of MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPV AFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQ FSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGEN SCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECI QCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADA GHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRR (SEQ ID NO: 483); the exemplary inducible promoter comprises the sequence of GGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTG GGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGG GGACTTTCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATAATGGAAGCTCGAATTC CAGACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC (SEQ ID NO: 479); and the exemplary targeted designed cytokine comprises the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELKGGGGSGGGGSGGGGSE VQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGSTY YADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYD MDYWGQGTQVTVSS (SEQ ID NO: 488).

[0079] FIG.19 is a series of graphs and corresponding table showing that humanized MSLN polypeptides (MSLN Polypeptide Nos 49-56) maintain similar affinity and biophysical properties as MSLN Polypeptide 48 (not humanized). Top panel: Each plot demonstrates affinity of each MSLN Polypeptide measured in nanomolar concentration of polypeptide bound to MSLN as a function of time for each MSLN Polypeptide shown. Bottom panel: Table showing quantification of physical characteristics for exemplary MSLN Polypeptides of the disclosure. Abbreviations: equilibrium-binding affinity (KD),^association constant (ka), dissociation constant (kdis), melting temperature (Tm), Phosphate Buffered Saline (PBS), and Size exclusion chromatography (SEC).

[0080] FIG.20 is a series of graphs showing a reduction in tumor volume (measured as cubic millimeters) as a function of days post tumor implant in a mouse xenograft model containing a subcutaneous tumor of human lung cancer (H1650) cells across three dosage levels (2x106cells, 1x106cells, and 0.5x 106cells) and CAR-T cells comprising one of four exemplary MSLN Polypeptides of the disclosure (MSLN Polypeptide Nos.48, 51, 54 and 56). In each CAR condition, the exemplary MSLN Polypeptide is combined with a spacer of the disclosure (Spacer No.1, comprising the sequence of VPCPVPP (SEQ ID NO: 159), without a linker sequence. Graphs show tumor volume in individual mice (5 per group). Mice dosed with indicated number of mock transduced T cells or CAR T at day 21 post tumor implant (vertical dotted line). Abbreviations: Not statistically significant (NS), * means statistical significance having a p-value of less than 0.05, ** means statistical significance having a p-value of less than 0.001 (less than 1E-3). DETAILED DESCRIPTION

[0081] The disclosure provides MSLN-binding polypeptides, receptors that comprise MSLN- binding polypeptides, nucleic acids encoding same, vectors comprising nucleic acid sequences and cells to contain or express one or more of these elements. Mesothelin (MSLN)

[0082] Mesothelin (MSLN) is a 40 kilodalton (kDa) protein expressed on the surface of mesothelial cells lining the peritoneum and binds to Mucin 16 (MUC16; also known as cancer antigen 125 (CA125)). Fig.1 shows the folded MSLN protein connected to a cell surface via a GPI-linker (linker also depicted in Fig.2). As shown in this figure, MSLN has been divided into three regions (I, II, and III). The GPI linkage begins at residue 606 of preprotein isoform 2 (see SEQ ID NO:

[0083] Within the expressed MSLN protein, Region I binds MUC16. Is GPI-anchored, unstructured region is about 19 amino acids in length, with GPI linkage at residue 606 of preproprotein isoform 2.

[0084] MSLN has three known isoforms. Isoform 1 and isoform 2 have a nine amino acid difference. Isoform 3 is shed from the cell surface. In some embodiments, distal epitopes generate better CARs than proximal for published binders.

[0085] This protein is often overexpressed on malignant cells. Evidence has linked MSLN to increased metastasis in various cancers. MSLN Polypeptides

[0086] MSLN polypeptides of the disclosure bind to one or more regions of MSLN (Regions depicted in Fig.1). In some embodiments, polypeptides of the disclosure bind to Region I of MSLN. In some embodiments, MSLN polypeptides of the disclosure numbered (1) through (26), and all polypeptides comprising the complementarity determining regions therein, bind to Region I of MSLN. In some embodiments, MSLN polypeptides of the disclosure numbered (30) through (37), and all polypeptides comprising the complementarity determining regions therein, bind to Region I of MSLN.

[0087] In some embodiments, polypeptides of the disclosure bind to Region II of MSLN. In some embodiments, polypeptides of the disclosure bind to Region III of MSLN. In some embodiments, MSLN polypeptides of the disclosure numbered (27) through (29), and all polypeptides comprising the complementarity determining regions therein, bind to Region III of MSLN.

[0088] In some embodiments, a MSLN binding polypeptide of the disclosure comprises one or more of a protein scaffold, an antibody, an antibody mimetic, and / or a functional fragment thereof. In some embodiments, a MSLN binding polypeptide of the disclosure comprises a sequence, which may be isolated or derived from any species, including but not limited to, human, non-human primate, rodent (including, but not limited to, mouse), and camelid species. In some embodiments, a targeting moiety comprises a sequence, which may be humanized, chimeric, recombinant, non-naturally occurring, modified (for example, to include synthetic nucleic acids or synthetic amino acids), optimized (for example, to reduce immunogenicity and / or aggregation during manufacturing).

[0089] In some embodiments of the disclosure, a MSLN binding polypeptide of the disclosure comprises an antibody, including, but not limited to, a monoclonal antibody, an antigen-binding fraction (Fab), a single-chain variable fraction (scFv), a domain antibody, one or more of a heavy chain (VH) and a light chain (VL) domain of an immunoglobulin (Ig) polypeptide or gene encoding the same, a heavy-chain antibody (a VH or a VHH), a camelid or camelid-like structured antibody, and a nanobody. In some embodiments, a MSLN binding polypeptide of the disclosure comprises an scFv, a VH, or a VHH.

[0090] In some embodiments of the disclosure, a MSLN binding polypeptide of the disclosure comprises an antibody mimetic, including, but not limited to, one or more of an engineered protein scaffold, a monobody, an affibody molecule, an adnectin molecule, an affimer molecule, an affitin molecule, an affilin molecule, an alphabody molecule, an anticalin molecule, an aptamer molecule, an atrimer molecule, an avimer molecule, a DARPin molecule, a fynomer, an armadillo repeat protein molecule, a Kunitz domain inhibitor molecule, a knottin molecule, a designed ankyrin repeat protein molecule, a nanofittin molecule and a centyrin molecule. Sequences Table 1

[0002] Table 2 Table 3

[0003] Table 4 Table 5A Table 5B: Exemplary Combinations of MSLN Polypeptides and Spacers Table 5C Exemplary Combinations of Spacers and Linkers Table 5D Exemplary Combinations of MSLN Polypeptides and Linkers

[0004] Table 6 Table 7

[0005] Table 8

[0006] Table 9A

[0007] Table 9B Exemplary Combinations of MSLN Polypeptides and EGFR Safety Switches (also referred to as EGFR-markers) EGFR Safety Switch

[0008]

[0091] Cells of the disclosure may be modified to express a cytokine, including but not limited to a de novo polypeptide (also referred to herein as a “designed cytokine” of the disclosure). Exemplary designed cytokines of the disclosure comprise one or more of sequences provided in Table 10 or a sequence having at least 70% identity to a sequence of provided in Table 10. Exemplary designed cytokines of the disclosure include, but are not limited to, those designed cytokines disclosed in PCT Publication No. WO 2023 / 137346, the contents of which are incorporated herein by reference in their entirety. Table 10A ^

[0092] In some embodiments of the Designed Cytokine of the disclosure, the polypeptide comprises the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK (SEQ ID NO: 459).

