Methods of enhancing engineered immune cell cytotoxicity

Abstract

Described herein are multicistronic expression systems that encode chimeric proteins, specifically membrane-cleavable chimeric systems and chimeric antigen receptors for administration in combination with the cytokine IL2 for treating a subject having cancer. Also described herein are nucleic acids, cells, and methods directed to the same.

Description

[0001] METHODS OF ENHANCING ENGINEERED IMMUNE CELL CYTOTOXICITY

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 600,011, filed November 16, 2023, which is hereby incorporated by reference in its entirety for all purposes.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on XXXX, is named STB-067WO and is XXXX bytes in size.

[0006] BACKGROUND

[0007] Cell-based therapy platforms provide promising avenues for treating a variety of diseases. One such promising platform is CAR based therapies in the treatment of cancer, including NK cell CAR-based therapies. Given their promise, improvements in cell-based therapies are needed. An active area of exploration is engineering cell-based therapies to produce and / or secrete effector molecules such as cytokines, a process referred to as armoring, that enhance the cell-based therapy. For example, unarmored CAR therapies have poor efficacy in solid tumors and armoring can impact the entire cancer immunity cycle and boost the activity of CAR. However, uncontrolled or unregulated armoring strategies can have negative impacts on treatment, such as off-target effects and toxicity in subjects. Thus, additional methods of controlling and regulating the armoring of cell-based therapies, such as regulating production and / or secretion of payload effector molecules, as well as optimizing cytokine supplementation for improving in vivo survival, expansion, and efficacy of cell-based therapies are required.

[0008] SUMMARY

[0009] Provided herein is a method of treating a subject known or suspected of having cancer, the method comprising: (A) administering a cell therapy to the subject, wherein the cell therapy comprises an immunoresponsive cell engineered to express a recombinant cytokine, wherein the immunoresponsive cell engineered to express the recombinant cytokine comprises an exogenous polynucleotide encoding a controlled release cytokine, oriented from N-terminal to C-terminal, having the formula: S - C - MT or MT - C - S wherein S comprises a secretable effector molecule, wherein the secretable effector molecule comprises the recombinant cytokine, C comprises a protease cleavage site, and MT comprises a cell membrane tethering domain, wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide; and (B) administering a pharmaceutically effective amount of IL-2.

[0010] In some aspects, the immunoresponsive cell comprises an exogenous polynucleotide encoding an inhibitory chimeric antigen receptor (iCAR) comprising: (i) an antigen-binding domain specific for endomucin (EMCN); (ii) one or more intracellular inhibitory domains that inhibit an immune response; and (iii) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

[0011] In some aspects, the immunoresponsive cell comprises an exogenous polynucleotide encoding a bivalent activating chimeric antigen receptor (aCAR) comprising: (i) an antigen- binding domain specific for FLT3; (ii) an antigen-binding domain specific for CD33; (iii) one or more intracellular signaling domains that stimulate an immune response; and (iv) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

[0012] Provided herein is method of treating a subject known or suspected of having cancer, the method comprising: (A) administering a cell therapy to the subject, wherein the cell therapy comprises an immunoresponsive cell engineered to express a recombinant cytokine; and (B) administering a pharmaceutically effective amount of IL-2.

[0013] In some aspects, the immunoresponsive cell is engineered to express the recombinant cytokine is from a multicistronic expression system.

[0014] In some aspects, the administration of IL-2 comprises multiple administrations.

[0015] In some aspects, the multiple administrations of IL-2 is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 administrations. In some aspects, wherein IL-2 is administered within 4, within 5, within 7, or within 8 hours following the start of the administration of the cell therapy. In some aspects, IL-2 is administered within 4 to 8 hours following the administration of the cell therapy. In some aspects, IL-2 is administered within 6 to 8 hours following the administration of the cell therapy. In some aspects, IL-2 is administered within 4 hours following the administration of the cell therapy. In some aspects, IL-2 is administered within 5 hours following the administration of the cell therapy. In some aspects, IL-2 is administered within 6 hours following the administration of the cell therapy. In some aspects, IL-2 is administered within 7 hours following the administration of the cell therapy. In some aspects, IL-2 is administered within 8 hours following the administration of the cell therapy. In some aspects, IL-2 is administered between 4 to 8 hours following the administration of the cell therapy. In some aspects, IL-2 is administered between 6 to 8 hours following the administration of the cell therapy. In some aspects, IL-2 is administered within 24 or 48 hours after the administering of the cell therapy.

[0016] In some aspects, the administration of the cell therapy comprises multiple administrations. In some aspects, IL-2 is administered at least 1, at least 2, a least 3 times a week following each administration of the cell therapy. In some aspects, IL-2 is administered at least 1 time a week following administration of each dose of a cell therapy. In some aspects, IL-2 is administered at least 2 times a week following administration of each dose of a cell therapy. In some aspects, IL-2 is administered at least 3 times a week following administration of each dose of a cell therapy. In some aspects, wherein the cell therapy is administered again at least on day 7 and / or day 14 following the initial administration of the cell therapy. In some aspects, the cell therapy is administered again at least on day 7 following the initial administration of the cell therapy. In some aspects, the cell therapy is administered again at least on day 7 and day 14 following the initial administration of the cell therapy. In some aspects, the cell therapy and the pharmaceutically effective amount of IL-2 are administered again on at least day 7 and / or day 14 following the initial administration of the cell therapy. In some aspects, the cell therapy and the pharmaceutically effective amount of IL-2 are administered again on at least day 7 following the initial administration of the cell therapy. In some aspects, wherein the cell therapy and the pharmaceutically effective amount of IL-2 are administered again on at least day 7 and day 14 following the initial administration of the cell therapy. In some aspects, the pharmaceutically effective amount of of IL-2 is between 1-10 MIU, optionally wherein the pharmaceutically effective amount of a dose of 3 MIU.

[0017] In some aspects, the recombinant cytokine comprises IL- 15 or a functional fragment thereof. In some aspects, the recombinant cytokine comprises crIL15.

[0018] In some aspects, the subject has, prior to administration of the cell therapy, been administered one or more lymphodepletion agents. In some aspects, the one or more lymphodepletion agents is administered 5 days prior to administration of the cell therapy. In some aspects, the one or more lymphodepletion agents is administered daily until administration of the cell therapy. In some aspects, the one or more lympodepletion agents comprises fludarabine, cytarabine (Ara-C), or combinations thereof. In some aspects, the one or more lymphodepletion agents are administered intravenously. In some aspects, fludarabine is administered at a dose of 30 mg / m2 and cytarabine (Ara-C) is administered at a dose of 2 g / m2.

[0019] In some aspects, the method of treatment comprises a reduction in tumor volume in the subject. In some aspects, the method of treatment comprises treatment of a solid tumor in the subject. In some aspects, the method of treatment comprises inducing an immune response in the subject.

[0020] In some aspects, the immunoresponsive cell engineered to express the recombinant cytokine comprises an exogenous polynucleotide encoding a controlled release cytokine, oriented from N-terminal to C-terminal, having the formula: S - C - MT or MT - C - S wherein S comprises a secretable effector molecule, wherein the secretable effector molecule comprises the recombinant cytokine, C comprises a protease cleavage site, and MT comprises a cell membrane tethering domain, wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.

[0021] In some aspects, the immunoresponsive cell comprises an exogenous polynucleotide encoding an inhibitory chimeric antigen receptor (iCAR) comprising: (i) an antigen-binding domain specific for endomucin (EMCN); (ii) one or more intracellular inhibitory domains that inhibit an immune response; and (iii) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

[0022] In some aspects, the immunoresponsive cell comprises an exogenous polynucleotide encoding a bivalent activating chimeric antigen receptor (aCAR) comprising: (i) an antigen- binding domain specific for FLT3; (ii) an antigen-binding domain specific for CD33; (iii) one or more intracellular signaling domains that stimulate an immune response; and (iv) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

[0023] In some aspects, the immunoresponsive cell comprises an engineered nucleic acid comprising: (A) an exogenous polynucleotide encoding a controlled release cytokine, oriented from N- terminal to C-terminal, having the formula: S - C - MT or MT - C - S wherein S comprises a secretable effector molecule, wherein the secretable effector molecule comprises the recombinant cytokine, C comprises a protease cleavage site, and MT comprises a cell membrane tethering domain, wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide; (B) an exogenous polynucleotide encoding an inhibitory chimeric antigen receptor (iCAR) comprising: (i) an antigen-binding domain specific for endomucin (EMCN); (ii) one or more intracellular inhibitory domains that inhibit an immune response; and (iii) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof; and (C) an exogenous polynucleotide encoding a bivalent activating chimeric antigen receptor (aCAR) comprising: (i) an antigen-binding domain specific for FLT3; (ii) an antigen-binding domain specific for CD33; (iii) one or more intracellular signaling domains that stimulate an immune response; and (iv) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

[0024] In some aspects, the exogenous polynucleotides encoding each of the membrane-cleavable chimeric protein, the iCAR, and the bivalent aCAR are linked together by a polynucleotide linker encoding a 2A ribosome skipping element, optionally wherein the 2A ribosome skipping element is selected from the group consisting of: a T2A ribosome skipping element, a E2A ribosome skipping element, a P2A ribosome skipping element, a F2A ribosome skipping element, ribosome skipping element fusions thereof, and combinations thereof, optionally wherein the ribosome skipping element fusion comprises an E2A / T2A ribosome skipping element, optionally wherein the E2A / T2A ribosome skipping element comprises the amino acid sequence QCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 221), optionally wherein the E2A / T2A ribosome skipping element is encoded by a polynucleotide sequence comprising the sequence CAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATCTAATCCTGG ACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACGTGGAGGAA AACCCTGGACCT (SEQ ID NO: 222) or the sequence CAGTGCACAAATTATGCACTGCTGAAGCTCGCCGGGGATGTCGAGAGTAACCCAGG ACCTGGAAGCGGAGAAGGTCGTGGTAGTCTACTAACGTGTGGTGATGTAGAAGAAA ATCCTGGACCT (SEQ ID NO: 223).

[0025] In some aspects, the membrane-cleavable chimeric protein, the iCAR, and the bivalent aCAR are encoded in order from 5’ to 3’: (i) membrane-cleavable chimeric protein; (ii) bivalent aCAR; and (iii) iCAR; or from 5’ to 3’: (i) membrane-cleavable chimeric protein; (ii) iCAR; and

[0026] (iii) bivalent aCAR.

[0027] In some aspects, the secretable effector molecule comprises a signal peptide or a signal- anchor sequence, optionally wherein the signal peptide comprises a native signal peptide native to the secretable effector molecule, optionally wherein the signal peptide comprises a non-native signal peptide or the signal-anchor sequence comprises a non-native signal-anchor sequence non-native to the secretable effector molecule, optionally wherein the non-native signal peptide or the non-native signal-anchor sequence is selected from the group consisting of: IgE, IL- 12, IL-2, optimized IL-2, trypsiongen-2, Gaussia luciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL-6, IL-8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin El, GROalpha, CXCL12, IL-21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa, optionally wherein the non-native signal peptide comprises an IgE signal peptide, optionally wherein the IgE signal peptide comprises the amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 228), optionally wherein the IgE signal peptide is encoded by a polynucleotide sequence comprising the sequence ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCT (SEQ ID NO: 229).

[0028] In some aspects, the protease cleavage site further comprises the N-terminal peptide linker, optionally selected from the group consisting of: SGGGGSGGGGSG(SEQ ID NO: 230); GGGSGGGGSGGGSLQ(SEQ ID NO: 231); GGS(SEQ ID NO: 232); GGSGGS(SEQ ID NO: 233); GGSGGSGGS(SEQ ID NO: 234); GGSGGSGGSGGS(SEQ ID NO: 235); GGSGGSGGSGGSGGS(SEQ ID NO: 236); GGGS(SEQ ID NO: 237) ; GGGSGGGS(SEQ ID NO: 238); GGGSGGGSGGGS(SEQ ID NO: 239); GGGSGGGSGGGSGGGS(SEQ ID NO:

[0029] 240); GGGSGGGSGGGSGGGSGGGS(SEQ ID NO: 241); GGGGS(SEQ ID NO: 242); GGGGSGGGGS(SEQ ID NO: 243); GGGGSGGGGSGGGGS(SEQ ID NO: 244); GGGGSGGGGSGGGGSGGGGS(SEQ ID NO: 245);

[0030] GGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO: 246); GSTSGSGKPGSGEGSTKG(SEQ ID NO: 247); and EAAAKEAAAKEAAAKEAAAK(SEQ ID NO: 248), optionally wherein the protease cleavage site further comprises the N-terminal peptide linker SGGGGSGGGGSG (SEQ ID NO: 230), optionally wherein the protease cleavage site further comprises a C-terminal peptide linker, optionally selected from the group consisting of: SGGGGSGGGGSG(SEQ ID NO: 230); GGGSGGGGSGGGSLQ(SEQ ID NO: 231);

[0031] GGS(SEQ ID NO: 232); GGSGGS(SEQ ID NO: 233); GGSGGSGGS(SEQ ID NO: 234); GGSGGSGGSGGS(SEQ ID NO: 235); GGSGGSGGSGGSGGS(SEQ ID NO: 236); GGGS (SEQ ID NO: 237); GGGSGGGS(SEQ ID NO: 238); GGGSGGGSGGGS(SEQ ID NO: 239); GGGSGGGSGGGSGGGS(SEQ ID NO: 240); GGGSGGGSGGGSGGGSGGGS(SEQ ID NO:

[0032] 241); GGGGS(SEQ ID NO: 242); GGGGSGGGGS(SEQ ID NO: 243);

[0033] GGGGSGGGGSGGGGS(SEQ ID NO: 244); GGGGSGGGGSGGGGSGGGGS(SEQ ID NO: 245); GGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO: 246);

[0034] GSTSGSGKPGSGEGSTKG(SEQ ID NO: 247); and EAAAKEAAAKEAAAKEAAAK(SEQ ID NO: 248), optionally wherein the protease cleavage site further comprises the C-terminal linker GGGSGGGGSGGGSLQ (SEQ ID NO: 231), optionally wherein the protease cleavage site comprises a Tumor Necrosis Factor-a Converting Enzyme (TACE)-specific cleavage site, optionally wherein the TACE-specific cleavage site comprises the amino acid sequence VTPEPIFSLI (SEQ ID NO: 191), optionally, wherein the TACE-specific cleavage site comprises the amino acid sequence SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ (SEQ ID NO: 250), optionally wherein the TACE-specific cleavage site is encoded by a polynucleotide sequence comprising the sequence TCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGTGACCCCTGAGCCTATCTT CAGCCTGATCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAA (SEQ ID NO: 251).

[0035] In some aspects, the cell membrane tethering domain comprises a transmembrane domain selected from the group consisting of: PDGFR-beta, CD8, CD28, CD3zeta-chain, CD4, 4- IBB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, LIR1, B7-1, and BTLA, optionally wherein the cell membrane tethering domain comprises a B7-1 transmembrane domain, optionally wherein the B7-1 transmembrane domain comprises the amino acid sequence LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 204), optionally wherein the B7-1 transmembrane domain is encoded by a polynucleotide sequence comprising the sequence TTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGC TGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCT GAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 252), optionally wherein the membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, has the formula S - C - MT, optionally wherein the membrane-cleavable chimeric protein comprises the amino acid sequence MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAM KCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS FVHIVQMFINTSSGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQLLPSWAITLISV NGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 226), optionally wherein the membrane-cleavable chimeric protein is encoded by a polynucleotide sequence comprising the sequence ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCTAAT TGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCA CATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGACCG CCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCC AGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAACAGCCTGTCCAG CAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAG AACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACACC TCATCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGTGACCCCTGAGCCTAT CTTCAGCCTGATCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTC AATTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCT GCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGG CTGAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 227).

[0036] In some aspects, the antigen-binding domain specific for EMCN comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the EMCN-VH comprises: a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 291), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 296), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 298), and the EMCN-VL comprises: a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 299), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 300), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 301), and wherein the amino acid sequences of the CDR-H1, the CDR-H2, the CDR-H3, the CDR-L1, the CDR-L2, and the CDR-L3 of the reference antibody are defined based on the Kabat numbering scheme; optionally wherein the EMCN-VH and the EMCN-VL are separated by a peptide linker, optionally wherein the antigen-binding domain specific for EMCN comprises the structure VH-L-VL or VL-L-VH, wherein L is the peptide linker, optionally wherein the peptide linker comprises the amino acid sequence SGGGGSGGGGSG(SEQ ID NO: 230); GGGSGGGGSGGGSLQ(SEQ ID NO: 231);

[0037] GGS(SEQ ID NO: 232); GGSGGS(SEQ ID NO: 233); GGSGGSGGS(SEQ ID NO: 234); GGSGGSGGSGGS(SEQ ID NO: 235); GGSGGSGGSGGSGGS(SEQ ID NO: 236);

[0038] GGGS(SEQ ID NO: 237) ; GGGSGGGS(SEQ ID NO: 238); GGGSGGGSGGGS(SEQ ID NO: 239); GGGSGGGSGGGSGGGS(SEQ ID NO: 240); GGGSGGGSGGGSGGGSGGGS(SEQ ID NO: 241); GGGGS(SEQ ID NO: 242); GGGGSGGGGS(SEQ ID NO: 243);

[0039] GGGGSGGGGSGGGGS(SEQ ID NO: 244); GGGGSGGGGSGGGGSGGGGS(SEQ ID NO: 245); GGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO: 246);

[0040] GSTSGSGKPGSGEGSTKG(SEQ ID NO: 247); or EAAAKEAAAKEAAAKEAAAK(SEQ ID NO: 248), optionally wherein the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 244), optionally wherein the peptide linker is encoded by a polynucleotide sequence comprising the sequence GGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCT (SEQ ID NO: 253).

[0041] In some aspects, the intracellular inhibitory domain comprises a LIR1 intracellular inhibitory domain, optionally wherein the LIR1 intracellular inhibitory domain comprises the amino acid sequence LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKH TQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEED RQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 285), optionally wherein the LIR1 intracellular inhibitory domain is encoded by a polynucleotide sequence comprising the sequence CTGCGGCACAGAAGGCAGGGCAAGCACTGGACAAGCACCCAGAGAAAGGCCGACT TTCAGCATCCTGCTGGCGCCGTTGGACCTGAGCCTACAGATAGAGGACTGCAGTGG CGGTCTAGCCCTGCCGCTGATGCCCAAGAGGAAAATCTTTACGCCGCCGTGAAGCA CACCCAGCCTGAGGATGGCGTGGAAATGGACACCAGATCTCCCCACGATGAGGACC CTCAGGCCGTGACATACGCAGAAGTGAAGCACTCCAGACCTCGGAGAGAGATGGC AAGCCCTCCATCTCCTCTGAGCGGCGAGTTCCTGGACACCAAAGACAGACAGGCCG AAGAGGACAGACAGATGGATACCGAAGCCGCCGCTTCTGAAGCCCCACAGGATGT GACATATGCCCAGCTGCATAGCCTGACACTGCGGAGAGAAGCCACAGAGCCTCCAC CTTCTCAAGAAGGCCCATCTCCTGCCGTGCCTTCCATCTATGCCACTCTGGCCATTC AC (SEQ ID NO: 286).

[0042] In some aspects, (A) the signal peptide is present in the iCAR, optionally wherein the signal peptide of the iCAR comprises a native signal peptide native or a non-native signal peptide, optionally wherein the non-native signal peptide or the non-native signal-anchor sequence is selected from the group consisting of: IgE, IL- 12, IL-2, optimized IL-2, trypsiongen- 2, Gaussia luciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL-6, IL-8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin El, GROalpha, CXCL12, IL-21, CD8, NKG2D, TNFR2, and GMCSF, optionally wherein the signal peptide of the iCAR comprises a CD8 signal peptide, optionally wherein the CD8 signal peptide comprises the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 137), optionally wherein the CD8 signal peptide is encoded by a polynucleotide sequence comprising the sequence ATGGCTCTGCCCGTGACAGCTTTGCTGCTGCCTTTGGCACTGCTGCTGCATGCTGCT AGACCA (SEQ ID NO: 416); and / or wherein (B) the hinge domain is present in the iCAR comprises, optionally wherein the hinge domain of the iCAR is selected from the group consisting of a human Ig (immunoglobulin) hinge, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge, a KIR2DS2 hinge, an LNGFR hinge, a LIR1 hinge, a PDGFR-beta extracellular linker, and combinations thereof, optionally wherein the hinge domain of the iCAR comprises a LIR1 hinge, optionally wherein the LIR1 hinge comprises the amino acid sequence HPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV (SEQ ID NO: 417), optionally wherein the LIR1 hinge comprises is encoded by a polynucleotide sequence comprising the sequence CACCCATCCGATCCTCTCGAGCTGGTGGTTTCTGGACCTTCTGGCGGCCCTAGCAGC CCTACAACAGGACCTACAAGCACAAGCGGCCCTGAGGACCAACCTCTGACACCAAC

[0043] AGGCAGCGATCCTCAGTCTGGACTGGGGAGACATCTGGGCGTT (SEQ ID NO: 418); optionally wherein the hinge domain of the iCAR comprises a CD8 hinge, optionally wherein the CD8 hinge comprises the amino acid sequence TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 271), optionally wherein the CD8 hinge comprises is encoded by a polynucleotide sequence comprising the sequence

[0044] ACAACAACACCCGCACCTCGGCCTCCAACTCCAGCTCCAACAATTGCACTGCAACC CCTGAGTCTGAGGCCCGAGGCCTGTAGGCCAGCAGCTGGCGGAGCTGTTCACACTA GAGGCCTGGACTTTGCCTGTGAC (SEQ ID NO: 283); and / or wherein (C) the transmembrane domain of the iCAR is present, optionally wherein the transmembrane domain of the iCAR comprises a transmembrane domain selected from the group consisting of: PDGFR- beta, CD8, CD28, CD3zeta-chain, CD4, 4- IBB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, LIR1, and BTLA, optionally wherein the transmembrane domain of the iCAR comprises a LIR1 transmembrane domain, optionally wherein the LIR1 transmembrane domain comprises the amino acid sequence

[0045] VIGILVAVILLLLLLLLLFLI (SEQ ID NO: 259), optionally wherein the LIR1 transmembrane domain is encoded by a polynucleotide sequence comprising the sequence

[0046] GTGATCGGCATTCTGGTCGCCGTGATCCTGCTCCTGTTGCTCCTGCTGCTTCTGTTCC TGATC (SEQ ID NO: 260).