[0093] In some embodiments of the Designed Cytokine of the disclosure, the polypeptide comprises the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK (SEQ ID NO: 353).

[0094] In some embodiments of the Designed Cytokine of the disclosure, the polypeptide comprises the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK (SEQ ID NO: 353).

[0095] In some embodiments of the Designed Cytokine of the disclosure, the polypeptide comprises the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTLTAGGSL SGDLKHLQNLSEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK (SEQ ID NO: 418).

[0096] In some embodiments of the Designed Cytokine of the disclosure, the polypeptide comprises the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSVDPEE LAKELQKLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK (SEQ ID NO: 396).

[0097] In some embodiments of the Designed Cytokine of the disclosure, the polypeptide comprises the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLK HLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK (SEQ ID NO: 459).

[0098] In some embodiments of the Designed Cytokine of the disclosure, the polypeptide is operably linked to a targeting moiety. In some embodiments, the polypeptide comprises a targeting moiety. In some embodiments, a fusion protein comprises the polypeptide and a targeting moiety. In some embodiments, the targeting moiety binds to a component of a tumor microenvironment (TME). In some embodiments, the targeting moiety binds to one or more of T-cell surface glycoprotein CD8 (also known as cluster of differentiation 8), Programmed cell death protein 1 (PD-1), Programmed death-ligand 1 (PD-L1; also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1) polypeptide), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), Cytotoxic T-lymphocyte protein 4 (CTLA4), Lymphocyte activation gene 3 protein (LAG3), T-cell immunoglobulin mucin receptor 3 (TIM3). In some embodiments, the targeting moiety binds CD8. In some embodiments, the targeting moiety binds PD-1. In some embodiments, the targeting moiety binds PD-Ll.

[0099] In some embodiments, the targeting moiety comprises an antibody, an antibody mimetic, or a functional fragment thereof. In some embodiments, the targeting moiety comprises one or more of a monoclonal antibody, an antigen-binding fraction (Fab), a single-chain variable fraction (scFv), a domain antibody, one or more of a heavy chain (VH) and a light chain (VL) domain of an immunoglobulin (Ig) polypeptide or gene encoding the same, a heavy -chain antibody (a VH or a VHH), a camelid or camelid-like structured antibody, and a nanobody. In some embodiments, the targeting moiety comprises a scFv. In some embodiments, the targeting moiety comprises a VHH. In some embodiments, a targeting moiety comprises one or more of a monoclonal antibody, an antigen-binding fraction (Fab), a single-chain variable fraction (scFv), a domain antibody, one or more of a heavy chain (VH) and a light chain (VL) domain of an immunoglobulin (Ig) polypeptide or gene encoding the same, a heavy -chain antibody (a VH or a VHH), a camelid or camelid-like structured antibody, and a nanobody that specifically or selectively binds to a target including, but not limited to, T-cell surface glycoprotein CDS(also known as cluster of differentiation 8), Programmed cell death protein l (PD- I), Programmed death-ligand l (PD-LI; also known as cluster of differentiation 274 (CD274) or B7 homolog I (87-H1) polypeptide), T- cell immunoreceptor with Ig and ITIM domains (TIGIT), Cytotoxic T- lymphocyte protein 4 (CTLA4), Lymphocyte activation gene 3 protein (LAGJ), and T-cell immunoglobulin mucin receptor 3 (TlM3).

[0100] In some embodiments of the disclosure, a targeting moiety comprises an antibody mimetic, including, but not limited to, one or more of an engineered protein scaffold, a monobody, an affibody molecule, an adnectin molecule, an affimer molecule, an affitin molecule, an affilin molecule, an alphabody molecule, an anticalin molecule, an aptamer molecule, an atrimer molecule, an avimer molecule, a DARPin molecule, a fynomer, an armadillo repeat protein molecule, a Kunitz domain inhibitor molecule, a knottin molecule, a designed ankyrin repeat protein molecule, a nanofittin molecule and a centyrin molecule. In some embodiments, a targeting moiety comprises an antibody mimetic that specifically or selectively binds to a target including, but not limited to, T-cell surface glycoprotein CDS(also known as cluster of differentiation 8), Programmed cell death protein l (PD- I), Programmed death-ligand l (PD-LI; also known as cluster of differentiation 274 (CD274) or B7 homolog I (87-H1) polypeptide), T- cell immunoreceptor with Ig and ITIM domains (TIGIT), Cytotoxic T- lymphocyte protein 4 (CTLA4), Lymphocyte activation gene 3 protein (LAGJ), and T-cell immunoglobulin mucin receptor 3 (TIM3).

[0101] In some embodiments, Designed Cytokines described herein include a targeting moiety that includes any one of the binders as described in Table 10B: Table 10B

[0009]

[0102] In some embodiments, the targeting moiety binds CD8 and comprises a VHH. In some embodiments, the targeting moiety binds CD8 and comprises a humanized VHH.

[0103] In some embodiments, Designed Cytokines and polypeptides of the disclosure, include a targeting moiety comprising any one of the sequences of Table 10C.

[0104] In some embodiments, targeting moieties of the disclosure comprise a humanized VHH that specifically binds to CD8. In preferred embodiments, targeting moieties of the disclosure comprise a humanized VHH provided in Table 10C. Table 10C ANTI-CD8a Humanized VHH Sequences

[0010] Table 10D: Exemplary Combinations of Designed Cytokines and CD8 Binders

[0011] 2232X X X X X X X X X X X X X X X X X X X242X X X X X X X X X X X X X X X X X X X252X X X X X X X X X X X X X X X X X X X262X X X X X X X X X X X X X X X X X X X272X X X X X X X X X X X X X X X X X X X282X X X X X X X X X X X X X X X X X X X292X X X X X X X X X X X X X X X X X X X2b49X X X X X X X X X X X X X X X X X X X 1

[0012] 3213X X X X X X X X X X X X X X X X X X X223X X X X X X X X X X X X X X X X X X X233X X X X X X X X X X X X X X X X X X X243X X X X X X X X X X X X X X X X X X X253X X X X X X X X X X X X X X X X X X X263X X X X X X X X X X X X X X X X X X X273X X X X X X X X X X X X X X X X X X X283X X X X X X X X X X X X X X X X X X X2 ^

[0013] Table 10D (Continued): Exemplary Combinations of Designed Cytokines and CD8 Binders