[0047] In some aspects, the iCAR comprises the amino acid sequence:

[0048] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWV RQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYY CTLRIKDWGQGTMVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCKS SQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEA EDVGVYYCLQGIHLPWTFGQGTKLEIKngaaHPSDPLELVVSGPSGGPSSPTTGPTSTSGP EDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKA

[0049] DFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDED PQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTY AQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 419), optionally wherein the iCAR is encoded by a polynucleotide sequence comprising the sequence

[0050] ATGGCTCTGCCCGTGACAGCTTTGCTGCTGCCTTTGGCACTGCTGCTGCATGCTGCT

[0051] AGACCAGAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATC

[0052] TCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGATACGATATGCACTG

[0053] GGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGTGATCTGGGGCAACG

[0054] GCAACACACACTACCACAGCGCCCTGAAGTCCCGGTTCACCATCTCCAGAGACAAC AGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGT

[0055] GTACTACTGCACCCTGAGAATCAAGGATTGGGGCCAGGGCACCATGGTCACCGTTT

[0056] CTTCTGGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCTGACGTG

[0057] GTCATGACACAGAGCCCTCTGAGCCTGCCTGTGACACTGGGACAGCCTGCCAGCAT

[0058] CAGCTGCAAGTCTAGCCAGTCTCTGGTGGCCAGCGACGAGAACACCTACCTGAACT

[0059] GGTTCCAGCAGAGGCCCGGACAGTCTCCTAGACGGCTGATCTACCAGGTGTCCAAG

[0060] CTGGATAGCGGCGTGCCCGATAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACC

[0061] CTGAAGATCAGCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTCTGCAAGG

[0062] CATCCATCTGCCTTGGACCTTTGGCCAGGGCACAAAGCTGGAAATCAAGAATGGCG

[0063] CTGCACACCCATCCGATCCTCTCGAGCTGGTGGTTTCTGGACCTTCTGGCGGCCCTA GCAGCCCTACAACAGGACCTACAAGCACAAGCGGCCCTGAGGACCAACCTCTGACA

[0064] CCAACAGGCAGCGATCCTCAGTCTGGACTGGGGAGACATCTGGGCGTTGTGATCGG

[0065] CATTCTGGTCGCCGTGATCCTGCTCCTGTTGCTCCTGCTGCTTCTGTTCCTGATCCTG

[0066] CGGCACAGAAGGCAGGGCAAGCACTGGACAAGCACCCAGAGAAAGGCCGACTTTC

[0067] AGCATCCTGCTGGCGCCGTTGGACCTGAGCCTACAGATAGAGGACTGCAGTGGCGG

[0068] TCTAGCCCTGCCGCTGATGCCCAAGAGGAAAATCTTTACGCCGCCGTGAAGCACAC CCAGCCTGAGGATGGCGTGGAAATGGACACCAGATCTCCCCACGATGAGGACCCTC

[0069] AGGCCGTGACATACGCAGAAGTGAAGCACTCCAGACCTCGGAGAGAGATGGCAAG

[0070] CCCTCCATCTCCTCTGAGCGGCGAGTTCCTGGACACCAAAGACAGACAGGCCGAAG AGGACAGACAGATGGATACCGAAGCCGCCGCTTCTGAAGCCCCACAGGATGTGACA

[0071] TATGCCCAGCTGCATAGCCTGACACTGCGGAGAGAAGCCACAGAGCCTCCACCTTC TCAAGAAGGCCCATCTCCTGCCGTGCCTTCCATCTATGCCACTCTGGCCATTCAC (SEQ ID NO: 420). In some aspects, the antigen-binding domain specific for FLT3 comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the FLT3-VH comprises: a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GGTFSSYAIS (SEQ ID NO: 360), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of GIIPIFGTANYAQKFQG (SEQ ID NO: 361), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of FALFGFREQAFDI (SEQ ID NO: 362), and the FLT3-VL comprises: a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASQSISSYLN (SEQ ID NO: 363), a light chain complementarity determining region 2 (CDR- L2) having the amino acid sequence of AASSLQS (SEQ ID NO: 364), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQSYSTPFT (SEQ ID NO: 365), and wherein the amino acid sequences of the CDR-H1, the CDR-H2, the CDR-H3, the CDR-L1, the CDR-L2, and the CDR-L3 of the reference antibody are defined based on the Kabat numbering scheme; optionally wherein: the FLT3-VH comprises the amino acid sequence EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTAN YAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCATFALFGFREQAFDIWGQGTTV TVSS (SEQ ID NO: 315); and the FLT3-VL comprises the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDLATYYCQQSYSTPFTFGPGTKVDIK (SEQ ID NO: 316), optionally wherein the FLT3-VH, and the FLT3-VL are separated by peptide linkers.

[0072] In some aspects, the antigen-binding domain specific for CD33 comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the CD33-VH comprises: a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of DYNMH (SEQ ID NO: 402), a heavy chain complementarity determining region 2 (CDR- H2) having the amino acid sequence of YIYPYNGGTGYNQKFKSKA (SEQ ID NO: 403), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of GRPAMDYWGQ (SEQ ID NO: 404), and the CD33-VL comprises: a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASES VDNYGISFMN (SEQ ID NO: 405), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of AASNQGS (SEQ ID NO: 406), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQSKEVPWT (SEQ ID NO: 407), and wherein the amino acid sequences of the CDR-H1, the CDR-H2, the CDR-H3, the CDR-L1, the CDR-L2, and the CDR-L3 of the reference antibody are defined based on the Kabat numbering scheme; optionally wherein: the CD33-VH comprises the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGG TGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTV SS (SEQ ID NO: 329); and the CD33-VL comprises the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQG SGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 330), optionally wherein the CD33-VH, and the CD33-VL are separated by peptide linkers.

[0073] In some aspects, the aCAR antigen binding domains comprises the structure (FLT3-VH) - LI - (CD33-VH) - L2 - (CD33-VL) - L3 - (FLT3-VL), wherein LI, L2, and L3 are a first, a second, and a third peptide linker, respectively; optionally wherein the LI, L2, and / or L3 are each independently selected from the group consisting of: SEQ ID Nos 230 - 248, optionally wherein the LI peptide linker is the amino acid sequence GGGGS (SEQ ID NO: 242) or GGGGSGGGGS (SEQ ID NO: 243), optionally wherein the LI peptide linker GGGGS (SEQ ID NO: 242) is encoded by a polynucleotide sequence comprising the sequence GGCGGCGGTGGCTCT (SEQ ID NO: 254) or the LI peptide linker GGGGSGGGGS (SEQ ID NO: 243) is encoded by a polynucleotide sequence comprising the sequence GGAGGCGGAGGATCTGGTGGTGGTGGATCT (SEQ ID NO: 256), optionally wherein the L2 peptide linker is the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 247), optionally wherein the L2 peptide linker is encoded by a polynucleotide sequence comprising the sequence GGCTCTACATCTGGCTCTGGCAAACCTGGAAGCGGCGAGGGATCTACCAAGGGC (SEQ ID NO: 249), optionally wherein the L2 peptide linker is the amino acid sequence GGGGSGGGGS (SEQ ID NO: 243), optionally wherein the L2 peptide linker is encoded by a polynucleotide sequence comprising the sequence GGTGGCGGAGGAAGTGGCGGCGGAGGCTCT (SEQ ID NO: 257), optionally wherein the L3 peptide linker is the amino acid sequence GGGGS (SEQ ID NO: 242)or GGGGSGGGGS (SEQ ID NO: 243), optionally wherein the L3 peptide linker GGGGS (SEQ ID NO: 242) is encoded by a polynucleotide sequence comprising the sequence GGTGGCGGCGGATCC (SEQ ID NO: 255) or the L3 peptide linker GGGGSGGGGS (SEQ ID NO: 243) is encoded by a polynucleotide sequence comprising the sequence GGCGGTGGCGGATCTGGCGGAGGTGGCAGT (SEQ ID NO: 258). In some aspects, the aCAR intracellular signaling domains that stimulate an immune response is selected from the group consisting of: CD3-zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278, FceRI, DAP10, DAP12, CD66d, CD97, CD2, ICOS, CD27, CD154, CD8, 0X40, 4-1BB, CD28, ZAP40, CD30, GITR, HVEM, DAP10, DAP12, MyD88, 2B4, CD40, PD-1, LFA-1, CD7, LIGHT, NKG2C, B7-H3, an MHC class I molecule, a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a SLAM protein, an activating NK cell receptor, BTLA, a Toll ligand receptor, CDS, ICAM-1, (CDlla / CD18), BAFFR, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 19, CD4, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, and combinations thereof, optionally wherein the aCAR intracellular signaling domains that stimulate an immune response comprise a CD28 co- stimulatory domain and a CD3^ signaling domain, optionally wherein the CD28 co-stimulatory domain comprises the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 287), optionally wherein the CD28 co-stimulatory domain is encoded by a polynucleotide sequence comprising the sequence AGAAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCTAGAC GGCCCGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGATTTCGCC GCCTACCGGTCC (SEQ ID NO: 288), optionally wherein the CD3(^ signaling domain comprises the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 289), optionally wherein the CD3(^ signaling domain is encoded by a polynucleotide sequence comprising the sequence AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACC AGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAG AGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGG AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGAT TGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGT CTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCC TCGC (SEQ ID NO: 290), optionally wherein

[0074] (A) the hinge domain of the aCAR is present, optionally wherein the hinge domain of the aCAR is selected from the group consisting of a human Ig (immunoglobulin) hinge, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge, a KIR2DS2 hinge, an LNGFR hinge, a LIR1 hinge, a PDGFR-beta extracellular linker, and combinations thereof, optionally wherein the hinge domain of the aCAR comprises a CD8 hinge, optionally wherein the CD8 hinge comprises the amino acid sequence

[0075] AESNSIMYFSHFVPVFEPAKPTTTPAPRPPTPAPTIASQPESERPEACRPAAGGAVHTRGE DFACD (SEQ ID NO: 272), optionally wherein the CD8 hinge comprises is encoded by a polynucleotide sequence comprising the sequence

[0076] GCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTGCCCGCC AAGCCTACAACAACCCCTGCTCCTAGACCACCTACACCAGCTCCTACAATCGCCAG CCAGCCTCTGTCTCTGAGGCCCGAAGCTTGTAGACCAGCTGCTGGCGGAGCCGTGC ATACAAGAGGACTGGATTTTGCCTGCGAC (SEQ ID NO: 284), and / or wherein (B) the transmembrane domain of the aCAR is present, optionally wherein the transmembrane domain of the aCAR is selected from the group consisting of a human Ig (immunoglobulin) hinge, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge, a KIR2DS2 hinge, an LNGFR hinge, a LIR1 hinge, a PDGFR-beta extracellular linker, and combinations thereof, optionally wherein the transmembrane domain of the aCAR comprises a CD8 hinge, optionally wherein the CD8 transmembrane comprises the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 206), optionally wherein the CD8 transmembrane comprises is encoded by a polynucleotide sequence comprising the sequence ATCTATATCTGGGCCCCTCTGGCTGGCACATGCGGAGTTCTGCTGCTCAGCCTGGTC ATCACCCTGTACTGCAACCACAGA (SEQ ID NO: 261); and / or wherein (C) the aCAR signal peptide of the aCAR is present, optionally wherein the signal peptide of the aCAR is selected from the group consisting of: IgE, IL- 12, IL-2, optimized IL-2, trypsiongen-2, Gaussia luciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL-6, IL-8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin El, GROalpha, CXCL12, IL-21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa, optionally wherein the signal peptide of the aCAR comprises a GM-CSFRa signal peptide, optionally wherein the GM-CSFRa signal peptide comprises the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 423), optionally wherein the GM-CSFRa signal peptide is encoded by a polynucleotide sequence comprising the sequence

[0077] ATGCTGCTGCTGGTTACATCTCTGCTGCTGTGCGAGCTGCCCCATCCTGCCTTTCTGC TTATTCCT (SEQ ID NO: 424), optionally wherein the aCAR comprises the amino acid sequence

[0078] MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVR QAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCA TFALFGFREQAFDIWGQGTTVTVSSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYT FTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSL RSEDTAVYYCARGRPAMDYWGQGTLVTVSSGSTSGSGKPGSGEGSTKGDIQMTQSPSS

[0079] LSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGS GSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKGGGGSDIQMTQSPSSLSA SVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL TISSLQPEDLATYYCQQSYSTPFTFGPGTKVDIKALSNSIMYFSHFVPVFLPAKPTTTPAP RPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL

[0080] YCNHRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA YKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 414), optionally wherein the aCAR comprises the amino acid sequence

[0081] MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVR QAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCA TFALFGFREQAFDIWGQGTTVTVSSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCK ASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAY MELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSGGGGSGGGGSDIQMTQSPSSLS

[0082] ASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGS GTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGT DFTLTISSLQPEDLATYYCQQSYSTPFTFGPGTKVDIKALSNSIMYFSHFVPVFLPAKPTT TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS

[0083] LVITLYCNHRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSA DAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 415).

[0084] In some aspects, the cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a Natural Killer T (NKT) cell, a myeloid cell, a macrophage, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, and induced pluripotent stem cell (iPSC), and an iPSC-derived cell, optionally wherein the cell is an NK cell.

[0085] In some aspects, the method comprising administering a therapeutically effective dose of any of the isolated cells disclosed herein, optionally wherein the subject has cancer, optionally wherein the isolated cell is derived from the subject or is allogeneic with reference to the subject.

[0086] In some aspects, the method comprising administering to a subject having a tumor a therapeutically effective dose of any of the isolated cells disclosed herein, optionally wherein the isolated cell is derived from the subject or is allogeneic with reference to the subject.

[0087] In some aspects, the method of making an engineered cell, comprises transducing an isolated cell with the multicistronic expression system disclosed herein, an expression vector disclosed herein or a polynucleotide disclosed herein.

[0088] Provided herein is a method of treating a subject known or suspected of having cancer, the method comprising: (A) administering a cell therapy to the subject, wherein the cell therapy comprises an immunoresponsive cell engineered to express a recombinant cytokine, wherein the immunoresponsive cell engineered to express the recombinant cytokine comprises an exogenous polynucleotide encoding a controlled release cytokine, oriented from N-terminal to C-terminal, having the formula: S - C - MT or MT - C - S wherein S comprises a secretable effector molecule, wherein the secretable effector molecule comprises the recombinant cytokine, C comprises a protease cleavage site, and MT comprises a cell membrane tethering domain, wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide; and (B) administering a pharmaceutically effective amount of an agent selected from the group consisting of: (i) cytarabine (Ara-C); (ii) decitabine; (iii) venetoclax; (iv) a combination of fludarabine and Ara-C; or (V) or a combination of decitabine and venetoclax.

[0089] In some aspects, the immunoresponsive cell comprises an exogenous polynucleotide encoding an inhibitory chimeric antigen receptor (iCAR) comprising: (i) an antigen-binding domain specific for endomucin (EMCN); (ii) one or more intracellular inhibitory domains that inhibit an immune response; and (iii) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof. In some aspects, the immunoresponsive cell comprises an exogenous polynucleotide encoding a bivalent activating chimeric antigen receptor (aCAR) comprising: (i) an antigen- binding domain specific for FLT3; (ii) an antigen-binding domain specific for CD33; (iii) one or more intracellular signaling domains that stimulate an immune response; and (iv) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

[0090] In some aspects, the agent comprises the combination of fludarabine and Ara-C.

[0091] In some aspects, the fludarabine is administered at a dose of 30 mg / m2 and the cytarabine (Ara- C) is administered at a dose of 2 g / m2. In some aspects, the agent is administered intravenously. In some aspects, wherein the agent is administered 5 days prior to administration of the cell therapy. In some aspects, when the Ara-C is administered, the Ara-C is administered at a dose of 2 g / m2. In some aspects, the agent comprises the combination of fludarabine and Ara-C, wherein the fludarabine is administered at a dose of 30 mg / m2 and the Ara-C is administered at a dose of 2 g / m2, and wherein the agent is administered 5 days prior to administration of the cell therapy.

[0092] BRIEF DESCRIPTION OF THE DRAWINGS

[0093] FIG. 1 provides a schematic illustrating components and organization of the overall FLT3 OR CD33 NOT EMCN logic-gated CAR-NK cell system.

[0094] FIG. 2 shows fold change in four lots of FLT3 OR CD33 NOT EMCN logic-gated CAR-NK or donor-matched non-engineered NK live cell counts over time (days) in culture.

[0095] FIG. 3 shows fold change in four lots of FLT3 OR CD33 NOT EMCN logic-gated CAR-NK or donor-matched non-engineered NK live cell counts over time (days) in culture with media without IL-2 supplementation or low dose IL-2 (10 lU / mL) containing media.

[0096] FIG. 4 shows fold change in four lots of FLT3 OR CD33 NOT EMCN logic-gated CAR-NK or donor-matched non-engineered NK live cell counts over time (days) in culture in media without IL-2 supplementation, low dose IL-2 (10 lU / mL) containing media, or high dose IL-2 (500 lU / mL) and IL 15 (10 ng / mL) containing media.

[0097] FIG. 5 provides a schematic of the experimental conditions of IL-2 treatment of FLT3 OR CD33 NOT EMCN logic-gated CAR-NK cells prior to preforming a cytotoxicity assay.

[0098] FIG. 6 shows the CD33 and FLT3 expression of different AML cell lines as measured by flow cytometry and the percent cytotoxicity of non-engineered NK cells or FLT3 OR CD33 NOT EMCN logic-gated CAR-NK cells in the presence of "IL-2 4 day” treatment against an AML cell line that did not express CD33 and FLT3 (KG-la). FIG. 7 shows percent cytotoxicity of FLT3 OR CD33 NOT EMCN logic-gated CAR-NK cells against different AML cell lines at effector: target (E:T) ratios of 2:1, 1:1 and 0.5:1 when treated with IL-2 as depicted in FIG. 5.

[0099] FIG. 8 provides a schematic of the experimental conditions of IL-2 treatment of ELT3 OR CD33 NOT EMCN logic-gated CAR-NK cells during a serial killing assay.

[0100] FIG. 9 shows percent cytotoxicity of FLT3 OR CD33 NOT EMCN logic-gated CAR-NK cells or donor-matched non-engineered NK cells against different AML cell lines over 3 rounds of serial killing.

[0101] FIG. 10 shows percent cytotoxicity of FLT3 OR CD33 NOT EMCN logic-gated CAR-NK cells against different AML cell lines over 3 rounds of serial killing in the presence or absence of IL-2 during the serial killing assay as depicted in FIG. 8.

[0102] DETAILED DESCRIPTION

[0103] The present disclosure provides methods of treating subjects, such as subjects known or suspected of having cancer, with a cell therapy in combination with a IL-2 cytokine therapy.

[0104] Cell therapies include immunoresponsive cells engineered to express a recombinant cytokine. Recombinant cytokines can include a controlled release cytokine. As used herein, the terms “membrane-cleavable,” “controlled-release,” and “calibrated release” are used interchangeably. In various embodiments, the controlled release protein disclosed herein is a controlled release cytokine. In certain embodiments, the cytokine is a controlled-release IL- 15 (crIL15) cytokine. Controlled release cytokines are described further herein (e.g., “Membrane Cleavable Chimeric Proteins”).

[0105] Recombinant cytokines can include a controlled release cytokine, oriented from N- terminal to C-terminal, having the formula: S - C - MT or MT - C - S, where S represents a secretable effector molecule that includes the recombinant cytokine, C represents a protease cleavage site, and MT represents a cell membrane tethering domain, and where S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.

[0106] Cell therapies can include multicistronic expression systems, i.e., more than one separate polypeptide (e.g., multiple chimeric proteins) can be produced from a single mRNA transcript. An illustrative non-limiting multicistronic system can include an polynucleotide encoding (i) a controlled-release cytokine; (ii) a bivalent aCAR; and (iii) an iCAR. The multicistronic systems include FLT3 OR CD33 NOT EMCN logic-gated CARs and a membrane-cleavable chimeric protein, controlled-release IL- 15 (crIL15).

[0107] Multicistronic expression systems can include engineered nucleic acids encoding: (A) an exogenous polynucleotide encoding a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula: S - C - MT or MT - C - S wherein S includes a secretable effector molecule, wherein the secretable effector molecule includes IL- 15, C includes a protease cleavage site , and MT includes a cell membrane tethering domain. S - C - MT or MT - C - S is configured to be expressed as a single polypeptide;

[0108] (B) an exogenous polynucleotide encoding an inhibitory chimeric antigen receptor (iCAR ) including: (i) an antigen-binding domain specific for endomucin (EMCN); (ii) one or more intracellular inhibitory domains that inhibit an immune response, and (iii) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof; and

[0109] (C) an exogenous polynucleotide encoding a bivalent activating chimeric antigen receptor (aCAR ) including: (i) an antigen-binding domain specific for FLT3; (ii) an antigen-binding domain specific for CD33; (iii) one or more intracellular signaling domains that stimulate an immune response, and (iv) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

[0110] Engineered nucleic acids can be multicistronic through the use of various linkers, e.g., a polynucleotide sequence encoding a first chimeric proteins can be linked to a nucleotide sequence encoding a second chimeric protein, such as in a first gene: I inker: second gene 5’ to 3’ orientation. A linker can encode a 2A ribosome skipping element, such as T2A. Other 2A ribosome skipping elements include, but are not limited to, E2A, P2A, and F2A. 2A ribosome skipping elements allow production of separate polypeptides encoded by the first and second genes are produced during translation. 2A ribosome skipping elements can include fusion peptides of 2A ribosome skipping elements, including, but not limited to, an E2A / T2A ribosome skipping element. In certain embodiments, the E2A / T2A ribosome skipping element comprises the amino acid sequence of GSGQCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 219). One exemplary nucleic acid encoding the E2A / T2A ribosome skipping element is GGTAGCGGCCAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATC TAATCCTGGACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACG TGGAGGAAAACCCTGGACCT (SEQ ID NO: 220). In certain embodiments, a nucleic acid encoding the E2A / T2A ribosome skipping element includes a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 220. In some embodiments, an engineered nucleic acid disclosed herein comprises an E2A / T2A ribosome skipping element. In certain embodiments, the E2A / T2A ribosome skipping element comprises the amino acid sequence of QCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 221). One exemplary nucleic acid encoding the E2A / T2A ribosome skipping element is

[0111] CAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATCTAATCCTGG ACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACGTGGAGGAA AACCCTGGACCT (SEQ ID NO: 222). Another exemplary nucleic acid encoding the E2A / T2A ribosome skipping element is

[0112] CAGTGCACAAATTATGCACTGCTGAAGCTCGCCGGGGATGTCGAGAGTAACCCAGG ACCTGGAAGCGGAGAAGGTCGTGGTAGTCTACTAACGTGTGGTGATGTAGAAGAAA ATCCTGGACCT (SEQ ID NO: 223). In certain embodiments, a nucleic acid encoding the E2A / T2A ribosome skipping element includes a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 222 or SEQ ID NO: 223.

[0113] A linker can encode a cleavable linker polypeptide sequence, such as a Furin cleavage site or a TEV cleavage site, wherein following expression the cleavable linker polypeptide is cleaved such that separate polypeptides encoded by the first and second genes are produced. A cleavable linker can include a polypeptide sequence, such as such a flexible linker (e.g., a Gly- Ser-Gly sequence), that further promotes cleavage.

[0114] A linker can encode an Internal Ribosome Entry Site (IRES), such that separate polypeptides encoded by the first and second genes are produced during translation. A linker can encode a splice acceptor, such as a viral splice acceptor.

[0115] A linker can be a combination of linkers, such as a Furin-2A linker that can produce separate polypeptides through 2A ribosome skipping followed by further cleavage of the Furin site to allow for complete removal of 2A residues. In some embodiments, a combination of linkers can include a Furin sequence, a flexible linker, and 2A linker. Accordingly, in some embodiments, the linker is a Furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, a linker of the present disclosure is a Furin-Gly-Ser-Gly-T2A fusion polypeptide.

[0116] In general, a multicistronic system can use any number or combination of linkers, to express any number of genes or portions thereof (e.g., an engineered nucleic acid can encode a first, a second, and a third chimeric protein, each separated by linkers such that separate polypeptides encoded by the first, second, and third chimeric proteins are produced). In general, the exogenous polynucleotides encoding each of the membrane-cleavable chimeric protein, the iCAR, and the bivalent aCAR can be encoded in any order. For example, the membrane-cleavable chimeric protein, the iCAR, and the bivalent aCAR can be encoded by the multicistronic system in order from 5’ to 3’: (i) membrane-cleavable chimeric protein; (ii) bivalent aCAR; and (iii) iCAR. In another example, the membrane-cleavable chimeric protein, the iCAR, and the bivalent aCAR can be encoded by the multicistronic system in order from 5’ to 3’: i) membrane-cleavable chimeric protein; (ii) iCAR; and (iii) bivalent aCAR.

[0117] Methods of Cell Therapy Treatment

[0118] Provided herein are methods that include delivering, or administering, to a subject (e.g., a human subject) engineered cells as provided herein as a cell therapy to produce in vivo at least one protein of interest produced by the engineered cells (e.g., a recombinant cytokine, such as controlled-release cytokines including crIL-15, and / or any of the chimeric proteins provided for herein, such as any of the multicistronic systems encoding each of (i) a membrane-cleavable chimeric protein; (ii) a bivalent aCAR; and (iii) an iCAR described herein, or the secreted effector molecules provided for herein following protease cleavage of the chimeric protein). Further provided herein are methods that include delivering, or administering, to a subject (e.g., a human subject) engineered cells as provided herein to produce in vivo at least two proteins of interest, e.g., at least two of the chimeric proteins provided for herein, such as each of (i) a membrane-cleavable chimeric protein; (ii) a bivalent aCAR; and (iii) an iCAR described herein, produced by the engineered cells.

[0119] Further provided herein are methods that include delivering, or administering, to a subject (e.g., a human subject) any of the delivery vehicles described herein, such as any of the delivery vehicles described herein comprising any of the proteins of interest described herein, e.g., any of the chimeric proteins provided for herein, such as each of (i) a membrane-cleavable chimeric protein; (ii) a bivalent aCAR; and (iii) an iCAR described herein. Further provided herein are methods that include delivering, or administering, to a subject (e.g., a human subject) any of the delivery vehicles described herein, such as any of the delivery vehicles described herein comprising two or more proteins of, e.g., at least two of the chimeric proteins provided for herein, such as each of (i) a membrane-cleavable chimeric protein; (ii) a bivalent aCAR; and (iii) an iCAR described herein.

[0120] In general, each of the cell therapies described herein are administered as part of therapy that also includes an IL-2 cytokine therapy, described in further detail herein (e.g., see “Combination Therapies”). In some embodiments, the engineered cells or delivery vehicles are administered via intravenous, intraperitoneal, intratracheal, subcutaneous, intratumoral, oral, anal, intranasal (e.g., packed in a delivery particle), or arterial (e.g., internal carotid artery) routes. Thus, the engineered cells or delivery vehicles may be administered systemically or locally (e.g., to a TME or via intratumoral administration). An engineered cell can be isolated from a subject, such as a subject known or suspected to have cancer. An engineered cell can be allogenic with reference to the subject being administered a treatment. Allogenic modified cells can be HLA- matched to the subject being administered a treatment. Delivery vehicles can be any of the lipid structure delivery systems described herein. Delivery vehicles can be any of the nanoparticles described herein.

[0121] Engineered cells or delivery vehicles can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. For example, engineered cells or delivery vehicles can be administered in combination with one or more IMiDs described herein. FDA-approved IMiDs can be administered in their approved fashion. In another example, engineered cells or delivery vehicles can be administered in combination with a checkpoint inhibitor therapy. Exemplary checkpoint inhibitors include, but are not limited to, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-PD-L2 antibodies, anti- CTLA-4 antibodies, anti-LAG-3 antibodies, anti-TIM-3 antibodies, anti-TIGIT antibodies, anti- VISTA antibodies, anti-KIR antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti- HVEM antibodies, anti-BTLA antibodies, anti-GAL9 antibodies, anti-A2AR antibodies, anti- phosphatidylserine antibodies, anti-CD27 antibodies, anti-TNFa antibodies, anti-TREMl antibodies, and anti-TREM2 antibodies. Illustrative immune checkpoint inhibitors include pembrolizumab (anti-PD-1; MK-3475 / Keytruda® - Merck), nivolumamb (anti-PD-1; Opdivo® - BMS), pidilizumab (anti-PD-1 antibody; CT-011 - Teva / CureTech), AMP224 (anti-PD-1; NCI), avelumab (anti-PD-Ll; Bavencio® - Pfizer), durvalumab (anti-PD-Ll; MEDI4736 / Imfinzi® - Medimmune / AstraZeneca), atezolizumab (anti-PD-Ll; Tecentriq® - Roche / Genentech), BMS- 936559 (anti-PD-Ll - BMS), tremelimumab (anti-CTLA-4; Medimmune / AstraZeneca), ipilimumab (anti-CTLA-4; Yervoy ® - BMS), lirilumab (anti-KIR; BMS), monalizumab (anti- NKG2A; Innate Pharma / AstraZeneca). In other examples, engineered cells or delivery vehicles can be administered in combination with TGFbeta inhibitors, VEGF inhibitors, or HPGE2. In another example, engineered cells or delivery vehicles can be administered in combination with an anti-CD40 antibody.

[0122] Some methods comprise selecting a subject (or patient population) having a tumor (or cancer) and treating that subject with engineered cells or delivery vehicles that modulate tumor- mediated immunosuppressive mechanisms. The engineered cells or delivery vehicles of the present disclosure may be used, in some instances, to treat cancer, such as ovarian cancer. Other cancers are described herein. For example, the engineered cells may be used to treat bladder tumors, brain tumors, breast tumors, cervical tumors, colorectal tumors, esophageal tumors, gliomas, kidney tumors, liver tumors, lung tumors, melanomas, ovarian tumors, pancreatic tumors, prostate tumors, skin tumors, thyroid tumors, and / or uterine tumors. The engineered cells or delivery vehicles of the present disclosure can be used to treat cancers with tumors located in the peritoneal space of a subject.

[0123] The methods provided herein also include delivering a preparation of engineered cells or delivery vehicles. A preparation, in some embodiments, is a substantially pure preparation, containing, for example, less than 5% (e.g., less than 4%, 3%, 2%, or 1%) of cells other than engineered cells. A preparation may comprise IxlO5cells / kg to IxlO7cells / kg cells. Preparation of engineered cells or delivery vehicles can include pharmaceutical compositions having one or more pharmaceutically acceptable carriers. For example, preparations of engineered cells or delivery vehicles can include any of the engineered viruses, such as an engineered AAV virus, or any of the engineered viral vectors, such as AAV vector, described herein.

[0124] In vivo Expression

[0125] The methods provided herein also include delivering a composition in vivo capable of producing the engineered cells described herein, e.g., capable of delivering any of the engineered nucleic acids described herein to a cell in vivo. Such compositions include any of the viral-mediated delivery platforms, any of the lipid structure delivery systems, any of the nanoparticle delivery systems, any of the genomic editing systems, or any of the other engineering delivery systems described herein capable of engineering a cell in vivo.

[0126] The methods provided herein also include delivering a composition in vivo capable of producing any of the proteins of interest described herein, e.g., any of the multicistronic systems encoding each of (i) a membrane-cleavable chimeric protein; (ii) a bivalent aCAR; and (iii) an iCAR described herein. The methods provided herein also include delivering a composition in vivo capable of producing two or more of the proteins of interest described herein. Compositions capable of in vivo production of proteins of interest include, but are not limited to, any of the engineered nucleic acids described herein. Compositions capable of in vivo production proteins of interest can be a naked mRNA or a naked plasmid.

[0127] Combination Therapies

[0128] In some embodiments, genetically modified cells of the present disclosure (e.g., immunoresponsive cells) expressing one or more proteins including an antigen-binding domain (e.g., scFv) of the present disclosure, such as a chimeric receptor of the present disclosure (e.g., an iCAR and / or an aCAR), and including a controlled-release cytokine (also referred to as a calibrated release cytokine or a membrane-cleavable cytokine herein) of the present disclosure (e.g., controlled-release crIL15) may be used in combination with other known agents and therapies.

[0129] In general, each of the cell therapies described herein (e.g., any of the engineered immunoresponsive cells described herein) are administered as part of therapy that also includes an IL-2 cytokine therapy.

[0130] In some embodiments, administration of the IL-2 cytokine can comprise multiple administrations. In some embodiments, the multiple administrations of IL-2 is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 administrations. The IL-2 cytokine can be administered once a day or more than once a day, e.g., twice a day, three times a day, or four times a day. The IL-2 cytokine can be administered for more than one day, e.g., the cytokine is administered for 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. For example, the IL-2 cytokine is administered once a day for 7 days.

[0131] In some embodiments, the cytokine IL-2 can be administered simultaneously or concurrently with the genetically modified cells, e.g., administered on the same day. In some embodiments, the IL-2 administration is within 4, within 5, within 6, within 7, or within 8 hours following the start of the administration of the genetically modified cells. In some embodiments, the IL-2 administration is within 4 hours following the start of the administration of the genetically modified cells. In some embodiments, the IL-2 administration is within 5 hours following the start of the administration of the genetically modified cells. In some embodiments, the IL-2 administration is within 6 hours following the start of the administration of the genetically modified cells. In some embodiments, the IL-2 administration is within 7 hours following the start of the administration of the genetically modified cells. In some embodiments, the IL-2 administration is within 8 hours following the start of the administration of the genetically modified cells. In some embodiments, the IL-2 administration is within 4 to 8 hours following the start of the administration of the genetically modified cells. In some embodiments, the IL-2 administration is within 6 to 8 hours following the start of the administration of the genetically modified cells. In some embodiments, the IL-2 administration is between 4 to 8 hours following the start of the administration of the genetically modified cells. In some embodiments, the IL-2 administration is between 6 to 8 hours following the start of the administration of the genetically modified cells. In some embodiments, the IL-2 cytokine can be administered within 24 or 48 hours after the administering of the genetically modified cells. The IL-2 cytokine may be prepared in the same pharmaceutical composition as the genetically modified cells, or may be prepared in a separate pharmaceutical composition. Alternatively, the IL-2 cytokine can be administered shortly after administration of the genetically modified cells, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the genetically modified cells. In some embodiments the administration of the genetically modified cells comprises multiple administrations. In some embodiments the administration of the genetically modified cells comprises multiple administrations and the cytokine IL-2 can be administered simultaneously or concurrently with the genetically modified cells, e.g., administered on the same day. In some embodiments where the IL-2 cytokine is administered in a dosing regimen that occurs over more than one day, the first day of the IL-2 cytokine dosing regimen can be on the same day as administration with the genetically modified cells, or the first day of the IL-2 cytokine dosing regimen can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the genetically modified cells. In one embodiment, on the first day, the genetically modified cells are administered to the subject, and on the second day, the IL-2 cytokine is administered once a day for the next 7 days. In some embodiments, the IL-2 cytokine is administered at least 1, at least 2, a least 3 times a week following each administration of the genetically modified cells. In some embodiments, the IL-2 cytokine is administered at least 1 time a week following each administration of the genetically modified cells. In some embodiments, the IL-2 cytokine is administered at least 2 times a week following each administration of the genetically modified cells. In some embodiments, the IL-2 cytokine is administered at least 3 times a week following each administration of the genetically modified cells. In some embodiments, the IL-2 cytokine is administered for a period of time after administration of the genetically modified cells, e.g., at least 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more after administration of the genetically modified cells. In one embodiment, the IL-2 cytokine is administered after assessment of the subject's response to the genetically modified cells.

[0132] In some embodiments, the genetically modified cells are administered again on day 7 and / or day 14 following the initial administration of the genetically modified cells. In some embodiments, the genetically modified cells are administered again on day 7 following the initial administration of the genetically modified cells. In some embodiments, the genetically modified cells are administered again on day 7 and day 14 following the initial administration of the genetically modified cells. In some embodiments, the genetically modified cells and the IL-2 cytokine are administered again on day 7 and / or day 14 following the initial administration of the genetically modified cells. In some embodiments, the genetically modified cells and the IL-2 cytokine are administered again on day 7 following the initial administration of the genetically modified cells. In some embodiments, the genetically modified cells and the IL-2 cytokine are administered again on day 7 and day 14 following the initial administration of the genetically modified cells.

[0133] In some embodiments, IL-2 cytokine is administered at a pharmaceutically effective dose. In some embodiments, the pharmaceutically effective dose of IL-2 is 3 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is at least about 1 - 10 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is at least about 1 - 9 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is at least about 1 - 8 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is at least about 1 - 7 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is at least about 1 - 6 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is at least about 1 - 5 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is at least about 1 - 4 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is at least about 1 - 3 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is at least about 1 - 2 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is at least about 2 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is at least about 3 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is at least about 4 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is at least about 5 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is at least about 6 MIU.

[0134] In some embodiments, the pharmaceutically effective dose of IL-2 is between 1 - 10 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is between 1 - 10 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is between 1 - 9 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is between 1 - 8 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is between 1 - 7 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is between 1 - 6 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is between 1 - 5 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is between 1 - 4 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is between 1 - 3 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is between 1 - 2 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is 2 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is 4 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is 5 MIU. In some embodiments, the pharmaceutically effective dose of IL-2 is 6 MIU.

[0135] In some embodiments, a combination therapy of the present disclosure comprises a genetically modified cells of the present disclosure that can be administered in combination with one or more additional therapeutic agents, including therapeutic agents in addition to IL-2. In some embodiments, the genetically modified cell and the one or more additional therapeutic agents, including IL-2 itself, can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the genetically modified can be administered first, and the one or more additional agents can be administered second, or the order of administration can be reversed. In some embodiments, the genetically modified cells are further modified to express one or more additional therapeutic agents.

[0136] In some embodiments, a genetically modified cell of the present disclosure may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents (e.g., cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506), antibodies, or other immunoablative agents (e.g., CAMPATH or anti-CD3 antibodies), cytoxin, fludarabme, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, irradiation, and peptide vaccines.

[0137] In some embodiments, a genetically modified cell of the present disclosure (e.g., immunoresponsive cells) may be used in combination with a lymphodepleting agent. Suitable lymphodepleting agents reduce or decrease lymphocytes, e.g., B cell lymphocytes and / or T cell lymphocytes, prior to immunotherapy. Examples of suitable lymphodepleting agents include, without limitation, fludarabine, cytarabine (Ara-C), cyclophosphamide, corticosteroids, alemtuzumab, total body irradiation (TBI), and any combination thereof. In some embodiments, the lymphodepletion agents are administered 5 days prior to administration of the genetically modified cells (e.g., any of the cell therapies described herein). In some embodiments, the lymphodepletion agents are administered daily until administration of the genetically modified cells. In some embodiments, the lymphodepletion agents are fludarabine administered at a dose of 30 mg / m2 and cytarabine (Ara-C) administered at a dose of 2 g / m2. In some embodiments, the lymphodepletion agents are fludarabine administered at a dose of 30 mg / m2 and cytarabine (Ara-C) administered at a dose of 2 g / m2 administered 5 days prior to administration of the genetically modified cells.

[0138] In some embodiments, a genetically modified cell of the present disclosure may be used in combination with a chemotherapeutic agent. Suitable chemotherapeutic agents include, without limitation, an anthracycline (e.g., doxorubicin), a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), an immune cell antibody (e.g., alemtuzamab, gemtuzumab, rituximab, tositumomab), an antimetabolite (e.g., folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors, such as fludarabine), an mTOR inhibitor, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib), an immunomodulator such as thalidomide or a thalidomide derivative (e.g., lenalidomide).

[0139] Examples of general chemotherapeutic agents suitable for use in combination therapies include, without limitation, anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5- deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Piatinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5- fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine (difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idaniycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90 / MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

[0140] Examples of suitable alkylating agents include, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard®. Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Rev immune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, Temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and Dacarbazine (DTIC-Dome®). Additional exemplary alkylating agents include, without limitation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® and Temodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®); Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®); Altretamme (also known as hexamethylmelamine (HMM), Hexalen®); Carmustine (BiCNU®); Bendamustine (Treanda®); Busulfan (Busulfex® and Myleran®); Carboplatin (Paraplatin®); Lomustine (also known as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® and Neosar®); Dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-Dome®); Aitretamine (also known as hexamethylmelamine (HMM), Hexalen®); Ifosfamide (Ifex®); Prednumustine; Procarbazine (Matulane®); Mechlorethamine (also known as nitrogen mustard, mustine and mechloroethamine hydrochloride, Mustargen®); Streptozocin (Zanosar®); Thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and Bendamustine HC1 (Treanda®).

[0141] Examples of suitable mTOR inhibitors include, without limitation, temsirolimus, ridaforolimus (deferolimus), AP23573, MK8669, everolimus (Afimtor® or RAD001), rapamycin (AY22989, Sirolmius®), and XL765.

[0142] Examples of suitable immunomodulators include, without limitation, afutuzumab, pegfilgrastim (Neulasta®), lenalidomide (CC-5013, Revlimid®), thalidomide (Thalomid®), actimid (CC4047), and IRX-2.