[0014] a29X X X X X X X X X 1 a39X X X X X X X X X 1 01X X X X X X X X X2b29X X X X X X X X X 1 b39X X X X X X X X X 1 a49X X X X X X X X X 1 59X X X X X X X X X1a69X X X X X X X X X 1 a79X X X X X X X X X 1 a89X X X X X X X X X 1

[0015] ^

[0016] Table 10D (Continued): Exemplary Combinations of Designed Cytokines and CD8 Binders Table 10E: Exemplary Combinations of Designed Cytokines and MSLN Polypeptides Table 10E (Continued): Exemplary Combinations of Designed Cytokines and MSLN Polypeptides

[0017] Table 10E (Continued): Exemplary Combinations of Designed Cytokines and MSLN Polypeptides

[0018] Table 10E (Continued): Exemplary Combinations of Designed Cytokines and MSLN Polypeptides Table 10F: Exemplary Combinations of MSLN Polypeptides and CD8 Binders

[0105] Table 10F (Continued): Exemplary Combinations of MSLN Polypeptides and CD8 Binders

[0019] Table 10G: Exemplary Combinations of MSLN Polypeptides and Promoters

[0020] Table 10H: Exemplary Combinations of Designed Cytokines and Promoters

[0021] Nucleic Acids

[0106] In some embodiments of the disclosure, the terms “Nucleic acid,” “nucleic acid molecule,” “nucleotide,” “nucleotide sequence,” “polynucleotide,” and grammatical variants thereof are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Single stranded nucleic acid sequences refer to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.

[0107] In some embodiments of the disclosure, “Nucleic acid,” and in particular a DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. In some embodiments of the disclosure, “Nucleic acid,” includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes. In discussing the structure of a particular double-stranded DNA molecules, sequences are provided according to the normal convention of writing the sequence left to right in the 5’ to 3’ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the messenger RNA or mRNA). Unless otherwise indicated, all nucleic acid and nucleotide sequences are written left to right in 5’ to 3’ orientation.

[0108] Nucleotides are referred to by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, ‘A’ represents adenine, ‘C’ represents cytosine, ‘G’ represents guanine, ‘T’ represents thymine, and ‘U’ represents uracil.

[0109] In some embodiments of the disclosure, the term “Polynucleotide” refers to polymers of nucleotides of any length or type, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”). It also includes modified, for example by alkylation and / or by capping, and unmodified forms of the polynucleotide. More particularly, “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose) and polyribonucleotides (containing D-ribose), including mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing nucleotide backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.

[0110] In some embodiments of the disclosure, a polynucleotide comprises a DNA sequence. In some embodiments of the disclosure, a polynucleotide comprises a DNA sequence inserted in a vector or a vector comprising a DNA sequence.

[0111] In some embodiments of the disclosure, a polynucleotide comprises an mRNA. In some embodiments, the mRNA is a synthetic mRNA or the mRNA comprises a synthetic nucleotide.

[0112] In some embodiments of the disclosure, a polynucleotide comprises at least one unnatural, non-naturally occurring or modified nucleic acid. In some embodiments, the polynucleotide comprises a plurality of unnatural, non-naturally occurring or modified nucleic acids. In some embodiments, all nucleic acids of a certain class are unnatural, non-naturally occurring or modified nucleic acids (e.g., all uridines in a polynucleotide can be replaced with an unnatural nucleobase, e.g., 5-methoxy uridine).

[0113] In some embodiments of the disclosure, “expression” refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0114] In some embodiments of the disclosure, “expression vector” refers to a plasmid, virus, or other nucleic acid designed for polypeptide expression in a cell. The vector or construct is used to introduce a gene into a host cell whereby the vector will interact with polymerases in the cell to express the protein encoded in the vector / construct. The expression vector may exist in the cell extrachromosomally or may be integrated into the chromosome. Expression vectors may include additional sequences which render the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). The polynucleotides of the disclosure may be provided as components of expression vectors.

[0115] In some embodiments of the disclosure, “cloning vector” refers to a plasmid, virus, or other nucleic acid designed for producing copies of a polynucleotide. Cloning vectors may contain transcription and translation initiation sequences, transcription and translation termination sequences and a polyadenylation signal. Such constructs will typically include a 5ƍ LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3ƍ LTR or a portion thereof. The polynucleotides of the disclosure may be provided as components of cloning vectors, which may be used to produce the polynucleotides of the disclosure.

[0116] In some embodiments of the disclosure, “encoding” or the like refers to the capacity of specific sequences of nucleotides in a polynucleotide (e.g. a gene, cDNA, or mRNA) to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0117] Unless otherwise specified, a nucleotide sequence “encoding an amino acid sequence,” e.g., a polynucleotide “encoding” a chimeric polypeptide, defined below of the present disclosure, includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Polypeptides

[0118] Amino acids are referred to by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. The amino acid residues are abbreviated as follows, where the abbreviations are shown in parentheses: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0119] Amino acid sequences are written left to right in amino to carboxy orientation.

[0120] In some embodiments of the disclosure, “Polypeptide” may refer to a sequence of amino acid subunits. In some embodiments, a “peptide” can be less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. “Polypeptide,” refers to proteins, polypeptides, and peptides of any length, size, structure, or function. “Polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acids of any length.

[0121] Polypeptides of the disclosure may comprise naturally or synthetically created or modified amino acids, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides in which one or more amino acid residues are artificial chemical analogs of a corresponding naturally occurring amino acid (including, for example, synthetic amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. Polypeptides also include gene products, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may comprise a single polypeptide or can be a multi-molecular complex such as a dimer, trimer, or tetramer. Polypeptides of the disclosure may comprise single-chain or multi-chain polypeptides. Most commonly disulfide linkages are found in multi-chain polypeptides.

[0122] The polypeptides of the disclosure may comprise L-amino acids + glycine, D-amino acids + glycine (which are resistant to L-amino acid-specific proteases in vivo), or a combination of D- and L-amino acids + glycine. Polypeptides described may be chemically synthesized or recombinantly expressed.

[0123] The polypeptides of the disclosure can include additional residues at the N-terminus, C- terminus, internal to the polypeptide, or a combination thereof; these additional residues are not included in determining the percent identity of the polypeptides of the disclosure relative to the reference polypeptide. Such residues may be any residues suitable for an intended use, including but not limited to tags.

[0124] In some embodiments of the disclosure, “chimeric polypeptide” may refer to any polypeptide comprised of a first amino acid sequence derived from a first source, bonded, covalently, or noncovalently, to a second amino acid sequence derived from a second source, wherein the first and second source are not the same. In some embodiments, a first source and a second source that are not the same can include two different biological entities, or two different proteins from the same biological entity, or a biological entity and a non-biological entity. A chimeric protein can include for example, a protein derived from at least 2 different biological sources. In some embodiments, the chimeric polypeptide may include sequences from similar proteins derived from two distinct species. In some embodiments, the chimeric polypeptide may include sequences from dissimilar proteins derived from the same species. A biological source can include any non-synthetically produced nucleic acid or amino acid sequence (e.g. a genomic or cDNA sequence, a plasmid or viral vector, a native virion or a mutant or analog of any of the above). A synthetic source can include a protein or nucleic acid sequence produced chemically and not by a biological system (e.g. solid phase synthesis of amino acid sequences). A chimeric protein can also include a protein derived from at least 2 different synthetic sources or a protein derived from at least one biological source and at least one synthetic source. A chimeric protein may also comprise a first amino acid sequence derived from a first source, covalently or noncovalently linked to a nucleic acid, derived from any source or a small organic or inorganic molecule derived from any source. The chimeric protein can comprise a linker molecule between the first and second amino acid sequence or between the first amino acid sequence and the nucleic acid, or between the first amino acid sequence and the small organic or inorganic molecule.

[0125] In some embodiments of the disclosure, a “fragment” of a polypeptide, or a “truncated polypeptide” may refer to an amino acid sequence of a polypeptide that is shorter than the sequence of a reference polypeptide (which may be a naturally-occurring sequence). In comparison to the reference polypeptide, the fragment may comprise an N- and / or C-terminal deletion. In comparison to the reference polypeptide, the fragment may comprise a deletion of any part of the sequence, whether or not the deletion is contiguous. A polypeptide in which internal amino acids have been deleted with respect to the naturally occurring sequence is also considered a fragment. The various polypeptide components of the disclosure may be provided as fragments or truncated versions of a reference protein.

[0126] In some embodiments of the disclosure, a “functional fragment” may refer to a polypeptide fragment that retains a function of the polypeptide. In some embodiments, a functional fragment of a bioactive peptide (e.g., an enzyme), retains the ability to catalyze a biological action because the functional fragment comprises a catalytic domain of the enzyme. Polypeptides of the disclosure may be provided as functional fragments or truncated versions.

[0127] In some embodiments of the disclosure, “amino acid substitution” may refer to replacing an amino acid residue present in a parent or reference sequence with another amino acid residue. In some embodiments, the parent or reference sequence comprises a wildtype sequence. An amino acid can be substituted, for example, via chemical peptide synthesis or through recombinant methods known in the art. For example, substituting an amino acid residue with an alternative amino acid residue is conducted by substituting the codon encoding the first amino acid with a codon encoding the second amino acid. Polypeptides of the disclosure may be provided with one or more amino acid substitutions.

[0128] In some embodiments of the disclosure, a “conservative amino acid substitution” is one in which one amino acid residue is replaced with an amino acid residue having a chemically similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), 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). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In some embodiments, a string of amino acids can be conservatively replaced with a chemically similar string that differs in order and / or composition of side chain family members. The various polypeptide components of the disclosure may be provided with conservative amino acid substitutions.

[0129] In some embodiments of the disclosure, non-conservative amino acid substitutions include those in which (i) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, Ile, Phe or Val), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly). The various polypeptide components of the disclosure may be provided with non-conservative amino acid substitutions. The likelihood that one of the foregoing non-conservative substitutions can alter functional properties of the protein is also correlated to the position of the substitution with respect to functionally important regions of the protein: some non-conservative substitutions can accordingly have little or no effect on biological properties. The various polypeptide components of the disclosure may be provided with non-conservative amino acid substitutions that do not significantly alter the functionality of the altered components. Sequence Analyses

[0130] In some embodiments of the disclosure, “identity” refers to the overall monomer conservation between polymeric molecules, e.g., between polypeptide molecules or polynucleotide molecules. “Identical” without any additional qualifiers, e.g., protein A is identical to protein B, implies the sequences are 100% identical (100% sequence identity). Describing two sequences as, e.g., “70% identical,” is equivalent to describing them as having, e.g., “70% sequence identity.”

[0131] When a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0132] In certain embodiments, the percentage identity (%ID) of a first amino acid (or nucleic acid) sequence to a second amino acid (or nucleic acid) sequence is calculated as %ID = 100 (Y / Z), where Y is the number of amino acid (or nucleobase) residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0133] Calculation of the percent identity of two polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes. For example, gaps can be introduced in one or both of a first and a second polypeptide sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes. In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The amino acids at corresponding amino acid positions are then compared.

[0134] Generation of a sequence alignment for the calculation of a percent sequence identity is not limited to binary sequence-sequence comparisons exclusively driven by primary sequence data. It will also be appreciated that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structures), functional data (e.g., location of mutations), or phylogenetic data. A suitable program that integrates heterogeneous data to generate a multiple sequence alignment is T-Coffee, available at www.tcoffee.org, and alternatively available, e.g., from the European Bioinformatics Institute (EBI) at website ebi.ac.uk / Tools / psa. It will also be appreciated that the final alignment used to calculate percent sequence identity can be curated either automatically or manually.

[0135] Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of programs available from the U.S. government’s National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the EBI. Sequence alignments can be conducted using methods known in the art such as MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE, etc. Different regions within a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, values from 80.11 to 80.14 are rounded down to 80.1, while values from 80.15 to 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.

[0136] In some embodiments of the disclosure, “non-naturally occurring” means a polypeptide or a polynucleotide sequence that does not exist in nature. In some embodiments, the non- naturally occurring sequence does not exist in nature because the sequence is altered relative to a naturally occurring sequence. In some embodiments, the non-naturally occurring sequence does not exist in nature because it is a combination of two known, naturally-occurring, sequences (e.g., chimeric polypeptide) that do not occur together in nature. In some embodiments, a non- naturally occurring polypeptide is a chimeric polypeptide. In some embodiments, a polypeptide or a polynucleotide is not naturally occurring because the sequence contains a portion (e.g., a fragment) that cannot be found in nature, i.e., a novel sequence. Any of the polynucleotides described herein may be provided as non-naturally occurring sequences, e.g., having sequences which are altered relative to native sequences or provided as polynucleotides which are linked to other polynucleotides in a manner that does not exist in nature. Any of the polypeptides described herein may be provided as non-naturally occurring sequences, e.g., having sequences which are altered relative to native sequences or provided as polypeptides which are linked to other polypeptides in a manner that does not exist in nature. Therapeutic Methods

[0137] In some embodiments of the disclosure, the term “therapeutically effective” may refer to imparting a beneficial effect on the recipient, e.g., providing some alleviation, mitigation, or decrease in at least one clinical symptom in the subject. Therapeutic effects of the disclosure need not be complete or curative, as long as some benefit is provided to the subject. For example, a therapeutic regimen that incorporates the polynucleotides, gene therapy vectors or cells of the disclosure with the small molecules of the disclosure may be structured such that the regimen is therapeutically effective as a whole.