[0143] Examples of suitable anthracyclines include, without limitation, doxorubicin (Adriamycin® and Rubex®); bleomycin (lenoxane®); daunorubicin (dauorubicin hydrochloride, daunomyem, and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposomal (daunorubicin citrate liposome, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®, Idamycin PES®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin; and desacet Iravidomycin.

[0144] Examples of suitable vinca alkaloids include, without limitation, vinorelbine tartrate (Navelbine®), Vincristine (Oncovin®), and Vindesine (Eldisine®)); vinblastine (also known as vinblastine sulfate, vincaleukoblastine and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbme®).

[0145] Examples of suitable proteosome inhibitors include, without limitation, bortezomib (Velcade®); carfilzomib; marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib (CEP- 18770); and ONX-0912.

[0146] In some embodiments, a genetically modified cell of the present disclosure is administered in combination with a CD20 inhibitor, e.g., an anti-CD20 antibody, or fragment thereof. Exemplary anti-CD20 antibodies include, without limitation, rituximab, ofatumumab, ocrelizumab, veltuzumab, obinutuzumab, TRU- 015 (Trubion Pharmaceuticals), ocaratuzumab, and Prol31921.

[0147] In some embodiments, a genetically modified cell of the present disclosure is administered in combination with an oncolytic virus. In some embodiments, oncolytic viruses are capable of selectively replicating in and triggering the death of or slowing the growth of a cancer cell. In some cases, oncolytic viruses have no effect or a minimal effect on non-cancer cells. Suitable oncolytic viruses include, without limitation, an oncolytic adenovirus, oncolytic Herpes Simplex Viruses, oncolytic retrovirus, oncolytic parvovirus, oncolytic vaccinia virus, oncolytic Sindbis virus, oncolytic influenza virus, or oncolytic RNA virus (e.g., oncolytic reovirus, oncolytic Newcastle Disease Virus (NDV), oncolytic measles virus, or oncolytic vesicular stomatitis virus (VSV)). In some embodiments, the oncolytic virus is a recombinant oncolytic virus.

[0148] In some embodiments, a genetically modified cell of the present disclosure is administered to a subject in combination with a protein tyrosine phosphatase inhibitor, e.g., a SHP-I inhibitor or a SHP-2 inhibitor. In one embodiment, a genetically modified cell of the present disclosure can be used in combination with a kinase inhibitor. Examples of suitable kinase inhibitors include, without limitation, CDK4 inhibitors, CDK4 / 6 inhibitors, BTK inhibitors, phosphatidylinositol 3 -kinase (PI3K) inhibitors, mTOR inhibitors, MNK inhibitors, and anaplastic lymphoma kinase (ALK) inhibitors.

[0149] In some embodiments, a genetically modified cell of the present disclosure is administered to a subject in combination with a modulator of myeloid-derived suppressor cells (MDSCs). MDSCs accumulate in the periphery and at the tumor site of many solid tumors. These cells suppress T cell responses, thereby hindering the efficacy of chimeric receptor- expressing cell therapy. Without being bound by theory, it is believed that administration of a MDSC modulator enhances the efficacy of a genetically modified cell of the present disclosure. Examples of suitable modulators of MDSCs include, without limitation, MCS110 and BLZ945.

[0150] In some embodiments, a genetically modified cell of the present disclosure is administered to a subject in combination with an agent that inhibits or reduces the activity of immunosuppressive plasma cells. Immunosuppressive plasma cells have been shown to impede T cell-dependent immunogenic chemotherapy, such as oxaliplatin (Shalapour et al., Nature 2015, 521 :94- 101). In one embodiment, immunosuppressive plasma cells can express one or more of IgA, interleukin (IL)- 10, and PD-L1.

[0151] In some embodiments, a genetically modified cell of the present disclosure is administered to a subject in combination with an interleukin- 15 (IL- 15) polypeptide, an interleukin- 15 receptor alpha (IL-I5Ra) polypeptide, or a combination of both an IL- 15 polypeptide and an IL-15Ra polypeptide. In some embodiments, a genetically modified cell of the present disclosure is further modified to express an interleukin- 15 (IL-15) polypeptide, an interleukin- 15 receptor alpha (IL-I5Ra) polypeptide, or a combination of both an IL- 15 polypeptide and an IL-15Ra polypeptide. In some embodiments, the interleukin- 15 (IL- 15) polypeptide expressed by the genetically modified cells is controlled-release IL- 15 (crIL15).

[0152] In some embodiments, a subject having cancer (e.g., AML) is administered a genetically modified cell of the present disclosure in combination with an agent, e.g., cytotoxic or chemotherapy agent, a biologic therapy (e.g., antibody, e.g., monoclonal antibody, or cellular therapy), or an inhibitor (e.g., kinase inhibitor). In some embodiments, the subject is administered a genetically modified cell of the present disclosure in combination with a cytotoxic agent, e.g., CPX-351 (Celator Pharmaceuticals), cytarabme, daunorubicin, vosaroxin (Sunesis Pharmaceuticals), sapacitabine (Cyclacel Pharmaceuticals), idarubicin, or mitoxantrone. CPX-351 is a liposomal formulation comprising cytarabine and daunorubicin at a 5: 1 molar ratio. In some embodiments, the subject is administered a chimeric receptor- expressing cell described herein in combination with a hypomethylating agent, e.g., a DNA methyltransferase inhibitor, e.g., azacytidine or decitabine. In some embodiments, the subject is administered a genetically modified cell of the present disclosure in combination with a biologic therapy, e.g., an antibody or cellular therapy, e.g., 225Ac-lintuzumab (Actimab-A; Actinium Pharmaceuticals), IPH2102 (Innate Pharma / Bristol Myers Squibb), SGN-CD33A (Seattle Genetics), or gemtuzumab ozogamicin (Mylotarg; Pfizer). In some embodiments, the subject is administered a genetically modified cell of the present disclosure in combination a FLT3 inhibitor, e.g., sorafenib (Bayer), midostaurin (Novartis), quizartinib (Daiichi Sankyo), crenoianib (Arog Pharmaceuticals), PLX3397 (Daiichi Sankyo), AKN-028 (Akinion Pharmaceuticals), or ASP2215 (Astelias). In some embodiments, the subject is administered a genetically modified cell of the present disclosure in combination with an isocitrate dehydrogenase (IDH) inhibitor, e.g., AG-221 (Celgene / Agios) or AG- 120 (Agios / Celgene). In some embodiments, the subject is administered a genetically modified cell of the present disclosure in combination with a cell cycle regulator, e.g., inhibitor of polo-like kinase 1 (Plkl), e.g., volasertib (Boehringer Ingelheim); or an inhibitor of cyclin-dependent kinase 9 (Cdk9), e.g., alvocidib (Tolero Pharmaceuticals / Sanofi Aventis). In some embodiments, the subject is administered a genetically modified cell of the present disclosure in combination with a B cell receptor signaling network inhibitor, e.g., an inhibitor of B-cell lymphoma 2 (Bel- 2), e.g., venetoclax (Abbvie / Roche); or an inhibitor of Button's tyrosine kinase (Btk), e.g., ibrutinib (Pharmacyclics / Johnson & Johnson Janssen Pharmaceutical). In some embodiments, the subject is administered a genetically modified cell of the present disclosure in combination with an inhibitor of Ml aminopeptidase; an inhibitor of histone deacetylase (HD AC), e.g., pracinostat (MEI Pharma); a multi-kinase inhibitor, e.g., rigosertib (Onconova Therapeutics / Baxter / SymBio); or a peptidic CXCR4 inverse agonist, e.g., BL-8040 (BioLineRx).

[0153] In some embodiments, a subject can be administered an agent which enhances the activity or fitness of a genetically modified cell of the present disclosure. For example, the agent may inhibit a molecule that modulates or regulates, e.g., inhibits, T cell function. In some embodiments, the molecule that modulates or regulates T cell function is an inhibitory molecule. In some embodiments, inhibitory molecules, such as Programmed Death 1 (PD-1) can decrease the ability of the genetically modified cell to mount an immune effector response. Examples of suitable inhibitory molecules include, without limitation, PD-1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LIR-1 (LILRB1), CD 160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF beta. Inhibition of a molecule that modulates or regulates, e.g., inhibits, T cell function, e.g., by inhibition at the DNA, RNA or protein level, can optimize the performance of genetically modified cells of the present disclosure. In some embodiments, an agent, e.g., an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription-activator like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), can be used to inhibit expression of an inhibitory molecule in the genetically modified cell. In one embodiment, the inhibitor is an shRNA. In some embodiments, a genetically modified cell of the present disclosure may be further modified to express an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription-activator like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), can be used to inhibit expression of an inhibitory molecule in the genetically modified cell.

[0154] In one embodiment, the agent that modulates or regulates, e.g., inhibits, T-cell function is inhibited within a genetically modified cell of the present disclosure. In such embodiments, a dsRNA molecule that inhibits expression of a molecule that modulates or regulates, e.g., inhibits, T-cell function is linked to the nucleic acid that encodes a component, e.g., all of the components, of a chimeric receptor of the present disclosure. In one embodiment, a nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T-cell function is operably linked to a promoter, e.g., a HI- or a U6-derived promoter such that the dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T-cell function is expressed, e.g., is expressed within a the genetically modified cell. In one embodiment the nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T-cell function is present on the same vector, e.g., a lentiviral vector, that comprises a nucleic acid molecule that encodes a component, e.g., all of the components, of the chimeric receptor. In such an embodiment the nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T-cell function is located on the vector, e.g., the lentiviral vector, 5'- or 3'- to the nucleic acid that encodes a component, e.g., all of the components, of the chimeric receptor. The nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T-cell function can be transcribed in the same or different direction as the nucleic acid that encodes a component, e.g., all of the components, of the chimeric receptor. In one embodiment the nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T-cell function is present on a vector other than the vector that comprises a nucleic acid molecule that encodes a component, e.g., all of the components, of the chimeric receptor. In one embodiment, the nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T-cell function it transiently expressed within the genetically modified cell. In one embodiment, the nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T-cell function is stably integrated into the genome of a genetically modified cell of the present disclosure.

[0155] In one embodiment, an agent that modulates or regulates, e.g., inhibits, T-cell function can be an antibody or antibody fragment that binds to an inhibitory molecule. For example, the agent can be an antibody or antibody fragment that binds to PD-1 , PD-L1, PD-L2 or CTLA4. In one embodiment, the agent is an antibody or antibody fragment that binds to TIM3. In one embodiment, the agent is an antibody or antibody fragment that binds to LAG3.

[0156] In some embodiments, the agent which enhances the activity of the genetically modified cell is a CEACAM inhibitor (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5 inhibitor). In one embodiment, the inhibitor of CEACAM is an anti-CEACAM antibody molecule. In one embodiment, the agent which enhances activity of a genetically modified cell of the present disclosure is miR- 17-92. In some embodiments, the agent which enhances the activity of the genetically modified cell is CD40L. In some embodiments, the agent which enhances the activity of the genetically modified cell is GM-CSF. In some embodiments, a genetically modified cell of the present disclosure is further modified to express an antibody or antibody fragment that binds to an inhibitory molecule of the present disclosure. In one embodiment, the agent which enhances activity of a genetically modified cell of the present disclosure is a cytokine. Cytokines have important functions related to immunoresponsive cell expansion, differentiation, survival, and homeostats. Cytokines that can be administered to the subject receiving a genetically modified cell of the present disclosure include, without limitation, IL-4, IL-7, IL-9, IIL-12, L-15, IL-18, and IL-21, or a combination thereof, and can include cytokines in addition to the IL-2 cytokine therapies described herein. The cytokine, inclusive of IL-2, can be administered once a day or more than once a day, e.g., twice a day, three times a day, or four times a day. The cytokine can be administered for more than one day, e.g., the cytokine is administered for 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. For example, the cytokine is administered once a day for 7 days. In some embodiments, a genetically modified cell of the present disclosure is further modified to express one or more cytokines, such as IL-4, IL-7, IL-9, IL-12, L-15, IL-18, and IL- 21, and can also include a genetically modified cell modified to express IL-2. Cells modified to express one or more cytokines can be the same cell modified to express a protein of interest described herein (e.g., a recombinant cytokine, such as conditional release cytokines including crIL-15, and / or any of the chimeric proteins provided for herein, such as any of the multicistronic systems encoding each of (i) a membrane-cleavable chimeric protein; (ii) a bivalent aCAR; and (iii) an iCAR described herein, or the secreted effector molecules provided for herein following protease cleavage of the chimeric protein). Cells modified to express one or more cytokines can be a different cell modified to express a protein of interest described herein.

[0157] In some embodiments, the cytokine can be administered simultaneously or concurrently with the genetically modified cells, e.g., administered on the same day. The cytokine may be prepared in the same pharmaceutical composition as the genetically modified cells, or may be prepared in a separate pharmaceutical composition. Alternatively, the cytokine can be administered shortly after administration of the genetically modified cells, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the genetically modified cells. In some embodiments where the cytokine is administered in a dosing regimen that occurs over more than one day, the first day of the cytokine dosing regimen can be on the same day as administration with the genetically modified cells, or the first day of the cytokine dosing regimen can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the genetically modified cells. In one embodiment, on the first day, the genetically modified cells are administered to the subject, and on the second day, a cytokine is administered once a day for the next 7 days. In some embodiments, the cytokine is administered for a period of time after administration of the genetically modified cells, e.g., at least 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more after administration of the genetically modified cells. In one embodiment, the cytokine is administered after assessment of the subject's response to the genetically modified cells.

[0158] Membrane Cleavable Chimeric Proteins

[0159] Multicistronic systems herein encode membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S, oriented from N-terminal to C-terminal and expressed as a single polypeptide. S refers to a secretable effector molecule. C refers to a protease cleavage site. MT refers to a cell membrane tethering domain. The membrane-cleavable chimeric protein is engineered such that secretion of the effector molecule can be regulated in a protease- dependent manner. Specifically, the membrane-cleavable chimeric protein is engineered such that secretion of the effector molecule can be regulated as part of a “Membrane-Cleavable” system, where incorporation of a protease cleavage site (“C”) and a cell membrane tethering domain (“MT”) allow for regulated secretion of an effector molecule in a protease-dependent manner. Without wishing to be bound by theory, the components of the Membrane-Cleavable system present in the membrane-cleavable chimeric protein generally regulate secretion through the below cellular processes:

[0160] MT: The cell membrane tethering domain contains a transmembrane domain (or a transmembrane-intracellular domain) that directs cellular-trafficking of the chimeric protein such that the protein is inserted into, or otherwise associated with, a cell membrane (“tethered”)

[0161] C: Following expression and localization of the chimeric protein into the cell membrane, the protease cleavage site directs cleavage of the chimeric protein such that the effector molecule is released (“secreted”) into the extracellular space. Generally, the protease cleavage site is protease-specific, including sites engineered to be protease- specific. The protease cleavage site can be selected or engineered to achieve optimal protein expression, cell-type specific cleavage, cell-state specific cleavage, and / or cleavage and release of the pay load at desired kinetics (e.g., ratio of membrane-bound to secreted chimeric protein levels)

[0162] Membrane-cleavable chimeric proteins are also described in International PCT Application No. WO2022098922, which is hereby incorporated by reference for all purposes.

[0163] In some aspects, membrane-cleavable chimeric proteins (or engineered nucleic acids encoding the membrane-cleavable chimeric proteins) are provided for herein having a protein of interest (e.g., any of the effector molecules described herein), a protease cleavage site, and a cell membrane tethering domain.

[0164] An “effector molecule,” refers to a molecule (e.g., a nucleic acid such as DNA or RNA, or a protein (polypeptide) or peptide) that binds to another molecule and modulates the biological activity of that molecule to which it binds. For example, an effector molecule may act as a ligand to increase or decrease enzymatic activity, gene expression, or cell signaling. Thus, in some embodiments, an effector molecule modulates (activates or inhibits) different immunomodulatory mechanisms. By directly binding to and modulating a molecule, an effector molecule may also indirectly modulate a second, downstream molecule.

[0165] In certain embodiments described herein (e.g., in general, for all membrane-cleavable chimeric proteins described herein), an effector molecule is a secretable effector molecule (e.g., referred to as “S” in the formula S - C - MT or MT - C - S for membrane-cleavable chimeric proteins described herein). Non-limiting examples of effector molecules include cytokines, chemokines, enzymes that modulate metabolite levels, growth factors, co-activation molecules, tumor microenvironment modifiers, ligands, peptides, enzymes, antibodies, antibodies or decoy molecules that modulate cytokines, homing molecules, and / or integrins.

[0166] The term “modulate” encompasses maintenance of a biological activity, inhibition (partial or complete) of a biological activity, and stimulation / activation (partial or complete) of a biological activity. The term also encompasses decreasing or increasing (e.g., enhancing) a biological activity. Two different effector molecules are considered to “modulate different tumor-mediated immunosuppressive mechanisms” when one effector molecule modulates a tumor-mediated immunosuppressive mechanism (e.g., stimulates T cell signaling) that is different from the tumor-mediated immunosuppressive mechanism modulated by the other effector molecule (e.g., stimulates antigen presentation and / or processing).

[0167] Modulation by an effector molecule may be direct or indirect. Direct modulation occurs when an effector molecule binds to another molecule and modulates activity of that molecule. Indirect modulation occurs when an effector molecule binds to another molecule, modulates activity of that molecule, and as a result of that modulation, the activity of yet another molecule (to which the effector molecule is not bound) is modulated.

[0168] In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism by at least one effector molecule results in an increase in an immuno stimulatory and / or anti- tumor immune response (e.g., systemically or in the tumor microenvironment) by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). For example, modulation of a tumor-mediated immunosuppressive mechanism may result in an increase in an immuno stimulatory and / or anti-tumor immune response by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%. In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism results in an increase in an immuno stimulatory and / or anti-tumor immune response 10-20%, 10-30%, 10- 40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20- 50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20-200%, 50-60%, 50-70%, 50-80%, 50- 90%, 50-100%, or 50-200%. It should be understood that “an increase” in an immuno stimulatory and / or anti-tumor immune response, for example, systemically or in a tumor microenvironment, is relative to the immunostimulatory and / or anti-tumor immune response that would otherwise occur, in the absence of the effector molecule(s).

[0169] In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism by at least one effector molecule results in an increase in an immuno stimulatory and / or anti- tumor immune response (e.g., systemically or in the tumor microenvironment) by at least 2 fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100 fold). For example, modulation of a tumor-mediated immunosuppressive mechanism may result in an increase in an immuno stimulatory and / or anti- tumor immune response by at least 3 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, or at least 100 fold. In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism results in an increase in an immuno stimulatory and / or anti- tumor immune response by 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100 fold.

[0170] Non-limiting examples of immuno stimulatory and / or anti-tumor immune mechanisms include T cell signaling, activity and / or recruitment, antigen presentation and / or processing, natural killer cell-mediated cytotoxic signaling, activity and / or recruitment, dendritic cell differentiation and / or maturation, immune cell recruitment, pro-inflammatory macrophage signaling, activity and / or recruitment, stroma degradation, immuno stimulatory metabolite production, stimulator of interferon genes (STING) signaling (which increases the secretion of IFN and Thl polarization, promoting an anti-tumor immune response), and / or Type I interferon signaling. An effector molecule may stimulate at least one (one or more) of the foregoing immuno stimulatory mechanisms, thus resulting in an increase in an immunostimulatory response. Changes in the foregoing immuno stimulatory and / or anti-tumor immune mechanisms may be assessed, for example, using in vitro assays for T cell proliferation or cytotoxicity, in vitro antigen presentation assays, expression assays (e.g., of particular markers), and / or cell secretion assays (e.g., of cytokines).

[0171] In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism by at least one effector molecule results in a decrease in an immunosuppressive response (e.g., systemically or in the tumor microenvironment) by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). For example, modulation of a tumor-mediated immunosuppressive mechanism may result in a decrease in an immunosuppressive response by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%. In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism results in a decrease in an immunosuppressive response 10- 20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20- 30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20-200%, 50-60%, 50- 70%, 50-80%, 50-90%, 50-100%, or 50-200%. It should be understood that “a decrease” in an immunosuppressive response, for example, systemically or in a tumor microenvironment, is relative to the immunosuppressive response that would otherwise occur, in the absence of the effector molecule(s).

[0172] In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism by at least one effector molecule results in a decrease in an immunosuppressive response (e.g., systemically or in the tumor microenvironment) by at least 2 fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100 fold). For example, modulation of a tumor-mediated immunosuppressive mechanism may result in a decrease in an immunosuppressive response by at least 3 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, or at least 100 fold. In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism results in a decrease in an immunosuppressive response by 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100 fold.

[0173] Non-limiting examples of immunosuppressive mechanisms include negative costimulatory signaling, pro-apoptotic signaling of cytotoxic cells (e.g., T cells and / or NK cells), T regulatory (Treg) cell signaling, tumor checkpoint molecule production / maintenance, myeloid-derived suppressor cell signaling, activity and / or recruitment, immunosuppressive factor / metabolite production, and / or vascular endothelial growth factor signaling. An effector molecule may inhibit at least one (one or more) of the foregoing immunosuppressive mechanisms, thus resulting in a decrease in an immunosuppressive response. Changes in the foregoing immunosuppressive mechanisms may be assessed, for example, by assaying for an increase in T cell proliferation and / or an increase in IFNy production (negative co- stimulatory signaling, Tregcell signaling and / or MDSC); Annexin V / PI flow staining (pro-apoptotic signaling); flow staining for expression, e.g., PDL1 expression (tumor checkpoint molecule production / maintenance); ELISA, LUMINEX®, RNA via qPCR, enzymatic assays, e.g., IDO tryptophan catabolism (immunosuppressive factor / metabolite production); and phosphorylation of PI3K, Akt, p38 (VEGF signaling). In some embodiments, effector molecules function additively: the effect of two effector molecules, for example, may be equal to the sum of the effect of the two effector molecules functioning separately. In other embodiments, effector molecules function synergistically: the effect of two effector molecules, for example, may be greater than the combined function of the two effector molecules.

[0174] Effector molecules that modulate tumor-mediated immunosuppressive mechanisms and / or modify tumor microenvironments may be, for example, secreted factors (e.g., cytokines, chemokines, antibodies, and / or decoy receptors that modulate extracellular mechanisms involved in the immune system), inhibitors (e.g., antibodies, antibody fragments, ligand TRAP and / or small blocking peptides), intracellular factors that control cell state (e.g., microRNAs and / or transcription factors that modulate the state of cells to enhance pro-inflammatory properties), factors packaged into exosomes (e.g., microRNAs, cytosolic factors, and / or extracellular factors), surface displayed factors (e.g., checkpoint inhibitors, TRAIL), and and / or metabolic genes (e.g., enzymes that produce / modulate or degrade metabolites or amino acids).

[0175] In some embodiments, at least one of the effector molecules stimulates an immuno stimulatory mechanism in the tumor microenvironment and / or inhibits an immunosuppressive mechanism in the tumor microenvironment.

[0176] In some embodiments, at least one of the effector molecules (a) stimulates T cell signaling, activity and / or recruitment, (b) stimulates antigen presentation and / or processing, (c) stimulates natural killer cell-mediated cytotoxic signaling, activity and / or recruitment, (d) stimulates dendritic cell differentiation and / or maturation, (e) stimulates immune cell recruitment, (f) stimulates pro-inflammatory macrophage signaling, activity and / or recruitment or inhibits anti-inflammatory macrophage signaling, activity and / or recruitment, (g) stimulates stroma degradation, (h) stimulates immuno stimulatory metabolite production, (i) stimulates Type I interferon signaling, (j) inhibits negative costimulatory signaling, (k) inhibits pro- apoptotic signaling of anti-tumor immune cells, (1) inhibits T regulatory (Treg) cell signaling, activity and / or recruitment, (m) inhibits tumor checkpoint molecules, (n) stimulates stimulator of interferon genes (STING) signaling, (o) inhibits myeloid-derived suppressor cell signaling, activity and / or recruitment, (p) degrades immunosuppressive factors / metabolites, (q) inhibits vascular endothelial growth factor signaling, and / or (r) directly kills tumor cells.

[0177] In some embodiments, effector molecules may be selected from the following non- limiting classes of molecules: cytokines, antibodies, chemokines, nucleotides, peptides, and enzymes. Non-limiting examples of the foregoing classes of effector molecules are listed in Table 1 and specific sequences encoding exemplary effector molecules are listed in Table 2. Effector molecules can be human, such as those listed in Table 1 or Table 2 or human equivalents of murine effector molecules listed in Table 1 or Table 2. Effector molecules can be human-derived, such as the endogenous human effector molecule or an effector molecule modified and / or optimized for function, e.g., codon optimized to improve expression, modified to improve stability, or modified at its signal sequence (see below). Various programs and algorithms for optimizing function are known to those skilled in the art and can be selected based on the improvement desired, such as codon optimization for a specific species (e.g., human, mouse, bacteria, etc.).

[0178] In some embodiments, the effector molecule comprises interleukin 12 (IL- 12), for example, p35 and p40 as a dimer that is generally referred to in the art as IL12p70. In some embodiments, the first effector molecule comprises an IL12p70 fusion protein. In some embodiments, the IL12p70 fusion protein is a human IL12p70 fusion protein. In some embodiments, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 203.

[0179] In some embodiments, the effector molecule comprises interleukin 15 (IL- 15). In some embodiments, the effector molecule consists of IL-15 (see, e.g., SEQ ID NO: 199). In some embodiments, the effector molecule comprises a fusion protein including IL- 15 and an extracellular portion of IL- 15 Receptor ex (IL-15RCX), such as the sushi domain as shown in SEQ ID NO: 201. An exemplary IL-15 / IL-15Ra sushi domain fusion is provided as SEQ ID NO: 202. In some embodiments, the effector molecule includes IL- 15 having the amino acid sequence NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCELLELQVISLESGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEELQSEVHIVQMEINTS (SEQ ID NO: 224). In some embodiments, IL- 15 is encoded by a polynucleotide sequence comprising the sequence AATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCAT GCACATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGA CCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGAC GCCAGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAACAGCCTGTC CAGCAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAG AAGAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAA CACCTCA (SEQ ID NO: 225). In some embodiments, IL- 15 is encoded by a polynucleotide sequence that includes a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to AATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCAT GCACATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGA CCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGAC GCCAGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAACAGCCTGTC CAGCAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAG AAGAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAA CACCTCA (SEQ ID NO: 225).

[0180] In some embodiments, the membrane-cleavable chimeric protein includes the amino acid sequence MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAM KCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS FVHIVQMFINTSSGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQLLPSWAITLISV NGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 226). In some embodiments, the membrane-cleavable chimeric protein is encoded by a polynucleotide sequence comprising the sequence

[0181] ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCTAAT TGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCA CATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGACCG CCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCC AGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAACAGCCTGTCCAG CAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAG AACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACACC TCATCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGTGACCCCTGAGCCTAT CTTCAGCCTGATCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTC AATTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCT GCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGG CTGAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 227). In some embodiments, the membrane-cleavable chimeric protein is encoded by a polynucleotide sequence that includes a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to

[0182] ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCTAAT TGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCA CATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGACCG CCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCC AGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAACAGCCTGTCCAG CAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAG AACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACACC TCATCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGTGACCCCTGAGCCTAT

[0183] CTTCAGCCTGATCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTC

[0184] AATTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCT

[0185] GCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGG CTGAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 227).

[0186] Table 1. Exemplary Effector Molecules

[0187] Table 2: Exemplary effector molecule sequences

[0188] Secretion Signals and Signal- Anchors

[0189] The one or more effector molecules of the chimeric proteins provided for herein can be secretable effector molecules having a secretion signal peptide (also referred to as a signal peptide or signal sequence) at the chimeric protein’s N-terminus (e.g., an effector molecule’s N- terminus for S - C - MT) that direct newly synthesized proteins destined for secretion or membrane localization (also referred to as membrane insertion) to the proper protein processing pathways. For chimeric proteins having the formula MT - C - S, a membrane tethering domain generally has a signal-anchor sequence (e.g., signal-anchor sequences of a Type II transmembrane protein) that direct newly synthesized proteins destined for membrane localization to the proper protein processing pathways. For chimeric proteins having the formula S - C - MT, a membrane tethering domain having a reverse signal-anchor sequence (e.g., signal-anchor sequences of certain Type III transmembrane proteins) can be used, generally without a separate secretion signal peptide, that direct newly synthesized proteins destined for membrane localization to the proper protein processing pathways.

[0190] In general, for all membrane-cleavable chimeric proteins described herein, the one or more effector molecules are secretable effector molecules (referred to as “S” in the formula S - C - MT or MT - C - S). In embodiments with two or more chimeric proteins, each chimeric protein can comprise a secretion signal. In embodiments with two or more chimeric proteins, each chimeric protein can comprise a secretion signal such that each effector molecule is capable of secretion from an engineered cell following cleavage of the protease cleavage site.

[0191] The secretion signal peptide operably associated with an effector molecule can be a native secretion signal peptide (e.g., the secretion signal peptide generally endogenously associated with the given effector molecule). The secretion signal peptide operably associated with an effector molecule can be a non-native secretion signal peptide native secretion signal peptide. Non-native secretion signal peptides can promote improved expression and function, such as maintained secretion, in particular environments, such as tumor microenvironments. Non-limiting examples of non-native secretion signal peptide are shown in Table 3.

[0192] A secretion signal peptide can be an IgE signal peptide. An IgE signal peptide can include the amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 228). An IgE signal peptide can be encoded by a polynucleotide sequence that includes the sequence ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCT (SEQ ID NO: 229). An IgE signal peptide can be encoded by a polynucleotide sequence that includes a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCT (SEQ ID NO: 229).

[0193] A secretion signal peptide of the membrane-cleavable chimeric protein (e.g., IL-15) can be an IgE signal peptide. An IgE signal peptide of the membrane-cleavable chimeric protein (e.g., IL- 15) can include the amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 228). An IgE signal peptide of the membrane-cleavable chimeric protein (e.g., IL- 15) can be encoded by a polynucleotide sequence that includes the sequence ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCT (SEQ ID NO: 229). An IgE signal peptide of the membrane-cleavable chimeric protein (e.g., IL- 15) can be encoded by a polynucleotide sequence that includes a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCT (SEQ ID NO: 229).

[0194] Table 3. Exemplary Signal Secretion Peptides

[0195] Protease Cleavage Site

[0196] In certain embodiments, the chimeric proteins provided for herein (e.g., in general, for all membrane-cleavable chimeric proteins described herein) contain a protease cleavage site (e.g., referred to as “C” in the formula S - C - MT or MT - C - S for membrane-cleavable chimeric proteins described herein). In general, the protease cleavage site can be any amino acid sequence motif capable of being cleaved by a protease. Examples of protease cleavage sites include, but are not limited to, a Type 1 transmembrane protease cleavage site, a Type II transmembrane protease cleavage site, a GPI anchored protease cleavage site, an ADAM8 protease cleavage site, an ADAM9 protease cleavage site, an ADAM10 protease cleavage site, an ADAM12 protease cleavage site, an ADAM 15 protease cleavage site, an ADAM 17 protease cleavage site, an ADAM 19 protease cleavage site, an ADAM20 protease cleavage site, an ADAM21 protease cleavage site, an ADAM28 protease cleavage site, an ADAM30 protease cleavage site, an ADAM33 protease cleavage site, a BACE1 protease cleavage site, a BACE2 protease cleavage site, a SIP protease cleavage site, an MT1-MMP protease cleavage site, an MT3-MMP protease cleavage site, an MT5-MMP protease cleavage site, a furin protease cleavage site, a PCSK7 protease cleavage site, a matriptase protease cleavage site, a matriptase-2 protease cleavage site, an MMP9 protease cleavage site, or an NS3 protease cleavage site.

[0197] One example of a protease cleavage site is a hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease cleavage site, including, but not limited to, a NS3 / NS4A, a NS4A / NS4B, a NS4B / NS5A, or a NS5A / NS5B cleavage site. For a description of NS3 protease and representative sequences of its cleavage sites for various strains of HCV, see, e.g., Hepatitis C Viruses: Genomes and Molecular Biology (S.L. Tan ed., Taylor & Francis, 2006), Chapter 6, pp. 163-206; herein incorporated by reference in its entirety. For example, the sequences of HCV NS4A / 4B protease cleavage site; HCV NS5A / 5B protease cleavage site; C-terminal degron with NS4A / 4B protease cleavage site; N-terminal degron with HCV NS5A / 5B protease cleavage site are provided. Representative NS3 sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. YP_001491553, YP_001469631, YP_001469632, NP_803144, NP.671491, YP_001469634, YP-001469630, YP_001469633, ADA68311, ADA68307, AFP99000, AFP98987, ADA68322, AFP99033, ADA68330, AFP99056, AFP99041, CBF60982, CBF60817, AHH29575, AIZ00747, AIZ00744, ABI36969, ABN05226, KF516075, KF516074, KF516056, AB826684, AB826683, JX171009, JX171008, JX171000, EU847455, EF154714, GU085487, JX171065, JX171063; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference.

[0198] Another example of a protease cleavage site is an ADAM 17- specific protease (also referred to as Tumor Necrosis Factor-a Converting Enzyme [TACE]) cleavage site. An ADAM 17- specific protease cleavage site can be an endogenous sequence of a substrate naturally cleaved by ADAM 17. An ADAM 17- specific protease cleavage site can be an engineered sequence capable of being cleaved by ADAM 17. An engineered ADAM 17- specific protease cleavage site can be an engineered for specific desired properties including, but not limited to, optimal expression of the chimeric proteins, specificity for ADAM 17, rate-of- cleavage by ADAM 17, ratio of secreted and membrane-bound chimeric protein levels, and cleavage in different cell states. A protease cleavage site can be selected for specific cleavage by ADAM 17. For example, certain protease cleavage sites capable of being cleaved by ADAM 17 are also capable of cleavage by additional ADAM family proteases, such as ADAM10. Accordingly, an ADAM 17- specific protease cleavage site can be selected and / or engineered such that cleavage by other proteases, such as ADAM 10, is reduced or eliminated. A protease cleavage site can be selected for rate-of-cleavage by ADAM 17. For example, it can be desirable to select a protease cleavage site demonstrating a specific rate-of-cleavage by ADAM 17, such as reduced cleavage kinetics relative to an endogenous sequence of a substrate naturally cleaved by ADAM 17. In such cases, in general, a specific rate-of-cleavage can be selected to regulate the rate of processing of the chimeric protein, which in turn regulates the rate of release / secretion of the payload effector molecule. Accordingly, an ADAM 17- specific protease cleavage site can be selected and / or engineered such that the sequence demonstrates a desired rate-of-cleavage by ADAM 17. A protease cleavage site can be selected for both specific cleavage by ADAM 17 and rate-of-cleavage by ADAM17. Exemplary ADAM17-specific protease cleavage sites, including those demonstrating particular specificity and rate-of-cleavage kinetics, are shown in Table 4A below with reference to the site of cleavage (P5-P1: N-terminal; Pl'-P5': C-terminal). Further details of ADAM 17 and ADAM10, including expression and protease cleavage sites, are described in Sharma, et al. (J Immunol October 15, 2017, 199 (8) 2865-2872), Pham et al. (Anticancer Res. 2017 Oct;37(10):5507-5513), Caescu et al. (Biochem J. 2009 Oct 23; 424(1): 79-88), and Tucher et al. (J. Proteome Res. 2014, 13, 4, 2205-2214), each herein incorporated by reference for purposes.

[0199] Table 4A - Various ADAM17 Protease Cleavage Site Sequences

[0200] In some embodiments, the protease cleavage site comprises a first region having the amino acid sequence of PRAE (SEQ ID NO: 176). In some embodiments, the protease cleavage site comprises a second region having the amino acid sequence of KGG (SEQ ID NO: 177). In some embodiments, the first region is located N-terminal to the second region. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 178), wherein Xi is A, Y, P, S, or F, and wherein X2is V, L, S, I, Y, T, or A. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 178), wherein Xi is A, Y, P, S, or F, and wherein X2is V, L, S, I, Y, or T. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAVKGG (SEQ ID NO: 179). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEALKGG (SEQ ID NO: 180). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEYSKGG (SEQ ID NO: 181). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEPIKGG (SEQ ID NO: 182). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAYKGG (SEQ ID NO: 183). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAESSKGG (SEQ ID NO: 184). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEFTKGG (SEQ ID NO: 185). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAAKGG (SEQ ID NO: 186).

[0201] In some embodiments, the protease cleavage site comprises the amino acid sequence of DEPHYSQRR (SEQ ID NO: 187). In some embodiments, the protease cleavage site comprises the amino acid sequence of PPLGPIFNPG (SEQ ID NO: 188). In some embodiments, the protease cleavage site comprises the amino acid sequence of PLAQAYRSS (SEQ ID NO: 189). In some embodiments, the protease cleavage site comprises the amino acid sequence of TPIDSSFNPD (SEQ ID NO: 190). In some embodiments, the protease cleavage site comprises the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191). The protease cleavage sites of SEQ ID NOs: 187, 189, and 191 are cleavable by ADAM 17.

[0202] In some embodiments, the protease cleavage site can include an N-terminal peptide linker, such as SGGGGSGGGGSG (SEQ ID NO: 230). In some embodiments, the protease cleavage site can include a C-terminal peptide linker, such as GGGSGGGGSGGGSLQ (SEQ ID NO: 231). In some embodiments, the protease cleavage site can include an N-terminal peptide linker and a C-terminal peptide linker, such as both table (SEQ ID NO: 230) and GGGSGGGGSGGGSLQ (SEQ ID NO: 231).

[0203] In some embodiments, the protease cleavage site can include a Tumor Necrosis Factor-a Converting Enzyme (TACE )-specific cleavage site (also referred to as ADAM 17), an N- terminal peptide linker, and a C-terminal peptide linker. In some embodiments, the protease cleavage site can include the TACE -specific cleavage site VTPEPIFSLI (SEQ ID NO: 191), an N-terminal peptide linker, and a C-terminal peptide linker. In some embodiments, the protease cleavage site can include the TACE -specific cleavage site, an N-terminal peptide linker, and a C-terminal peptide linker and have the amino acid sequence SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ (SEQ ID NO: 250). In some embodiments, the protease cleavage site can include the TACE -specific cleavage site, an N- terminal peptide linker, and a C-terminal peptide linker encoded by a polynucleotide sequence comprising the sequence TCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGTGACCCCTGAGCCTATCTT CAGCCTGATCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAA (SEQ ID NO: 251). In some embodiments, the protease cleavage site can include the TACE - specific cleavage site, an N-terminal peptide linker, and a C-terminal peptide linker encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to TCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGTGACCCCTGAGCCTATCTT CAGCCTGATCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAA (SEQ ID NO: 251).

[0204] In some embodiments, the protease cleavage site comprises the amino acid sequence of ITQGLAVSTISSFF (SEQ ID NO: 198), which is a cleavage site that is native to CD16 and is cleavable by ADAM 17.

[0205] The protease cleavage site can be C-terminal of the secretable effector molecule. The protease cleavage site can be N-terminal of the secretable effector molecule. In general, for all membrane-cleavable chimeric proteins described herein, the protease cleavage site is either: (1) C-terminal of the secretable effector molecule and N-terminal of the cell membrane tethering domain (in other words, the protease cleavage site is in between the secretable effector molecule and the cell membrane tethering domain); or (2) N-terminal of the secretable effector molecule and C-terminal of the cell membrane tethering domain (also between the secretable effector molecule and the cell membrane tethering domain with domain orientation inverted). The protease cleavage site can be connected to the secretable effector molecule by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the effector molecule or protease cleavage site. The protease cleavage site can be connected to the cell membrane tethering domain by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane tethering domain or protease cleavage site. A polypeptide linker can be any amino acid sequence that connects a first polypeptide sequence and a second polypeptide sequence. A polypeptide linker can be a flexible linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, GSG linkers (e.g.. [GS]4GG [SEQ ID NO: 504 ]), A(EAAAK)3A (SEQ ID NO: 505 ), and Whitlow linkers (e.g., a “KEGS”(SEQ ID NO: 506) linker such as the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), an eGK linker such as the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 185), and linkers described in more detail in Issued U.S. Pat. No. 5,990,275 herein incorporated by reference). Additional exemplary polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, and SEQ ID NO: 197. Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition etc.) and are known to those skilled in the art. In the Membrane-Cleavable system, following expression and localization of the chimeric protein into the cell membrane, the protease cleavage site directs cleavage of the chimeric protein such that the effector molecule is released (“secreted”) into the extracellular space of a cell.

[0206] In general, a protease that cleaves the protease cleavage site is a protease specific for that specific protease cleavage site. For example, in the case of a disintegrin and metalloproteinase (“ADAM”) family protease, the protease that cleaves a specific ADAM protease cleavage site is generally limited to the ADAM protease(s) that specifically recognize the specific ADAM protease cleavage site motif. A protease cleavage site can be selected and / or engineered such that cleavage by undesired proteases is reduced or eliminated. Proteases can be membrane- bound or membrane-associated. Proteases can be secreted, e.g., secreted in a specific cellular environment, such as a tumor microenvironment (“TME”).

[0207] A protease that cleaves the protease cleavage site of the chimeric protein can be expressed in the same cell that expresses the chimeric protein. A protease that cleaves the protease cleavage site of the chimeric protein can be endogenous to a cell expressing the chimeric protein. In other words, a cell engineered to express the chimeric protein can endogenously express the protease specific for the protease cleavage site present in the chimeric protein. Endogenous expression of the protease refers to both expression under generally homeostatic conditions (e.g., a cell generally considered to be healthy), and also to differential expression under non-homeostatic conditions (e.g., upregulated expression in a tumor cell). The protease cleavage site can be selected based on the known proteases endogenously expressed by a desired cell population. In such cases, in general, the cleavage of the protease cleavage site (and thus release / secretion of a payload) can be restricted to only those cells of interest due to the cell-restricted protease needing to come in contact with the protease cleavage site of chimeric protein expressed in the same cell. For example, and without wishing to be bound by theory, ADAM 17 is believed to be restricted in its endogenous expression to NK cell and T cells. Thus, selection of an ADAM17-specific protease cleavage site may restrict the cleavage of the protease cleavage site to NK cell and T cells co-expressing the chimeric protein. In other examples, a protease cleavage site can be selected for a specific tumor- associated protease known to be expressed in a particular tumor population of interest (e.g., in a specific tumor cell engineered to express the chimeric protein). Protease and / or expression databases can be used to select an appropriate protease cleavage site, such as selecting a protease cleavage site cleaved by a tumor-associated proteases through consulting Oncomine (www.oncomine.org), the European Bioinformatic Institute (www.ebi.ac.uk) in particular (www.ebi.ac.uk / gxa), PMAP (www.proteolysis.org), ExPASy Peptide Cutter (ca.expasy.org / tools / peptide cutter) and PMAP.Cut DB (cutdb.burnham.org), each of which is incorporated by reference for all purposes.