[0138] In some embodiments of the disclosure, the term “therapeutically effective amount” refers to a dose or an amount of a nucleic acid, vector, polypeptide, composition, pharmaceutical composition or cell of the disclosure sufficient to impart a therapeutically effective benefit on the recipient. For example, polynucleotides, gene therapy vectors or cells of the disclosure may be administered in a therapeutically effective amount. A subject who has been administered polynucleotides, gene therapy vectors or cells of the disclosure may subsequently be administered a therapeutically effective amount of a small molecule of the disclosure, i.e., an amount sufficient to impart a beneficial effect on the recipient given the previous administration of polynucleotides, gene therapy vectors or cells. Cell Therapy

[0139] In some embodiments of the disclosure, the term “stem cell” may refer to an undifferentiated or partially differentiated cell that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell.

[0140] In some embodiments of the disclosure, the term “Pluripotent stem cell” (PSC) may refer to a cell that can maintain an undifferentiated state indefinitely and can differentiate into most, if not all cells of the body.

[0141] In some embodiments of the disclosure, the term “Induced pluripotent stem cell” (iPS or iPSC) may refer to a pluripotent stem cell that can be generated directly from a somatic cell. This includes, but is not limited to, specialized cells such as skin or blood cells derived from an adult.

[0142] In some embodiments of the disclosure, the term “multipotent” may refer to a cell that can develop into more than one cell type but is more limited than a pluripotent cell. For example, adult stem cells and cord blood stem cells may be considered as multipotent.

[0143] In some embodiments of the disclosure, the term “hematopoietic cell” may refer to a cell that arises from a hematopoietic stem cell (HSC). Hematopoietic cells of the disclosure include, but is not limited to, myeloid progenitor cells, lymphoid progenitor cells, megakaryocytes, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, macrophages, thrombocytes, monocytes, natural killer cells, T lymphocytes, B lymphocytes and plasma cells.

[0144] In some embodiments of the disclosure, the term “T-lymphocyte” or “T-cell” may refer to a hematopoietic cell that normally develops in the thymus. T-lymphocytes or T-cells include, but are not limited to, natural killer T cells, regulatory T cells, helper T cells, cytotoxic T cells, memory T cells, gamma delta T cells, and mucosal invariant T cells.

[0145] In some embodiments of the disclosure, the term “mesenchyme” may refer to a type of animal tissue comprising loose cells embedded in a mesh of proteins and fluid, i.e., the extracellular matrix. Mesenchyme directly gives rise to most of the body’s connective tissues including bones, cartilage, lymphatic system, and circulatory system.

[0146] In some embodiments of the disclosure, the term “mesenchymal cell” may refer to a cell that is derived from a mesenchymal tissue. In some embodiments, cells of the disclosure may be mesenchymal cells.

[0147] In some embodiments of the disclosure, the term “mesenchymal stromal cell” (MSC) may refer to a spindle shaped plastic-adherent cell isolated from bone marrow, adipose, and other tissue sources, with multipotent differentiation capacity in vitro. For example, a mesenchymal stromal cell can differentiate into osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells), and adipocytes (fat cells which give rise to marrow adipose tissue). The term mesenchymal stromal cell is suggested in the scientific literature to replace the term “mesenchymal stem cell”. In some cases, cells of the disclosure may be mesenchymal stromal cells.

[0148] In some embodiments of the disclosure, an “autologous cell” is a cell obtained from the same individual to whom it may be administered as a therapy (the cell is autologous to the subject). Autologous cells of the disclosure include, but are not limited to, hematopoietic cells and stem cells, such as hematopoietic stem cells.

[0149] In some embodiments of the disclosure, an allogeneic cell is a cell obtained from an individual who is not the intended recipient of the cell as a therapy (the cell is allogeneic to the subject). Allogeneic cells of the disclosure may be selected from immunologically compatible donors with respect to the subject of the methods of the disclosure. Allogeneic cells of the disclosure may be modified to produce “universal” allogeneic cells, suitable for administration to any subject without unintended immunogenicity. Allogeneic cells of the disclosure include, but are not limited to, hematopoietic cells and stem cells, such as hematopoietic stem cells.

[0150] In some embodiments of the disclosure, the term “Transfect” or “transform” or “transduce” may refer to a process by which exogenous nucleic acid is transferred or introduced into a host cell. In some embodiments, a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid or progeny of the cell.

[0151] In some embodiments of the disclosure, the term “Cell therapy” may refer to the provision or delivery of cells into a recipient for therapeutic purposes. Formulations

[0152] In some embodiments of the disclosure, “pharmaceutically acceptable” refers to compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio. For example, the small molecules, polynucleotides, polypeptides, gene therapy vectors or cells of the disclosure may be administered as part of a composition together with other pharmaceutically acceptable components, including pharmaceutically acceptable carriers.

[0153] In some embodiments of the disclosure, the term “pharmaceutically acceptable salts” refers to derivatives of the small molecules of the disclosure wherein the specified compound is converted to an acid or base salt thereof. Such pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluensulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, and the like. For example, the small molecules of the disclosure may be provided as pharmaceutically acceptable salts.

[0154] In some embodiments of the disclosure, the term “excipients” refer to pharmacologically inert ingredients that are not active in the body. See, for example, Hancock, B. C., Moss, G. P., and Goldfarb, D. J. (2020). Handbook of pharmaceutical excipients. London: Pharmaceutical Press, the entire disclosure of which is incorporated herein by reference. The small molecules of the disclosure may be mixed with pharmaceutically acceptable carriers, diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, polymers, disintegrating agents, glidants, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, lubricating agents, acidifying agents, and dispensing agents, depending on the nature of the mode of administration and dosage forms. Such ingredients, including pharmaceutically acceptable carriers and excipients that may be used to formulate oral dosage forms. Pharmaceutically acceptable carriers include water, ethanol, polyols, vegetable oils, fats, waxes polymers, including gel forming and non-gel forming polymers, and suitable mixtures thereof. Examples of excipients include starch, pregelatinized starch, Avicel, lactose, milk sugar, sodium citrate, calcium carbonate, dicalcium phosphate, and lake blend. Examples of disintegrating agents include starch, alginic acids, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc, as well as high molecular weight polyethylene glycols. For example, the small molecules, polynucleotides, gene therapy vectors or cells of the disclosure may be provided and administered in compositions that include pharmaceutically acceptable excipients. Definitions

[0155] In some embodiments of the disclosure, the term “subject” refers to any mammal, including without limitation, humans.

[0156] The terms "a", "an" and "the" include their plural forms unless the context clearly dictates otherwise.

[0157] The term “and” is used interchangeably with “or” unless expressly stated otherwise.

[0158] The term "And / or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, "and / or" as used in a phrase such as "A and / or B," includes "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, "and / or," as used in a phrase such as "A, B, and / or C," is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0159] Numeric ranges are inclusive of the numbers defining the range. Where a range of values is stated, each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, as is each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the disclosure. Thus, ranges are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0160] Where a value is explicitly stated, it is to be understood that values which are about the same quantity or amount as the stated value are also within the scope of the disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of a disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of a disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0161] Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0162] Singular or plural words also include the plural and singular number, respectively. Thus, for example, where the specification describes a gene of interest, the disclosure includes polynucleotides with a single gene of interest or multiple genes of interest.

[0163] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form.

[0164] Headings are included herein for reference and to aid in locating the various sections. These headings are not intended to limit the scope of the concepts described with respect to the headings. Such concepts may have applicability throughout the present specification.

Claims

CLAIMS What is claimed is:

1. A polypeptide comprising: (a) a complementarity determining region 1 (CDR1) comprising one or more of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, and 85; (b) a complementarity determining region 2 (CDR2) comprising one or more of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, and 86; and (c) a complementarity determining region 3 (CDR3) comprising one or more of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84 and 87.

2. The polypeptide of claim 1, comprising: (1) (a) a CDR1 comprising SEQ ID NO: 1 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 2 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 3 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (2) (a) a CDR1 comprising SEQ ID NO: 4 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 5 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 6 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (3) (a) a CDR1 comprising SEQ ID NO: 7 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 8 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 9 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (4) (a) a CDR1 comprising SEQ ID NO: 10 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 11 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 12 or a sequence having at least 90% thereto;wherein the polypeptide specifically binds to mesothelin (MSLN); (5) (a) a CDR1 comprising SEQ ID NO: 13 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 14 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 15 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (6) (a) a CDR1 comprising SEQ ID NO: 16 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 17 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 18 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (7) (a) a CDR1 comprising SEQ ID NO: 19 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 20 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 21 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (8) (a) a CDR1 comprising SEQ ID NO: 22 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 23 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 24 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (9) (a) a CDR1 comprising SEQ ID NO: 25 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 26 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 27 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (10) (a) a CDR1 comprising SEQ ID NO: 28 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 29 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 30 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (11) (a) a CDR1 comprising SEQ ID NO: 31 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 32 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 33 or a sequence having at least 90% thereto;wherein the polypeptide specifically binds to mesothelin (MSLN); (12) (a) a CDR1 comprising SEQ ID NO: 34 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 35 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 36 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (13) (a) a CDR1 comprising SEQ ID NO: 37 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 38 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 39 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (14) (a) a CDR1 comprising SEQ ID NO: 40 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 41 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 42 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (15) (a) a CDR1 comprising SEQ ID NO: 43 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 44 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 45 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (16) (a) a CDR1 comprising SEQ ID NO: 46 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 47 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 48 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (17) (a) a CDR1 comprising SEQ ID NO: 49 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 50 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 51 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (18) (a) a CDR1 comprising SEQ ID NO: 52 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 53 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 54 or a sequence having at least 90% thereto;wherein the polypeptide specifically binds to mesothelin (MSLN); (19) (a) a CDR1 comprising SEQ ID NO: 55 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 56 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 57 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (20) (a) a CDR1 comprising SEQ ID NO: 58 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 59 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 60 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (21) (a) a CDR1 comprising SEQ ID NO: 61 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 62 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 63 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (22) (a) a CDR1 comprising SEQ ID NO: 64 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 65 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 66 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (23) (a) a CDR1 comprising SEQ ID NO: 67 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 68 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 69 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (24) (a) a CDR1 comprising SEQ ID NO: 70 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 71 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 72 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (25) (a) a CDR1 comprising SEQ ID NO: 73 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 74 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 75 or a sequence having at least 90% thereto;wherein the polypeptide specifically binds to mesothelin (MSLN); (26) (a) a CDR1 comprising SEQ ID NO: 76 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 77 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 78 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (27) (a) a CDR1 comprising SEQ ID NO: 79 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 80 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 81 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); (28) (a) a CDR1 comprising SEQ ID NO: 82 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 83 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 84 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN); or (29) (a) a CDR1 comprising SEQ ID NO: 85 or a sequence having at least 90% thereto; (b) a CDR2 comprising SEQ ID NO: 86 or a sequence having at least 90% thereto; and (c) a CDR3 comprising SEQ ID NO: 87 or a sequence having at least 90% thereto; wherein the polypeptide specifically binds to mesothelin (MSLN).

3. The polypeptide of claim 2, comprising: (1) a sequence comprising SEQ ID NO: 88 or a sequence having at least 70% thereto; (2) a sequence comprising SEQ ID NO: 89 or a sequence having at least 70% thereto; (3) a sequence comprising SEQ ID NO: 90 or a sequence having at least 70% thereto; (4) a sequence comprising SEQ ID NO: 91 or a sequence having at least 70% thereto; (5) a sequence comprising SEQ ID NO: 92 or a sequence having at least 70% thereto; (6) a sequence comprising SEQ ID NO: 93 or a sequence having at least 70% thereto; (7) a sequence comprising one or more of SEQ ID NO: 94 or 117-128, or a sequence having at least 70% thereto; (8) a sequence comprising SEQ ID NO: 95 or a sequence having at least 70% thereto; (9) a sequence comprising SEQ ID NO: 96 or a sequence having at least 70% thereto; (10) a sequence comprising SEQ ID NO: 97 or a sequence having at least 70% thereto; (11) a sequence comprising SEQ ID NO: 98 or a sequence having at least 70% thereto;(12) a sequence comprising SEQ ID NO: 99 or a sequence having at least 70% thereto; (13) a sequence comprising SEQ ID NO: 100 or a sequence having at least 70% thereto; (14) a sequence comprising SEQ ID NO: 101 or a sequence having at least 70% thereto; (15) a sequence comprising SEQ ID NO: 102 or a sequence having at least 70% thereto; (16) a sequence comprising SEQ ID NO: 103 or a sequence having at least 70% thereto; (17) a sequence comprising SEQ ID NO: 104 or a sequence having at least 70% thereto; (18) a sequence comprising SEQ ID NO: 105 or a sequence having at least 70% thereto; (19) a sequence comprising SEQ ID NO: 106 or a sequence having at least 70% thereto; (20) a sequence comprising SEQ ID NO: 107 or a sequence having at least 70% thereto; (21) a sequence comprising SEQ ID NO: 108 or a sequence having at least 70% thereto; (22) a sequence comprising SEQ ID NO: 109 or a sequence having at least 70% thereto; (23) a sequence comprising SEQ ID NO: 110 or a sequence having at least 70% thereto; (24) a sequence comprising one or more of SEQ ID NO: 111 or 129-134, or a sequence having at least 70% thereto; (25) a sequence comprising SEQ ID NO: 112 or a sequence having at least 70% thereto; (26) a sequence comprising SEQ ID NO: 113 or a sequence having at least 70% thereto; (27) a sequence comprising SEQ ID NO: 114 or a sequence having at least 70% thereto; (28) a sequence comprising SEQ ID NO: 115 or a sequence having at least 70% thereto; or (29) a sequence comprising SEQ ID NO: 116 or a sequence having at least 70% thereto.