[0208] A protease that cleaves the protease cleavage site of the chimeric protein can be heterologous to a cell expressing the chimeric protein. For example, a cell engineered to express the chimeric protein can also be engineered to express a protease not generally expressed by the cell that is specific for the protease cleavage site present in the chimeric protein. A cell engineered to express both the chimeric protein and the protease can be engineered to express each from separate engineered nucleic acids or from a multicistronic systems (multicistronic and multi-promoter systems are described in greater detail in the Section herein titled “Multicistronic and Multiple Promoter Systems”). Heterologous proteases and their corresponding protease cleavage site can be selected as described above with reference to endogenous proteases.

[0209] A protease that cleaves the protease cleavage site of the chimeric protein can be expressed on a separate distinct cell than the cell that expresses the chimeric protein. For example, the protease can be generally expressed in a specific cellular environment, such as a tumor microenvironment. In such cases, in general, the cleavage of the protease cleavage site can be restricted to only those cellular environments of interest (e.g., a tumor microenvironment) due to the environment-restricted protease needing to come in contact with the protease cleavage site. In embodiments having membrane-cleavable chimeric proteins, in general, the secretion of the effector molecule can be restricted to only those cellular environments of interest (e.g., a tumor microenvironment) due to the environment-restricted protease needing to come in contact with the protease cleavage site. A protease that cleaves the protease cleavage site of the chimeric protein can be endogenous to the separate distinct cell. A protease that cleaves the protease cleavage site of the chimeric protein can be heterologous to the separate distinct cell. For example, the separate distinct cell can be engineered to express a protease not generally expressed by the separate distinct cell.

[0210] Proteases include, but are not limited to, a Type 1 transmembrane protease, a Type II transmembrane protease, a GPI anchored protease, an ADAM8 protease, an ADAM9 protease, an ADAM 10 protease, an ADAM 12 protease, an ADAM 15 protease, an ADAM 17 protease, an ADAM 19 protease, an ADAM20 protease, an ADAM21 protease, an ADAM28 protease, an ADAM30 protease, an ADAM33 protease, a BACE1 protease, a BACE2 protease, a SIP protease, an MT1-MMP protease, an MT3-MMP protease, an MT5-MMP protease, a furin protease, a PCSK7 protease, a matriptase protease, a matriptase-2 protease, and an MMP9 protease. A protease can be an NS3 protease. A protease can be an ADAM 17 protease.

[0211] Proteases can be tumor associated proteases, such as, a cathepsin, a cysteine protease, an aspartyl protease, a serine protease, or a metalloprotease. Specific examples of tumor associated proteases include Cathepsin B, Cathepsin L, Cathepsin S, Cathepsin D, Cathepsin E, Cathepsin A, Cathepsin G, Thrombin, Plasmin, Urokinase, Tissue Plasminogen Activator, Metalloproteinase 1 (MMP1), MMP2, MMP3, MMP4, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP20, MMP21, MMP23, MMP24, MMP25, MMP26, MMP28, ADAM, AD AMTS, CD 10 (CALLA), or prostate specific antigen.

[0212] Proteases can also include, but are not limited to, proteases (including proteases with exemplary cognate protease cleavage sites) described in International Application Pub. No. WO2023205614, which is hereby incorporated by reference for all purposes.

[0213] Protease enzymatic activity can be regulated. For example, certain proteases can be inactivated by the presence or absence of a specific agent (e.g., that binds to the protease, such as specific small molecule inhibitors). Such proteases can be referred to as a “repressible protease.” Exemplary inhibitors for certain proteases are described in International Application Pub. No. WO2023205614, which is hereby incorporated by reference for all purposes. For example, an NS3 protease can be repressed by a protease inhibitor including, but not limited to, simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir. In another example, protease activity can be regulated through regulating expression of the protease itself, such as engineering a cell to express a protease using an inducible promoter system (e.g., Tet On / Off systems) or cell-specific promoters (promoters that can be used to express a heterologous protease are described in more detail in the Section herein titled “Promoters”). A protease can also contain a degron, such as any of the degrons described herein, and can be regulated using any of the degron systems described herein.

[0214] Protease enzymatic activity can also be regulated through selection of a specific protease cleavage site. For example, a protease cleavage site can be selected and / or engineered such that the sequence demonstrates a desired rate-of-cleavage by a desired protease, such as reduced cleavage kinetics relative to an endogenous sequence of a substrate naturally cleaved by the desired protease. As another example, a protease cleavage site can be selected and / or engineered such that the sequence demonstrates a desired rate-of-cleavage in a cell-state specific manner. For example, various cell states (e.g., following cellular signaling, such as immune cell activation) can influence the expression and / or localization of certain proteases. As an illustrative example, ADAM 17 protein levels and localization is known to be influenced by signaling, such as through Protein kinase C (PKC) signaling pathways (e.g., activation by the PKC activator Phorbol-12-myristat-13-acetat [PMA]). Accordingly, a protease cleavage site can be selected and / or engineered such that cleavage of the protease cleavage site and subsequent release of an effector molecule is increased or decreased, as desired, depending on the protease properties (e.g., expression and / or localization) in a specific cell state. As another example, a protease cleavage site (particularly in combination with a specific membrane tethering domain) can be selected and / or engineered for optimal protein expression of the chimeric protein.

[0215] Cell Membrane Tethering Domain

[0216] The membrane-cleavable chimeric proteins provided for herein contain a cell-membrane tethering domain (referred to as “MT” in the formula S - C - MT or MT - C - S). In general, the cell-membrane tethering domain can be any amino acid sequence motif capable of directing the chimeric protein to be localized to (e.g., inserted into), or otherwise associated with, the cell membrane of the cell expressing the chimeric protein. The cell-membrane tethering domain can be a transmembrane-intracellular domain. The cell-membrane tethering domain can be a transmembrane domain. The cell-membrane tethering domain can be an integral membrane protein domain (e.g., a transmembrane domain). The cell-membrane tethering domain can be derived from a Type I, Type II, or Type III transmembrane protein. The cell-membrane tethering domain can include post-translational modification tag, or motif capable of post-translational modification to modify the chimeric protein to include a post-translational modification tag, where the post-translational modification tag allows association with a cell membrane. Examples of post-translational modification tags include, but are not limited to, lipid-anchor domains (e.g., a GPI lipid- anchor, a myristoylation tag, or palmitoylation tag). Examples of cell- membrane tethering domains include, but are not limited to, a transmembrane-intracellular domain and / or transmembrane domain derived from PDGFR-beta, CD8, CD28, CD3zeta-chain, CD4, 4-1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, or BTLA. The cell membrane tethering domain can be a cell surface receptor or a cell membrane-bound portion thereof.

[0217] In some embodiments, the cell-membrane tethering domain comprises a transmembrane domain derived from a B7-1 polypeptide. In some embodiments, the B7-1 transmembrane domain comprises the sequence LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 204). In some embodiments, the B7-1 transmembrane domain is encoded by a polynucleotide sequence having the sequence TTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGC TGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCT GAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 252). In some embodiments, the B7-1 transmembrane domain is encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to TTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGC TGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCT GAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 252).

[0218] In some embodiments, the cell-membrane tethering domain of the membrane-cleavable chimeric protein (e.g., IL- 15) includes a transmembrane domain derived from a B7-1 polypeptide. In some embodiments, the B7-1 transmembrane domain of the membrane- cleavable chimeric protein (e.g., IL-15) includes the sequence LLPSWAITLISVNGIEVICCLTYCEAPRCRERRRNERLRRESVRPV (SEQ ID NO: 204). In some embodiments, the B7-1 transmembrane domain of the membrane-cleavable chimeric protein (e.g., IL-15) is encoded by a polynucleotide sequence having the sequence TTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGC TGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCT GAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 252). In some embodiments, the B7-1 transmembrane domain of the membrane-cleavable chimeric protein (e.g., IL-15) is encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to TTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGC TGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCT GAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 252).

[0219] In some embodiments, the cell-membrane tethering domain comprises a transmembrane domain derived from a CD8 polypeptide. Any suitable CD8 polypeptide may be used. Exemplary CD8 polypeptides include, without limitation, NCBI Reference Nos. NP_001139345 and AAA92533.1. Examples of CD8 transmembrane domains include IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 205), IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 206), and IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 207). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 205). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 206). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 207). In some embodiments, the cell- membrane tethering domain comprises a hinge and transmembrane domain derived from CD8.In some embodiments, the CD8 hinge comprises the sequence TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 271). In some embodiments, the CD8 hinge comprises the sequence AAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI WAPEAGTCGVEEESEVITEYCNHRN (SEQ ID NO: 209).

[0220] In general, for all membrane-cleavable chimeric proteins described herein, the cell membrane tethering domain is either: (1) C-terminal of the protease cleavage site and N- terminal of any intracellular domain, if present (in other words, the cell membrane tethering domain is in between the protease cleavage site and, if present, an intracellular domain); or (2) N-terminal of the protease cleavage site and C-terminal of any intracellular domain, if present (also between the protease cleavage site and, if present, an intracellular domain with domain orientation inverted). In embodiments featuring a degron associated with the chimeric protein, the degron domain is the terminal cytoplasmic-oriented domain, specifically relative to the cell membrane tethering (in other words, the cell membrane tethering domain is in between the protease cleavage site and the degron). The cell membrane tethering domain can be connected to the protease cleavage site by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of cell membrane tethering domain or protease cleavage site. The cell membrane tethering domain can be connected to an intracellular domain, if present, by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane tethering domain or the intracellular domain. The cell membrane tethering domain can be connected to the degron, if present, by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane tethering domain or degron. A polypeptide linker can be any amino acid sequence that connects a first polypeptide sequence and a second polypeptide sequence. A polypeptide linker can be a flexible linker (e.g., a Gly- Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS]4GG [SEQ ID NO: 504 ]), A(EAAAK)3A (SEQ ID NO: 505 ), and Whitlow linkers (e.g., a “KEGS” (SEQ ID NO: 506) linker such as the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), an eGK linker such as the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 185), and linkers described in more detail in Issued U.S. Pat. No. 5,990,275 herein incorporated by reference). Additional polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, and SEQ ID NO: 197. Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition etc.) and are known to those skilled in the art.

[0221] In general, the cell-membrane tethering domain is oriented such that the secreted effector molecule and the protease cleavage site are extracellularly exposed following insertion into, or association with, the cell membrane, such that the protease cleavage site is capable of being cleaved by its respective protease and releasing (“secreting”) the effector molecule into the extracellular space.

[0222] Degron Systems and Domains

[0223] In some embodiments, any of the proteins described herein can include a degron domain including, but not limited to, a protease, a transcription factor, a promoter or constituent of a promoter system (e.g., an ACP), and / or any of the membrane-cleavable chimeric protein described herein. In general, the degron domain can be any amino acid sequence motif capable of directing regulated degradation, such as regulated degradation through a ubiquitin-mediated pathway. In the presence of an immunomodulatory drug (IMiD), the degron domain directs ubiquitin-mediated degradation of a degron-fusion protein.

[0224] The degron domain can be a cereblon (CRBN) polypeptide substrate domain capable of binding CRBN in response to an immunomodulatory drug (IMiD) including, but not limited to, IKZF1, IKZF3, CKla, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827, or a fragment thereof that is capable of drug-inducible binding of CRBN. The CRBN polypeptide substrate domain can be a chimeric fusion product of native CRBN polypeptide sequences, such as a IKZF3 / ZFP91 / IKZF3 chimeric fusion product having the amino acid sequence of FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRD AL (SEQ ID NO: 175). Degron domains, and in particular CRBN degron systems, are described in more detail in International Application Pub. No. WO2019 / 089592A1, herein incorporated by reference for all purposes. Other examples of degron domains include, but are not limited to HCV NS4 degron, PEST (two copies of residues 277-307 of human IKBOI; SEQ ID NO: 161), GRR (residues 352-408 of human pl05; SEQ ID NO: 162), DRR (residues 210-295 of yeast Cdc34; SEQ ID NO: 163), SNS (tandem repeat of SP2 and NB (SP2-NB-SP2 of influenza A or influenza B; e.g., SEQ ID NO: 164), RPB (four copies of residues 1688-1702 of yeast RPB; SEQ ID NO: 165), SPmix (tandem repeat of SP1 and SP2 (SP2-SP1-SP2-SP1-SP2 of influenza A virus M2 protein; SEQ ID NO: 166), NS2 (three copies of residues 79-93 of influenza A virus NS protein; SEQ ID NO: 167), ODC (residues 106-142 of ornithine decarboxylase; SEQ ID NO: 168), Nek2A, mouse ODC (residues 422-461; SEQ ID NO: 169), mouse ODC_DA (residues 422-461 of mODC including D433A and D434A point mutations), an APC / C degron, a COP1 E3 ligase binding degron motif, a CRL4-Cdt2 binding PIP degron, an actinfilin-binding degron, a KEAP1 binding degron, a KLHL2 and KLHL3 binding degron, an MDM2 binding motif, an N-degron, a hydroxyproline modification in hypoxia signaling, a phytohormone-dependent SCF- LRR-binding degron, an SCF ubiquitin ligase binding phosphodegron, a phytohormone- dependent SCF-LRR-binding degron, a DSGxxS (SEQ ID NO: 509) phospho-dependent degron, an Siah binding motif, an SPOP SBC docking motif, or a PCNA binding PIP box.

[0225] Regulated degradation can be drug-inducible. Drugs capable of mediating / regulating degradation can be small-molecule compounds. Drugs capable of mediating / regulating degradation can include an “immunomodulatory drug” (IMiD). In general, as used herein, IMiDs refer to a class of small-molecule immunomodulatory drugs containing an imide group. Cereblon (CRBN) is known target of IMiDs and binding of an IMiD to CRBN or a CRBN polypeptide substrate domain alters the substrate specificity of the CRBN E3 ubiquitin ligase complex leading to degradation of proteins having a CRBN polypeptide substrate domain (e.g., any of secretable effector molecules or other proteins of interest described herein). For degron domains having a CRBN polypeptide substrate domain, examples of imide-containing IMiDs include, but are not limited to, a thalidomide, a lenalidomide, or a pomalidomide. The IMiD can be an FDA- approved drug.

[0226] Chimeric proteins described herein can contain a degron domain (e.g., referred to as “D” in the formula S - C - MT - D or D - MT - C - S for membrane-cleavable chimeric proteins described herein). In the absence of an IMiD, degron / ubiquitin-mediated degradation of the chimeric protein does not occur. Following expression and localization of the chimeric protein into the cell membrane, the protease cleavage site directs cleavage of the chimeric protein such that the effector molecule is released (“secreted”) into the extracellular space. In the presence of an immunomodulatory drug (IMiD), the degron domain directs ubiquitin-mediated degradation of the chimeric protein such that secretion of the effector molecule is reduced or eliminated. In general, for membrane-cleavable chimeric proteins fused to a degron domain, the degron domain is the terminal cytoplasmic-oriented domain, specifically relative to the cell membrane tethering domain, e.g., the most C-terminal domain in the formula S - C - MT - D or the most N-terminal domain in the formula D - MT - C - S. The degron domain can be connected to the cell membrane tethering domain by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane tethering domain or the degron domain. A polypeptide linker can be any amino acid sequence that connects a first polypeptide sequence and a second polypeptide sequence. A polypeptide linker can be a flexible linker (e.g., a Gly- Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS]4GG [SEQ ID NO: 504 ]), A(EAAAK)3A (SEQ ID NO: 505 ), and Whitlow linkers (e.g., a “KEGS”(SEQ ID NO: 506) linker such as the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), an eGK linker such as the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 185), and linkers described in more detail in Issued U.S. Pat. No. 5,990,275 herein incorporated by reference). Additional polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, and SEQ ID NO: 197. Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition etc.) and are known to those skilled in the art. In general, the degron is oriented in relation to the cell membrane tethering domain such that the degron is exposed to the cytosol following localization to the cell membrane such that the degron domain is capable of mediating degradation (e.g., exposure to the cytosol and cytosol ) and is capable of mediating ubiquitin- mediated degradation.

[0227] For degron-fusion proteins, the degron domain can be N-terminal or C-terminal of the protein of interest, e.g., the effector molecule. The degron domain can be connected to the protein of interest by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the protein of interest or the degron domain. A polypeptide linker can be any amino acid sequence that connects a first polypeptide sequence and a second polypeptide sequence. A polypeptide linker can be a flexible linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS]4GG [SEQ ID NO: 504 ]), A(EAAAK)3A (SEQ ID NO: 505 ), and Whitlow linkers (e.g., a “KEGS” (SEQ ID NO: 506) linker such as the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), an eGK linker such as the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 185), and linkers described in more detail in Issued U.S. Pat. No. 5,990,275 herein incorporated by reference). Additional polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, and SEQ ID NO: 197. Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition etc.) and are known to those skilled in the art. A polypeptide linker can be cleavable, e.g., any of the protease cleavage sites described herein.

[0228] Homing Molecules

[0229] A “tumor microenvironment” is the cellular environment in which a tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix (ECM) (see, e.g., Pattabiraman, D.R. & Weinberg, R.A. Nature Reviews Drug Discovery 13, 497-512 (2014); Balkwill, F.R. et al. J Cell Sci 125, 5591-5596, 2012; and Li, H. et al. J Cell Biochem 101(4), 805-15, 2007).

[0230] In some embodiments, engineered nucleic acids are configured to produce at least one homing molecule. For example, in membrane-cleavable chimeric proteins described herein containing a secreted effector molecule, the secreted effector molecule can be a homing molecule. “Homing,” refers to active navigation (migration) of a cell to a target site (e.g., a cell, tissue (e.g., tumor), or organ). A “homing molecule” refers to a molecule that directs cells to a target site. In some embodiments, a homing molecule functions to recognize and / or initiate interaction of an engineered cell to a target site. Non-limiting examples of homing molecules include CXCR1, CCR9, CXCR2, CXCR3, CXCR4, CCR2, CCR4, FPR2, VEGFR, IL6R, CXCR1, CSCR7, and PDGFR.

[0231] In some embodiments, a homing molecule is a chemokine receptor (cell surface molecule that binds to a chemokine). Chemokines are small cytokines or signaling proteins secreted by cells that can induce directed chemotaxis in cells. Chemokines can be classified into four main subfamilies: CXC, CC, CX3C and XC, all of which exert biological effects by binding selectively to chemokine receptors located on the surface of target cells. In some embodiments, engineered nucleic acids are configured to produce CXCR4, a chemokine receptor which allows engineered cells to home along a chemokine gradient towards a stromal cell-derived factor 1 (also known as SDF1, C-X-C motif chemokine 12, and CXCL12 )- expressing cell, tissue, or tumor. Non-limiting examples of chemokine receptors that may be encoded by the engineered nucleic acids of the present disclosure include: CXC chemokine receptors (e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7), CC chemokine receptors (CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, and CCR11), CX3C chemokine receptors (e.g., CX3CR1, which binds to CX3CL1), and XC chemokine receptors (e.g., XCR1). In some embodiments, a chemokine receptor is a G protein-linked transmembrane receptor, or a member of the tumor necrosis factor (TNF) receptor superfamily (including but not limited to TNFRSF1A, TNFRSF1B). In some embodiments, engineered nucleic acids are configured to produce CXCL8, CXCL9, and / or CXCL10 (promote T-cell recruitment), CCL3 and / or CXCL5, CCL21 (Thl recruitment and polarization).

[0232] In some embodiments, engineered nucleic acids are configured to produce G-protein coupled receptors (GPCRs) that detect N-formylated-containing oligopeptides (including but not limited to FPR2 and FPRL1).

[0233] In some embodiments, engineered nucleic acids are configured to produce receptors that detect interleukins (including but not limited to IL6R).

[0234] In some embodiments, engineered nucleic acids are configured to produce receptors that detect growth factors secreted from other cells, tissues, or tumors (including but not limited to FGFR, PDGFR, EGFR, and receptors of the VEGF family, including but not limited to VEGF-C and VEGF-D). In some embodiments, a homing molecule is an integrin. Integrins are transmembrane receptors that facilitate cell-extracellular matrix (ECM) adhesion. Integrins are obligate heterodimers having two subunits: a (alpha) and P (beta). The a subunit of an integrin may be, without limitation: ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, IGTA7, ITGA8, ITGA9, IGTA10, IGTA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGA2B, ITGAX. The p subunit of an integrin may be, without limitation: ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, and ITGB8. Engineered nucleic acids can be configured to produce any combination of the integrin a and P subunits.

[0235] In some embodiments, a homing molecule is a matrix metalloproteinase (MMP). MMPs are enzymes that cleave components of the basement membrane underlying the endothelial cell wall. Non-limiting examples of MMPs include MMP-2, MMP-9, and MMP. In some embodiments, engineered nucleic acids are configured to produce an inhibitor of a molecule (e.g., protein) that inhibits MMPs. For example, engineered nucleic acids can be configured to express an inhibitor (e.g., an RNAi molecule) of membrane type 1 MMP (MT1-MMP) or TIMP metallopeptidase inhibitor 1 (TIMP-1).