4. The polypeptide of any one of claims 1-3, wherein a VHH comprises the polypeptide.

5. The polypeptide of any one of claims 1-4, wherein the polypeptide does not comprise one or more of polypeptide (4), polypeptide (10), polypeptide (11), polypeptide (12), polypeptide (15) and polypeptide (20).

6. A polypeptide comprising: a light chain variable region comprising: (a) a CDR1 comprising SASSSVSYMH (SEQ ID NO: 135); (b) a CDR2 comprising RWIYDTSKLAS (SEQ ID NO: 136); and (c) a CDR3 comprising QQWSGYPLT (SEQ ID NO: 137); and a heavy chain variable region comprising: (d) a CDR1 comprising GYSFTGYTMN (SEQ ID NO: 138); (e) a CDR2 comprising IGLITPYNGASS (SEQ ID NO: 139); and (f) a CDR3 comprising ARGGYDGRGFDY (SEQ ID NO: 140);wherein the light chain comprises a sequence having at least 70% identity to SEQ ID NO: 141 and wherein the heavy chain comprises a sequence having at least 70% identity to SEQ ID NO:

142.

7. A polypeptide comprising (30) a light chain variable region comprising SEQ ID NO: 143 and a heavy chain variable region comprising SEQ ID NO: 144; (31) a light chain variable region comprising SEQ ID NO: 145 and a heavy chain variable region comprising SEQ ID NO: 146; (32) a light chain variable region comprising SEQ ID NO: 147 and a heavy chain variable region comprising SEQ ID NO: 148; (33) a light chain variable region comprising SEQ ID NO: 149 and a heavy chain variable region comprising SEQ ID NO: 150; (34) a light chain variable region comprising SEQ ID NO: 151 and a heavy chain variable region comprising SEQ ID NO: 152; (35) a light chain variable region comprising SEQ ID NO: 153 and a heavy chain variable region comprising SEQ ID NO: 154; (36) a light chain variable region comprising SEQ ID NO: 155 and a heavy chain variable region comprising SEQ ID NO: 156; and (37) a light chain variable region comprising SEQ ID NO: 157 and a heavy chain variable region comprising SEQ ID NO:

158.

8. The polypeptide of any one of claims 1-7, wherein the polypeptide has been modified to decrease immunogenicity.

9. The polypeptide of any one of claims 1-8, wherein the polypeptide has been modified to increase stability.

10. The polypeptide of any one of claims 1-9, wherein the polypeptide has been humanized.

11. The polypeptide of any one of claims 1-10, wherein the polypeptide further comprises a spacer sequence.

12. The polypeptide of claim 11, wherein the spacer sequence comprises (a) one or more of SEQ ID NOs: 159-177 (b) a sequence having at least 90% identity to a sequence of (a), or (c) a sequence producing a protein having a length of between 25 and 250 angstroms, inclusive of the endpoints.

13. The polypeptide of claim 11, wherein the spacer sequence comprises (a) SEQ ID NOs: 164, (b) a sequence having at least 90% identity to a sequence of (a), or (c) a sequence producing a protein having a length of between 40 and 50 angstroms, inclusive of the endpoints.

14. The polypeptide of claim 11, wherein the spacer sequence comprises (a) SEQ ID NOs: 165, (b) a sequence having at least 90% identity to a sequence of (a), or (c) a sequence producing a protein having a length of between 50 and 60 angstroms, inclusive of the endpoints.

15. The polypeptide of claim 11, wherein the spacer sequence comprises (a) SEQ ID NOs: 166, (b) a sequence having at least 90% identity to a sequence of (a), or (c) a sequence producing a protein having a length of between 50 and 60 angstroms, inclusive of the endpoints.

16. The polypeptide of claim 11, wherein the spacer sequence comprises (a) SEQ ID NOs: 167, (b) a sequence having at least 90% identity to a sequence of (a), or(c) a sequence producing a protein having a length of between 60 and 70 angstroms, inclusive of the endpoints.

17. The polypeptide of claim 11, wherein the spacer sequence comprises (a) SEQ ID NOs: 168, (b) a sequence having at least 90% identity to a sequence of (a), or (c) a sequence producing a protein having a length of between 70 and 80 angstroms, inclusive of the endpoints.

18. The polypeptide of any one of claims 11-17, wherein the spacer sequence further comprises a linker sequence.

19. The polypeptide of claim 18, wherein the linker sequence comprises one or more of a small molecule, a DNA sequence, an RNA sequence, and an amino acid sequence.

20. The polypeptide of claim 18 or 19, wherein the linker sequence comprises an amino acid sequence.

21. The polypeptide of any one of claims 18-20, wherein the linker comprises a Glycine (G) and / or a Serine (S).

22. The polypeptide of any one of claims 18-21, wherein the linker comprises GGGSG (SEQ ID NO: 178).

23. The polypeptide of any one of claims 18-22, wherein the linker has a length of between 15 and 20 Angstroms.

24. The polypeptide of any one of claims 18-22, wherein the linker has a length of about 18 Angstroms.

25. A chimeric receptor comprising a polypeptide of any one of claims 1-24.

26. The chimeric receptor of claim 25, wherein the chimeric receptor is a Chimeric Antigen Receptor (CAR).

27. The chimeric receptor of claim 26, wherein the CAR comprises (a) an extracellular domain comprising the polypeptide of any one of claims 1-24, (b) a transmembrane domain, and (c) an intracellular domain.

28. The chimeric receptor of claim 27, wherein the transmembrane domain comprises a sequence isolated or derived from a sequence encoding human CD3-zeta, human CD4, human CD8, human CD28 and / or human ICOS.

29. The chimeric receptor of claim 27 or 28, wherein the intracellular domain comprises a sequence isolated or derived from a human CD3-zeta sequence.

30. The chimeric receptor of any one of claims 27-29, wherein the intracellular domain further comprises at least one co-stimulatory domain.

31. The chimeric receptor of any one of claims 27-30, wherein the at least one co- stimulatory domain comprises a sequence isolated or derived from a human CD28 sequence.

32. The chimeric receptor of any one of claims 27-31, wherein the at least one co- stimulatory domain comprises a sequence isolated or derived from a human 41BB sequence or an OX40 sequence.

33. The chimeric receptor of any one of claims 27-32, wherein the intracellular domain comprises a sequence isolated or derived from a cytokine inducer.

34. The chimeric receptor of claim 33, wherein the cytokine inducer comprises an IL-12 cytokine inducer.

35. The chimeric receptor of claim 33 or 34, wherein the cytokine inducer binds to a sequence isolated or derived from a Nuclear factor of activated T cells (NFAT) sequence.

36. The chimeric receptor of any one of claims 27-32, wherein the intracellular domain comprises a sequence isolated or derived from an IL-2 receptor (IL-2R).

37. The chimeric receptor of claim 36, wherein the IL-2R comprises IL-2R beta.

38. A nucleic acid sequence encoding the polypeptide of any one of claims 1-24 or the chimeric receptor of any one of claims 25-37.