[0236] In some embodiments, a homing molecule is a ligand that binds to selectin (e.g., hematopoietic cell E- / L- selectin ligand (HCELL), Dykstran c / al., Stem Cells. 2016 Oct;34(10):2501-2511) on the endothelium of a target tissue, for example.

[0237] The term “homing molecule” also encompasses transcription factors that regulate the production of molecules that improve / enhance homing of cells.

[0238] In some embodiments, a homing molecule includes an antibody, such as anti-integrin alpha4,beta7 or anti-MAdCAM.

[0239] Chimeric Antigen Receptors (CARs)

[0240] Certain aspects of the present disclosure relate to chimeric receptors that have any one of the antigen- specific binding domains described herein (e.g., EMCN-specific, FLT3-specific, and / or CD33-specific) and are capable of specifically binding to a protein, an antigen-derived antigen, or an antigen-derived epitope.

[0241] In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). In general, CARs are chimeric proteins that include an antigen-binding domain and polypeptide molecules that are heterologous to the antigen-binding domain, such as peptides heterologous to an antibody that an antigen-binding domain may be derived from. Polypeptide molecules that are heterologous to the antigen-binding domain can include, but are not limited to, a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, or combinations thereof. In some embodiments, CARs are engineered receptors that graft or confer a specificity of interest (e.g., EMCN, FLT3, or CD33) onto an immune effector cell. In certain embodiments, CARs can be used to graft the specificity of an antibody onto an immunoresponsive cell, such as a T cell. In some embodiments, CARs of the present disclosure comprise an extracellular antigen-binding domain (e.g., an scFv) fused to a transmembrane domain, fused to one or more intracellular signaling domains.

[0242] In some embodiments, the chimeric antigen receptor is an activating chimeric antigen receptor (aCAR and also generally referred to as CAR unless otherwise specified). In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce activation of the immunoresponsive cell. In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce stimulation of the immunoresponsive cell. In some embodiments, activation of an immunoresponsive cell results in killing of target cells. In some embodiments, activation of an immunoresponsive cell results in cytokine or chemokine expression and / or secretion by the immunoresponsive cell. In some embodiments, stimulation of an immunoresponsive cell results in cytokine or chemokine expression and / or secretion by the immunoresponsive cell. In some embodiments, stimulation of an immunoresponsive cell induces differentiation of the immunoresponsive cell. In some embodiments, stimulation of an immunoresponsive cell induces proliferation of the immunoresponsive cell. In some embodiments, activation and / or stimulation of the immunoresponsive cell can be combinations of the above responses.

[0243] The number of ABDs in a binding molecule, such as the chimeric proteins described herein, defines the “valency” of the binding molecule. A binding molecule having a single ABD is “monovalent”. A binding molecule having a plurality of ABDs is said to be “multivalent”. A multivalent binding molecule having two ABDs is “bivalent.” A multivalent binding molecule having three ABDs is “bivalent.” A multivalent binding molecule having four ABDs is “tetravalent.” In various multivalent embodiments, all of the plurality of ABDs have the same recognition specificity and can be referred to as a “monospecific multivalent” binding molecule. In other multivalent embodiments, at least two of the plurality of ABDs have different recognition specificities. Such binding molecules are multivalent and “multispecific.” In multivalent embodiments in which the ABDs collectively have two recognition specificities, the binding molecule is “bispecific.” In multivalent embodiments in which the ABDs collectively have three recognition specificities, the binding molecule is “trispecific.” In multivalent embodiments in which the ABDs collectively have a plurality of recognition specificities for different epitopes present on the same antigen, the binding molecule is “multiparatopic.” Multivalent embodiments in which the ABDs collectively recognize two epitopes on the same antigen are “biparatopic.”

[0244] In various multivalent embodiments, multivalency of the binding molecule improves the avidity of the binding molecule for a specific target. As described herein, “avidity” refers to the overall strength of interaction between two or more molecules, e.g. a multivalent binding molecule for a specific target, wherein the avidity is the cumulative strength of interaction provided by the affinities of multiple ABDs. Avidity can be measured by the same methods as those used to determine affinity, as described above. In certain embodiments, the avidity of a binding molecule for a specific target is such that the interaction is a specific binding interaction, wherein the avidity between two molecules has a KD value below 10-6M, 10-7M, 10-8M, 10-9M, or 10-10M. In certain embodiments, the avidity of a binding molecule for a specific target has a KD value such that the interaction is a specific binding interaction, wherein the one or more affinities of individual ABDs do not have has a KD value that qualifies as specifically binding their respective antigens or epitopes on their own. In certain embodiments, the avidity is the cumulative strength of interaction provided by the affinities of multiple ABDs for separate antigens on a shared specific target or complex, such as separate antigens found on an individual cell. In certain embodiments, the avidity is the cumulative strength of interaction provided by the affinities of multiple ABDs for separate epitopes on a shared individual antigen.

[0245] In some embodiments, an aCAR can be a bivalent-bispecific CAR. In some embodiments, an aCAR can be a bivalent-bispecific CAR expressed on a cell of the present disclosure (e.g., an immunoresponsive cell) as OR-logic gates to increase the potential targets of an activating chimeric receptor (e.g., an OR-logic gate targeting multiple tumor targets). A bivalent-bispecific aCAR can include an antigen-binding domain specific for FLT3 and an antigen-binding domain specific for CD33. A bivalent-bispecific aCAR can include i) an antigen-binding domain specific for FLT3; (ii) an antigen-binding domain specific for CD33; (iii) one or more intracellular signaling domains that stimulate an immune response, and (iv) one or more polypeptides including, but not limited to, a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

[0246] A CAR of the present disclosure may be a first, second, or third generation CAR. "First generation" CARs comprise a single intracellular signaling domain, generally derived from a T cell receptor chain. "First generation" CARs generally have the intracellular signaling domain from the CD3-zeta (CD3ζ) chain, which is the primary transmitter of signals from endogenous TCRs. "First generation" CARs can provide de novo antigen recognition and cause activation of both CD4+and CD8+T cells through their CD3^ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. "Second generation" CARs add a second intracellular signaling domain from one of various co-stimulatory molecules (e.g., CD28, 4- IBB, ICOS, 0X40) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. "Second generation" CARs provide both co- stimulation (e.g., CD28 or 4- IBB) and activation (CD3^). Preclinical studies have indicated that "Second Generation" CARs can improve the anti-tumor activity of immunoresponsive cell, such as a T cell. "Third generation" CARs have multiple intracellular co-stimulation signaling domains (e.g., CD28 and 4- IBB) and an intracellular activation signaling domain (CD3^).

[0247] In some embodiments, the chimeric antigen receptor is a chimeric inhibitory receptor (iCAR). In some embodiments, the one or more chimeric inhibitory receptors bind antigens that are expressed on a non-tumor cell derived from a tissue selected from the group consisting of brain, neuronal tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, urinary bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

[0248] In some embodiments, a chimeric inhibitory receptor (e.g., an EMCN-specific chimeric inhibitory receptor) may be used, for example, with one or more activating chimeric receptors (e.g., activating chimeric TCRs or CARs, such as FLT3 and / or CD33 aCARs) expressed on a cell of the present disclosure (e.g., an immunoresponsive cell) as NOT-logic gates to control, modulate, or otherwise inhibit one or more activities of the one or more activating chimeric receptors. For instance, if a healthy cell expresses both an antigen that is recognized by a tumor- targeting chimeric receptor and an antigen that is recognized by an chimeric inhibitory receptor, an immunoresponsive cell expressing the tumor antigen may bind to the healthy cell. In such a case, the inhibitory chimeric antigen will also bind its cognate ligand on the healthy cell and the inhibitory function of the chimeric inhibitory receptor will reduce, decrease, prevent, or inhibit the activation of the immunoresponsive cell via the tumor-targeting chimeric receptor (“NOT- logic gating”). In some embodiments, a chimeric inhibitory receptor of the present disclosure may inhibit one or more activities of a cell of the present disclosure (e.g., an immunoresponsive cell). In some embodiments, an immunoresponsive cell may comprise one or more tumor- targeting chimeric receptors and one or more chimeric inhibitory receptors that targets an antigen that is not expressed, or generally considered to be expressed, on the tumor (e.g., EMCN). Combinations of tumor- targeting chimeric receptors and chimeric inhibitory receptors in the same immunoresponsive cell may be used to reduce on-target off-tumor toxicity.

[0249] In some embodiments, the extracellular antigen-binding domain of a CAR of the present disclosure binds to one or more antigens (e.g., EMCN, FLT3, or CD33) with a dissociation constant (Ka) of about 2 x 10'7M or less, about 1 x 10'7M or less, about 9 x 10'8M or less, about 1 x 10'8M or less, about 9 x 10'9M or less, about 5 x 10'9M or less, about 4 x 10'9M or less, about 3 x 10'9M or less, about 2 x 10'9M or less, or about 1 x 10'9M or less. In some embodiments, the Ka ranges from about is about 2 x 10'7M to about 1 x 10'9M.

[0250] Binding of the extracellular antigen-binding domain of a CAR of the present disclosure can be determined by, for example, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), FACS analysis, a bioassay (e.g., growth inhibition), bio-layer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., Biacore®), or a Western Blot assay. Each of these assays generally detect the presence of protein- antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody or scFv) specific for the complex of interest. For example, the scFv can be radioactively labeled and used in an RIA assay. The radioactive isotope can be detected by such means as the use of a y counter or a scintillation counter or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a secondary antibody specific for the extracellular antigen-binding domain and wherein the secondary antibody is labeled (e.g., radioactively or with a fluorescent marker).

[0251] In some embodiments, CARs of the present disclosure comprise an extracellular antigen- binding domain that binds to EMCN, FLT3, or CD33. In some embodiments, CARs of the present disclosure comprise an extracellular antigen-binding domain that binds to an EMCN protein, an EMCN-derived antigen, or an EMCN-derived epitope. In some embodiments, CARs of the present disclosure comprise an extracellular antigen-binding domain that binds to a FLT3 protein, a FLT3-derived antigen, or a FLT3-derived epitope. In some embodiments. In some embodiments, CARs of the present disclosure comprise an extracellular antigen-binding domain that binds to an CD33 protein, a CD33-derived antigen, or a CD33-derived epitope. CARs of the present disclosure comprise an extracellular antigen-binding domain, a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the extracellular antigen- binding domain comprises a Fab fragment, which may be crosslinked. In certain embodiments, the extracellular binding domain is a F(ab)2 fragment. Extracellular Antigen-binding Domain

[0252] Antigen-binding domains of the present disclosure can include any domain that binds to the antigen including, without limitation, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a bispecific antibody, a conjugated antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a single- domain antibody (sdAb) such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody, and to an alternative scaffold known in the art to function as antigen-binding domain, such as a recombinant fibronectin domain, a T cell receptor (TCR), a recombinant TCR with enhanced affinity, or a fragment thereof, e.g., single chain TCR, and the like. In some instances, it is beneficial for the antigen-binding domain to be derived from the same species in which the CAR will ultimately be used in.

[0253] In some embodiments, the extracellular antigen-binding domain comprises an antibody. In certain embodiments, the antibody is a human antibody. In certain embodiments, the antibody is a chimeric antibody. In some embodiments, the extracellular antigen-binding domain comprises an antigen -binding fragment of an antibody.

[0254] In some embodiments, the extracellular antigen-binding domain comprises a F(ab) fragment. In certain embodiments, the extracellular antigen-binding domain comprises a F(ab') fragment.

[0255] In some embodiments, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the extracellular antigen-binding domain comprises two single chain variable fragments (scFvs). In some embodiments, each of the two scFvs binds to a distinct epitope on the same antigen. In some embodiments, the extracellular antigen-binding domain comprises a first scFv and a second scFv. In some embodiments, the first scFv and the second scFv bind distinct epitopes on the same antigen. In certain embodiments, the scFv is a mammalian scFv. In certain embodiments, the scFv is a chimeric scFv. In certain embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL).

[0256] In certain embodiments, the VH and VL are separated by a peptide linker. In certain embodiments, the peptide linker comprises any of the amino acid sequences shown in Table 6.

[0257] In certain embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. In some embodiments, each of the one or more scFvs comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. When there are two or more scFv linked together, each scFv can be linked to the next scFv with a peptide linked. In some embodiments, each of the one or more scFvs is separated by a peptide linker.

[0258] In some embodiments, a peptide linker includes the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 244). In some embodiments, a peptide linker between antigen binding domains of an iCAR comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 244). In some embodiments, a peptide linker is encoded by a polynucleotide sequence comprising the sequence GGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCT (SEQ ID NO: 253). In some embodiments, a nucleic acid encoding peptide linker includes a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to GGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCT (SEQ ID NO: 253).

[0259] In some embodiments, a peptide linker includes the amino acid sequence GGGGS (SEQ ID NO: 242). In some embodiments, a peptide linker between antigen binding domains of an aCAR comprises the amino acid sequence GGGGS (SEQ ID NO: 242). In some embodiments, a peptide linker is encoded by a polynucleotide sequence comprising the sequence GGCGGCGGTGGCTCT (SEQ ID NO: 254) or GGTGGCGGCGGATCC (SEQ ID NO: 255). In some embodiments, a nucleic acid encoding peptide linker includes a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to GGCGGCGGTGGCTCT (SEQ ID NO: 254) or GGTGGCGGCGGATCC (SEQ ID NO: 255).

[0260] In some embodiments, a peptide linker includes the amino acid sequence GGGGSGGGGS (SEQ ID NO: 243). In some embodiments, a peptide linker between antigen binding domains of an aCAR comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 243). In some embodiments, a peptide linker is encoded by a polynucleotide sequence comprising the sequence GGAGGCGGAGGATCTGGTGGTGGTGGATCT (SEQ ID NO: 256), GGTGGCGGAGGAAGTGGCGGCGGAGGCTCT (SEQ ID NO: 257), or GGCGGTGGCGGATCTGGCGGAGGTGGCAGT (SEQ ID NO: 258). In some embodiments, a nucleic acid encoding peptide linker includes a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to GGAGGCGGAGGATCTGGTGGTGGTGGATCT (SEQ ID NO: 256), GGTGGCGGAGGAAGTGGCGGCGGAGGCTCT (SEQ ID NO: 257), or GGCGGTGGCGGATCTGGCGGAGGTGGCAGT (SEQ ID NO: 258). In some embodiments, a peptide linker includes the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 247). In some embodiments, a peptide linker between antigen binding domains of an aCAR comprises the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 247). In some embodiments, a peptide linker is encoded by a polynucleotide sequence comprising the sequence

[0261] GGCTCTACATCTGGCTCTGGCAAACCTGGAAGCGGCGAGGGATCTACCAAGGGC (SEQ ID NO: 249). In some embodiments, a nucleic acid encoding peptide linker includes a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to GGCTCTACATCTGGCTCTGGCAAACCTGGAAGCGGCGAGGGATCTACCAAGGGC (SEQ ID NO: 249).

[0262] Table 6. Peptide Linkers In some embodiments, the immune effector cell comprises a first chimeric receptor and a second chimeric receptor. The antigen-binding domain of the first chimeric receptor and the antigen-binding domain of the second chimeric receptor can be an appropriate antigen biding domain described herein or known in the art. For example, the first or second antigen-binding domain can be one or more antibodies, antigen-binding fragments of an antibody, F(ab) fragments, F(ab') fragments, single chain variable fragments (scFvs), or single-domain antibodies (sdAbs). In some embodiments, the antigen-binding domain of the first chimeric receptor and / or the second chimeric receptor comprises two single chain variable fragments (scFvs). In some embodiments, each of the two scFvs binds to a distinct epitope on the same antigen. In some embodiments, the antigen-binding domain of the first chimeric receptor can be specific for EMCN and the chimeric receptor can be specific for a second distinct antigen, such as a cancer antigen (e.g., an antigen expressed on a myeloid cell, such as an AML cell).

[0263] In some embodiments, the extracellular antigen-binding domain comprises a single- domain antibody (sdAb). In certain embodiments, the sdAb is a humanized sdAb. In certain embodiments, the sdAb is a chimeric sdAb.

[0264] In some embodiments, a CAR of the present disclosure may comprise two or more antigen-binding domains, three or more antigen-binding domains, four or more antigen-binding domains, five or more antigen-binding domains, six or more antigen-binding domains, seven or more antigen-binding domains, eight or more antigen-binding domains, nine or more antigen- binding domains, or ten or more antigen-binding domains. In some embodiments, each of the two or more antigen-binding domains binds the same antigen. In some embodiments, each of the two or more antigen-binding domains binds a different epitope of the same antigen. In some embodiments, each of the two or more antigen-binding domains binds a different antigen.

[0265] In some embodiments, the CAR comprises two antigen -binding domains. In some embodiments, the two antigen-binding domains are attached to one another via a flexible linker. In some embodiments, each of the two-antigen-binding domains may be independently selected from an antibody, an antigen-binding fragment of an antibody, an scFv, a sdAb, a recombinant fibronectin domain, a T cell receptor (TCR), a recombinant TCR with enhanced affinity, and a single chain TCR. In some embodiments, the CAR comprising two antigen-binding domains is a bispecific CAR or a tandem CAR (tanCAR).

[0266] In certain embodiments, the bispecific CAR or tanCAR comprises an antigen-binding domain comprising a bispecific antibody or antibody fragment (e.g., scFv). In some embodiments, within each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, the VH can be upstream or downstream of the VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VHi) upstream of its VL (VLi) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL2) upstream of its VH (VH2), such that the overall bispecific antibody molecule has the arrangement VH1-VL1-VL2-VH2. In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VLi) upstream of its VH (VHi) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH2) upstream of its VL (VL2), such that the overall bispecific antibody molecule has the arrangement VLi VH1-VH2-VL2. In some embodiments, a linker is disposed between the two antibodies or antibody fragments (e.g., scFvs), for example, between VLi and VL2 if the construct is arranged as VH1-VL1-VL2-VH2, or between VHi and VH2 if the construct is arranged as VL1-VH1-VH2- VL2. The linker may be a linker as described herein, e.g., a (Gly4-Ser)n linker, wherein n is 1 , 2, 3, 4, 5, or 6. In general, the linker between the two scFvs should be long enough to avoid mispairing between the domains of the two scFvs. In some embodiments, a linker is disposed between the VL and VH of the first scFv. In some embodiments, a linker is disposed between the VL and VH of the second scFv. In constructs that have multiple linkers, any two or more of the linkers may be the same or different. Accordingly, in some embodiments, a bispecific CAR or tanCAR comprises VLs, VHs, and may further comprise one or more linkers in an arrangement as described herein.

[0267] In an illustrative non-limiting example, a CD33 / FLT3 bispecific bivalent aCAR can have antigen binding domains encoded in the following order: (FLT3-VH) - LI - (CD33-VH) - L2 - (CD33-VL) - L3 - (FLT3-VL), wherein LI, L2, and L3 are a first, a second, and a third peptide linker, respectively. LI, L2, and L3 can be the same peptide linker. LI, L2, and L3 can be the same peptide linker encoded by the same polynucleotide sequence. LI, L2, and L3 can be the same peptide linker encoded by different polynucleotide sequences. LI and L3 can be the same peptide linker and L2 a different peptide linker. LI and L3 can be the same peptide linker encoded by the same polynucleotide sequence. LI and L3 can be the same peptide linker encoded by different polynucleotide sequences. LI, L2, and L3 can each be different peptide linkers.

[0268] CAR Transmembrane Domains

[0269] In some embodiments, the transmembrane domain of a CAR of the present disclosure (e.g., the EMCN- specific, FLT3- specific, and / or CD33-specific CARs described herein) comprises a hydrophobic alpha helix that spans at least a portion of a cell membrane. It has been shown that different transmembrane domains can result in different receptor stability. After antigen recognition, receptors cluster and a signal is transmitted to the cell. In some embodiments, the transmembrane domain of a CAR of the present disclosure can comprise the transmembrane domain of a CD8 polypeptide, a CD28 polypeptide, a CD3-zeta polypeptide, a CD4 polypeptide, a 4- IBB polypeptide, an 0X40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a LIR-1 (LILRB1) polypeptide, or can be a synthetic peptide, or any combination thereof.