39. The nucleic acid sequence of claim 38 comprising a regulatory sequence capable of driving expression of the nucleic acid sequence in a mammalian cell.

40. The nucleic acid sequence of claim 39, wherein the regulatory sequence comprises one or more of a promoter, a response element, an enhancer and any combination thereof.

41. The nucleic acid sequence of claim 39 or 40, wherein the regulatory sequence comprises two or more response elements or repeated response elements, optionally, arranged in tandem.

42. The nucleic acid sequence of any one of claims 39-41, wherein the regulatory sequence comprises a constitutive promoter.

43. The nucleic acid sequence of any one of claims 39-41, wherein the regulatory sequence comprises an inducible promoter.

44. The nucleic acid sequence of any one of claims 39-43, wherein the regulatory sequence comprises a minimal promoter.

45. The nucleic acid sequence of any one of claims 39-44, wherein the regulatory sequence comprises a promoter comprising one or more transcription factor binding motifs.

46. The nucleic acid sequence of any one of claims 39-45, wherein the regulatory sequence comprises a promoter comprising one or more concatemerized sequences or motifs.

47. A vector comprising the nucleic acid of any one of claims 38-46.

48. The vector of claim 47, wherein the vector is an expression vector.

49. The vector of claim 47, wherein the vector is a delivery vector.

50. The vector of any one of claims 47-49, wherein the vector is a non-viral vector.

51. The vector of any one of claims 47-49, wherein the vector is a viral vector.

52. A cell comprising one or more of (a) a polypeptide of any one of claims 1-24; (b) a chimeric receptor of any one of claims 25-37; (c) a nucleic acid sequence of any one of claims 38-46; and (d) a vector of any one of claims 47-51.

53. The cell of claim 52, wherein the cell is an immune cell.

54. The cell of claim 53, wherein the cell is a stem cell or a T-cell precursor cell.

55. The cell of claim 54, wherein the cell is a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), a transdifferentiated cell, a dedifferentiated cell or a manufactured cell.

56. The cell of claim 53, wherein the cell is a somatic cell or a differentiated cell.

57. The cell of claim 53, wherein the cell is a T cell, a Natural Killer (NK) cell, or a macrophage.

58. The cell of any one of claims 52-57, wherein the cell is isolated or derived from one or more of bone marrow, peripheral circulating blood, umbilical cord blood and placental blood.

59. The cell of any one of claims 52-58, wherein the cell has been cryopreserved.

60. The cell of any one of claims 52-59, wherein the cell has been modified to become an allogeneic cell.

61. The cell of any one of claims 52-60, wherein the cell has been modified to produce an allogeneic cell.

62. The cell of any one of claims 52-61, wherein the cell is capable of expressing an exogenous protein capable of binding to or signaling through one or more cytokine receptor(s).

63. The cell of claim 62, wherein the exogenous protein comprises a cytokine.

64. The cell of any one of claims 52-63, wherein the cell is capable of expressing a safety switch.

65. The cell of claim 64, wherein the safety switch comprises a truncated EGFR (EGFRt).

66. The cell of claim 65, wherein the EGFRt comprises a sequence of SEQ ID NO:

229.

67. The cell of claim 64, wherein the safety switch comprises an EGFRopt sequence.

68. The cell of claim 67, wherein the EGFRopt sequence comprises a sequence of one or more of SEQ ID NOs: 230-244.

69. A composition comprising one or more of: (a) a polypeptide of any one of claims 1-24; (b) a chimeric receptor of any one of claims 25-37; (c) a nucleic acid sequence of any one of claims 38-46; (d) a vector of any one of claims 47-51; and (e) a cell of any one of claims 52-68.

70. A pharmaceutical composition comprising one or more of: (a) a polypeptide of any one of claims 1-24, a chimeric receptor of any one of claims 25- 37, a nucleic acid sequence of any one of claims 38-46, a vector of any one of claims 47-51, a cell of any one of claims 52-68, and a composition of claim 69; and (b) a pharmaceutically acceptable carrier.

71. The pharmaceutical composition of claim 70, wherein the pharmaceutical composition is formulated for systemic administration.

72. The pharmaceutical composition of claim 71, wherein the systemic administration comprises an intravenous injection or an intravenous infusion.

73. The pharmaceutical composition of claim 70, wherein the pharmaceutical composition is formulated for local administration.

74. The pharmaceutical composition of claim 73, wherein the local administration comprises an intratumoral injection or infusion, an intraspinal injection or infusion, an intracerebellar injection or infusion, or intracisternal injection or infusion.

75. A use of one or more of a polypeptide of any one of claims 1-24, a chimeric receptor of any one of claims 25-37, a nucleic acid sequence of any one of claims 38-46, a vector of any one of claims 47-51, a cell of any one of claims 52-68, a composition of claim 69 and a pharmaceutical composition of any one of claims 70-74 in the manufacture of a medicament for treating a disease or disorder.

76. A use of one or more of a polypeptide of any one of claims 1-24, a chimeric receptor of any one of claims 25-37, a nucleic acid sequence of any one of claims 38-46, a vector of any one of claims 47-51, a cell of any one of claims 52-68, a composition of claim 69 or a pharmaceutical composition of any one of claims 70-74 for treating a disease or disorder.

77. The use of claim 75 or 76, wherein the disease or disorder comprises a proliferative disorder.

78. The use of claim 77, wherein the proliferative disorder is a metastatic disorder.

79. The use of claim 78, wherein the metastatic disorder comprises a solid tumor.

80. A method of treating a disease or disorder comprising administering an effective amount of one or more of a polypeptide of any one of claims 1-24, a chimeric receptor of any one of claims 25-37, a nucleic acid sequence of any one of claims 38-46, a vector of any one of claims 47-51, a cell of any one of claims 52-68, a composition of claim 69 or a pharmaceutical composition of any one of claims 70-74 to a subject in need thereof, thereby treating the disease or disorder.

81. The method of claim 80, wherein the subject is at risk of developing the disease or disorder.

82. The method of claim 80, wherein the subject has been diagnosed with the disease or disorder.

83. The method of claim any one of claims 80-82, wherein the disease or disorder comprises a proliferative disorder.

84. The method of claim 83, wherein the proliferative disorder is a metastatic disorder.

85. The method of claim 84, wherein the metastatic disorder comprises a solid tumor.

86. The method of claim any one of claims 80-85, wherein treating the disease or disorder comprises one or more of: (a) decreasing the severity of a sign or symptom of the disease or disorder; (b) improving a function negatively affected by the disease or disorder; (c) improving the subject’s prognosis; (d) increasing the subject’s duration of progression-free survival (PFS); (e) reducing a quantity of cells affected by the disease or disorder; (f) inducing a remission form the disease or disorder; (g) delaying or preventing a relapse of the disease or disorder; (h) delaying or preventing a second presentation of the disease or disorder; (i) increasing a population of memory cells capable of treating a relapse or second presentation of the disease or disorder.

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