[0270] In some embodiments, the transmembrane domain is derived from a CD8 polypeptide. Any suitable CD8 polypeptide may be used. Exemplary CD8 polypeptides include, without limitation, NCBI Reference Nos. NP_001139345 and AAA92533.1. In some embodiments, the transmembrane domain is derived from a CD28 polypeptide. Any suitable CD28 polypeptide may be used. Exemplary CD28 polypeptides include, without limitation, NCBI Reference Nos. NP_006130.1 and NP_031668.3. In some embodiments, the transmembrane domain is derived from a CD3-zeta polypeptide. Any suitable CD3-zeta polypeptide may be used. Exemplary CD3-zeta polypeptides include, without limitation, NCBI Reference Nos. NP_932170.1 and NP_001106862.1. In some embodiments, the transmembrane domain is derived from a CD4 polypeptide. Any suitable CD4 polypeptide may be used. Exemplary CD4 polypeptides include, without limitation, NCBI Reference Nos. NP_000607.1 and NP_038516.1. In some embodiments, the transmembrane domain is derived from a 4- IBB polypeptide. Any suitable 4- 1BB polypeptide may be used. Exemplary 4- IBB polypeptides include, without limitation, NCBI Reference Nos. NP_001552.2 and NP_001070977.1. In some embodiments, the transmembrane domain is derived from an 0X40 polypeptide. Any suitable 0X40 polypeptide may be used. Exemplary 0X40 polypeptides include, without limitation, NCBI Reference Nos. NP_003318.1 and NP_035789.1. In some embodiments, the transmembrane domain is derived from an ICOS polypeptide. Any suitable ICOS polypeptide may be used. Exemplary ICOS polypeptides include, without limitation, NCBI Reference Nos. NP_036224 and NP_059508. In some embodiments, the transmembrane domain is derived from a CTLA-4 polypeptide. Any suitable CTLA-4 polypeptide may be used. Exemplary CTLA-4 polypeptides include, without limitation, NCBI Reference Nos. NP_005205.2 and NP_033973.2. In some embodiments, the transmembrane domain is derived from a PD-1 polypeptide. Any suitable PD-1 polypeptide may be used. Exemplary PD-1 polypeptides include, without limitation, NCBI Reference Nos. NP_005009 and NP_032824. In some embodiments, the transmembrane domain is derived from a LAG-3 polypeptide. Any suitable LAG-3 polypeptide may be used. Exemplary LAG-3 polypeptides include, without limitation, NCBI Reference Nos. NP_002277.4 and NP_032505.1. In some embodiments, the transmembrane domain is derived from a 2B4 polypeptide. Any suitable 2B4 polypeptide may be used. Exemplary 2B4 polypeptides include, without limitation, NCBI Reference Nos. NP_057466.1 and NP_061199.2. In some embodiments, the transmembrane domain is derived from a BTLA polypeptide. Any suitable BTLA polypeptide may be used. Exemplary BTLA polypeptides include, without limitation, NCBI Reference Nos. NP_861445.4 and NP_001032808.2. Any suitable LIR-1 (LILRB1) polypeptide may be used. Exemplary LIR-1 (LILRB1) polypeptides include, without limitation, NCBI Reference Nos. NP_001075106.2 and NP_001075107.2.

[0271] In some embodiments, the transmembrane domain comprises a polypeptide comprising an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the sequence of NCBI Reference No. NP_001139345, AAA92533.1, NP_006130.1, NP_031668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP.038516.1, NP_001552.2, NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_005009, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2, or fragments thereof. In some embodiments, the homology may be determined using standard software such as BLAST or EASTA. In some embodiments, the polypeptide may comprise one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions. In some embodiments, the polypeptide can have an amino acid sequence that is a consecutive portion of NCBI Reference No. NP_001139345, AAA92533.1, NP_006130.1, NP_031668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP.038516.1, NP_001552.2, NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_005009, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2 that is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, or at least 240 amino acids in length.

[0272] Eurther examples of suitable polypeptides from which a transmembrane domain may be derived include, without limitation, the transmembrane region(s) of the alpha, beta or zeta chain of the T-cell receptor, CD27, CD3 epsilon, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, CD2, CD27, LFA-1 (CDl la, CD18), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, IL2R beta, IL2R gamma, IL7R0C, ITGA1, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CD 11b, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKG2D, and NG2C.

[0273] In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 205). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 206). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 207).

[0274] In some embodiments, an iCAR transmembrane domain includes a LIR1 transmembrane domain. In some embodiments, an iCAR transmembrane domain includes a LIR1 transmembrane domain having the sequence VIGILVAVILLLLLLLLLFLI (SEQ ID NO: 259). In some embodiments, an iCAR transmembrane domain includes a LIR1 transmembrane domain encoded by a polynucleotide sequence having the sequence GTGATCGGCATTCTGGTCGCCGTGATCCTGCTCCTGTTGCTCCTGCTGCTTCTGTTCC TGATC (SEQ ID NO: 260). In some embodiments, an iCAR transmembrane domain includes a LIR1 transmembrane domain encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to GTGATCGGCATTCTGGTCGCCGTGATCCTGCTCCTGTTGCTCCTGCTGCTTCTGTTCC TGATC (SEQ ID NO: 260).

[0275] In some embodiments, an aCAR transmembrane domain includes a CD8 transmembrane domain. In some embodiments, an aCAR transmembrane domain includes a CD8 transmembrane domain having the sequence IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 206). In some embodiments, an aCAR transmembrane domain includes a CD8 transmembrane domain encoded by a polynucleotide sequence having the sequence ATCTATATCTGGGCCCCTCTGGCTGGCACATGCGGAGTTCTGCTGCTCAGCCTGGTC ATCACCCTGTACTGCAACCACAGA (SEQ ID NO: 261). In some embodiments, an aCAR transmembrane domain includes a CD8 transmembrane domain encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to ATCTATATCTGGGCCCCTCTGGCTGGCACATGCGGAGTTCTGCTGCTCAGCCTGGTC ATCACCCTGTACTGCAACCACAGA (SEQ ID NO: 261). Spacer Region

[0276] In some embodiments, a CAR of the present disclosure (e.g., the EMCN- specific, FLT3- specific, and / or CD33-specific CARs described herein) can also comprise a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region may be flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition. In some embodiments, the spacer region may be a hinge from a human protein. For example, the hinge may be a human Ig (immunoglobulin) hinge, including without limitation an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge. In some embodiments, the spacer region may comprise an IgG4 hinge, an IgG2 hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, an LNGFR hinge, or a PDGFR-beta extracellular linker. In some embodiments, the spacer region is localized between the antigen-binding domain and the transmembrane domain. In some embodiments, a spacer region may comprise any of the amino acid sequences listed in Table 7, or an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the amino acid sequences listed in Table 7. In some embodiments, nucleic acids encoding any of the spacer regions of the present disclosure may comprise any of the nucleic acid sequences listed in Table 8, or a nucleic acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the nucleic acid sequences listed in Table 8.

[0277] In some embodiments, an aCAR hinge domain includes a CD8 hinge. In some embodiments, an aCAR hinge domain includes a CD8 hinge having the amino acid sequence ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFACD (SEQ ID NO: 272). In some embodiments, an aCAR hinge domain includes a CD8 hinge encoded by a polynucleotide sequence having the sequence GCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTGCCCGCC AAGCCTACAACAACCCCTGCTCCTAGACCACCTACACCAGCTCCTACAATCGCCAG CCAGCCTCTGTCTCTGAGGCCCGAAGCTTGTAGACCAGCTGCTGGCGGAGCCGTGC ATACAAGAGGACTGGATTTTGCCTGCGAC (SEQ ID NO: 284). In some embodiments, an aCAR hinge domain includes a CD8 hinge encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to GCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTGCCCGCC AAGCCTACAACAACCCCTGCTCCTAGACCACCTACACCAGCTCCTACAATCGCCAG CCAGCCTCTGTCTCTGAGGCCCGAAGCTTGTAGACCAGCTGCTGGCGGAGCCGTGC ATACAAGAGGACTGGATTTTGCCTGCGAC (SEQ ID NO: 284). In some embodiments, an iCAR hinge domain includes a CD8 hinge. In some embodiments, an iCAR hinge domain includes a CD8 hinge having the amino acid sequence TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 271). In some embodiments, an iCAR hinge domain includes a CD8 hinge encoded by a polynucleotide sequence having the sequence ACAACAACACCCGCACCTCGGCCTCCAACTCCAGCTCCAACAATTGCACTGCAACC CCTGAGTCTGAGGCCCGAGGCCTGTAGGCCAGCAGCTGGCGGAGCTGTTCACACTA GAGGCCTGGACTTTGCCTGTGAC (SEQ ID NO: 283). In some embodiments, an iCAR hinge domain includes a CD8 hinge encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to ACAACAACACCCGCACCTCGGCCTCCAACTCCAGCTCCAACAATTGCACTGCAACC CCTGAGTCTGAGGCCCGAGGCCTGTAGGCCAGCAGCTGGCGGAGCTGTTCACACTA GAGGCCTGGACTTTGCCTGTGAC (SEQ ID NO: 283).

[0278] In some embodiments, an iCAR hinge domain includes a LIR1 hinge. In some embodiments, an iCAR hinge domain includes a LIR1 hinge having the amino acid sequence HPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV (SEQ ID NO: 417). In some embodiments, an iCAR hinge domain includes a LIR1 hinge encoded by a polynucleotide sequence having the sequence CACCCATCCGATCCTCTCGAGCTGGTGGTTTCTGGACCTTCTGGCGGCCCTAGCAGC CCTACAACAGGACCTACAAGCACAAGCGGCCCTGAGGACCAACCTCTGACACCAAC AGGCAGCGATCCTCAGTCTGGACTGGGGAGACATCTGGGCGTT (SEQ ID NO: 418). In some embodiments, an iCAR hinge domain includes a LIR1 hinge encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to CACCCATCCGATCCTCTCGAGCTGGTGGTTTCTGGACCTTCTGGCGGCCCTAGCAGC CCTACAACAGGACCTACAAGCACAAGCGGCCCTGAGGACCAACCTCTGACACCAAC AGGCAGCGATCCTCAGTCTGGACTGGGGAGACATCTGGGCGTT (SEQ ID NO: 418). Table 7. Spacer Amino Acid Sequences

[0279] Table 8. Spacer Nucleic Acid Sequences

[0280] In some embodiments, a CAR of the present disclosure may further include a short oligopeptide or polypeptide linker that is between 2 amino acid residues and 10 amino acid residues in length, and that may form the linkage between the transmembrane domain and the cytoplasmic region of the CAR. A non-limiting example of a suitable linker is a glycine-serine doublet. In some embodiments, the linker comprises the ammo acid sequence of GGCKJSGGCKJS (SEQ ID NO: 208).

[0281] In some aspects, the transmembrane domain further comprises at least a portion of an extracellular domain of the same protein. Intracellular Signaling Domains

[0282] In some embodiments, a CAR of the present disclosure (e.g., the EMCN- specific, FLT3- specific, and / or CD33-specific CARs described herein) comprises one or more cytoplasmic domains or regions. The cytoplasmic domain or region of the CAR may include an intracellular signaling domain.

[0283] Examples of suitable intracellular signaling domains that may be used in CARs of the present disclosure include, without limitation, cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to modulate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0284] Without wishing to be bound by theory, it is believed that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary and / or co- stimulatory signal is thus also typically required for full activation. Accordingly, T cell activation may be mediated by two distinct classes of cytoplasmic signaling sequences, those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic domain, e.g., a co-stimulatory domain). In addition, T cell signaling and function (e.g., an activating signaling cascade) can be negatively regulated by inhibitory receptors present in a T cell through intracellular inhibitory co-signaling domains.

[0285] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure can include an inhibitory intracellular signaling domains. Examples of inhibitory intracellular domains that can be used include PD-1, CTLA4, TIGIT, BTLA, and LIR-1 (LILRB1), TIM3, KIR3DL1, NKG2A, LAG3, SLAP1, SLAP2, Dok-1, Dok-2, LAIR1, GRB-2, CD200R, SIRPa, HAVR, GITR, PD-L1, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, CD94, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, and SIGLEC-10. Table 9 and Table 10 provide the amino acid and nucleotide sequneces, respectively, of the exemplary inhibitory intracellular signaling domains. In some embodiments, the inhibitory intracellular signaling domain includes one or more intracellular inhibitory co-signaling domains (e.g., see Table 9 and Table 10. In some embodiments, the one or more intracellular inhibitory co-signaling domains are linked to other domains (e.g., a transmembrane domain) through a peptide linker (e.g., see Table 6) or a spacer or hinge sequence (e.g., see Tables 7 and 8). In some embodiments, when two or more intracellular inhibitory co-signaling domains are present, the two or more intracellular inhibitory co-signaling domains can be linked through a peptide linker (e.g., see Table 6) or a spacer or hinge sequence (e.g., see Tables 7 and 8). In some embodiments, the intracellular inhibitory co-signaling domain is an inhibitory domain. In some embodiments, the one or more intracellular inhibitory co-signaling domains of a chimeric protein comprises one or more ITIM-containing protein, or fragment(s) thereof. ITIMs are conserved amino acid sequences found in cytoplasmic tails of many inhibitory immune receptors. Examples of ITIM- containing proteins include, but are not limited to, PD-1, TIGIT, BTLA, and LIR-1 (LILRB1), TIM3, KIR3DL1, NKG2A, LAG3, LAIR1, SIRPa, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, CD94, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, and SIGLEC-10.

[0286] Table 9 - Exemplary inhibitory intracellular signaling domain amino acid sequences

[0287] Table 10 - Exemplary inhibitory intracellular signaling domain nucleic acid sequences

[0288] In some embodiments, an iCAR includes a LIR1 intracellular inhibitory domain. In some embodiments, an iCAR includes a LIR1 intracellular inhibitory domain having the amino acid sequence LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKH TQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEED RQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 285). In some embodiments, an iCAR includes a LIR1 intracellular inhibitory domain encoded by a polynucleotide sequence comprising the sequence CTGCGGCACAGAAGGCAGGGCAAGCACTGGACAAGCACCCAGAGAAAGGCCGACT TTCAGCATCCTGCTGGCGCCGTTGGACCTGAGCCTACAGATAGAGGACTGCAGTGG CGGTCTAGCCCTGCCGCTGATGCCCAAGAGGAAAATCTTTACGCCGCCGTGAAGCA CACCCAGCCTGAGGATGGCGTGGAAATGGACACCAGATCTCCCCACGATGAGGACC CTCAGGCCGTGACATACGCAGAAGTGAAGCACTCCAGACCTCGGAGAGAGATGGC AAGCCCTCC ATCTCCTCTGAGCGGCGAGTTCCTGGAC ACC AAAGACAGAC AGGCCG AAGAGGACAGACAGATGGATACCGAAGCCGCCGCTTCTGAAGCCCCACAGGATGT GACATATGCCCAGCTGCATAGCCTGACACTGCGGAGAGAAGCCACAGAGCCTCCAC CTTCTCAAGAAGGCCCATCTCCTGCCGTGCCTTCCATCTATGCCACTCTGGCCATTC AC (SEQ ID NO: 286). In some embodiments, an iCAR includes a LIR1 intracellular inhibitory domain encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to CTGCGGCACAGAAGGCAGGGCAAGCACTGGACAAGCACCCAGAGAAAGGCCGACT TTCAGCATCCTGCTGGCGCCGTTGGACCTGAGCCTACAGATAGAGGACTGCAGTGG CGGTCTAGCCCTGCCGCTGATGCCCAAGAGGAAAATCTTTACGCCGCCGTGAAGCA CACCCAGCCTGAGGATGGCGTGGAAATGGACACCAGATCTCCCCACGATGAGGACC CTCAGGCCGTGACATACGCAGAAGTGAAGCACTCCAGACCTCGGAGAGAGATGGC AAGCCCTCCATCTCCTCTGAGCGGCGAGTTCCTGGACACCAAAGACAGACAGGCCG AAGAGGACAGACAGATGGATACCGAAGCCGCCGCTTCTGAAGCCCCACAGGATGT GACATATGCCCAGCTGCATAGCCTGACACTGCGGAGAGAAGCCACAGAGCCTCCAC CTTCTCAAGAAGGCCCATCTCCTGCCGTGCCTTCCATCTATGCCACTCTGGCCATTC AC (SEQ ID NO: 286).

[0289] In some embodiments, the one or more intracellular inhibitory co-signaling domains comprise one or more non-ITIM scaffold proteins, or a fragment(s) thereof. In some embodiments, the one or more non-ITIM scaffold proteins, or fragments thereof, are selected from GRB-2, Dok-1, Dok-2, SLAP, LAG3, HAVR, GITR, and PD-L1.

[0290] The inhibitory intracellular signaling domain can further include an enzymatic inhibitory domain. In some embodiments, the enzymatic inhibitory domain comprises an enzyme catalytic domain. In some embodiments, the enzyme catalytic domain is derived from an enzyme selected from the group consisting of: CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c- CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, and RasGAP. Examples of enzymatic regulation of signaling is described in more detail in Pavel Otahal et al. (Biochim Biophys Acta. 2011 Feb;1813(2):367-76), Kosugi A., et al. (Involvement of SHP-1 tyrosine phosphatase in TCR- mediated signaling pathways in lipid rafts, Immunity, 2001 Jun; 14(6): 669-80), and Stanford, et al. (Regulation of TCR signaling by tyrosine phosphatases: from immune homeostasis to autoimmunity, Immunology, 2012 Sep; 137(1): 1-19), each of which is incorporated herein by reference for all purposes.

[0291] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure can comprise a primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way or in an inhibitory way. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine -based activation motifs (IT AMs). Examples of suitable ITAM- containing primary intracellular signaling domains that that may be used in the CARs of the present disclosure include, without limitation, those of CD3-zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FceRI, DAP10, DAP12, and CD66d.

[0292] In some embodiments, a CAR of the present disclosure comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3-zeta polypeptide. A CD3-zeta polypeptide of the present disclosure may have an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the sequence of NCBI Reference No. NP_932170 or NP_001106864.2, or fragments thereof. In some embodiments, the CD3-zeta polypeptide may comprise one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions. In some embodiments, the polypeptide can have an amino acid sequence that is a consecutive portion of NCBI Reference No. NP_932170 or NP_001106864.2 that is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, or at least 160, at least 170, or at least 180 amino acids in length.

[0293] In other embodiments, a primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain. In one embodiment, a primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and / or truncated ITAM-containing primary intracellular signaling domain. In one embodiment, a primary signaling domain comprises one, two, three, four or more ITAM motifs.

[0294] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure can comprise the CD3-zeta signaling domain by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a CAR of the present disclosure. For example, the intracellular signaling domain of the CAR can comprise a CD3-zeta chain portion and a costimulatory signaling domain. The costimulatory signaling domain may refer to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule of the present disclosure is a cell surface molecule other than an antigen receptor or its ligands that may be required for an efficient response of lymphocytes to an antigen. Examples of suitable costimulatory molecules include, without limitation, CD97, CD2, ICOS, CD27, CD154, CD8, 0X40, 4-1BB, CD28, ZAP40, CD30, GITR, HVEM, DAP10, DAP12, MyD88, 2B4, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, an MHC class I molecule, a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, CDS, ICAM-1, (CDl la / CD18), BAFFR, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD 18, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, and the like.

[0295] In some embodiments, aCAR intracellular signaling domains include a CD28 co- stimulatory domain. In some embodiments, aCAR intracellular signaling domains include a CD28 co-stimulatory domain having the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 287). In some embodiments, aCAR intracellular signaling domains include a CD28 co-stimulatory domain encoded by a polynucleotide sequence comprising the sequence AGAAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCTAGAC GGCCCGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGATTTCGCC GCCTACCGGTCC (SEQ ID NO: 288). In some embodiments, aCAR intracellular signaling domains include a CD28 co-stimulatory domain encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to AGAAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCTAGAC GGCCCGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGATTTCGCC GCCTACCGGTCC (SEQ ID NO: 288).

[0296] In some embodiments, aCAR intracellular signaling domains include a CD3(^ signaling domain. In some embodiments, aCAR intracellular signaling domains include a CD3(^ signaling domain having the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 289). In some embodiments, aCAR intracellular signaling domains include a CD3(^ signaling domain encoded by a polynucleotide sequence having the sequence AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACC AGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAG AGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGG AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGAT TGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGT CTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCC TCGC (SEQ ID NO: 290). In some embodiments, aCAR intracellular signaling domains include a CD3(^ signaling domain encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACC AGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAG AGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGG AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGAT TGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGT CTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCC TCGC (SEQ ID NO: 290).

[0297] In some embodiments, aCAR intracellular signaling domains include a CD28 co- stimulatory domain and a CD3^ signaling domain. In some embodiments, aCAR intracellular signaling domains include a CD28 co-stimulatory domain having the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 287) and a CD3(^ signaling domain having the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 289).

[0298] In some embodiments, the intracellular signaling sequences within the cytoplasmic portion of a CAR of the present disclosure may be linked to each other in a random or specified order. In some embodiments, a short oligopeptide or polypeptide linker, for example, between 2 amino acids and 10 amino acids (e.g., 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids) in length may form the linkage between intracellular signaling sequences. In one embodiment, a glycine- serine doublet can be used as a suitable linker. In one embodiment, a single ammo acid, e.g., an alanine or a glycine, can be used as a suitable linker. In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains, e.g., two costimulatory signaling domains, three costimulatory signaling domains, four costimulatory signaling domains, five costimulatory signaling domains, six costimulatory signaling domains, seven costimulatory signaling domains, eight costimulatory signaling domains, nine costimulatory signaling domains, 10 costimulatory signaling domains, or more costimulatory signaling domains. In one embodiment, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the two or more costimulatory signaling domains are separated by a linker of the present disclosure (e.g., any of the linkers described in Table 6). In one embodiment, the linker is a glycine residue. In another embodiment, the linker is an alanine residue.

[0299] In some embodiments, a cell of the present disclosure expresses a CAR that includes an antigen-binding domain, a transmembrane domain, a primary signaling domain, and one or more costimulatory signaling domains.

[0300] In some embodiments, the transmembrane domain is derived from the same protein as one of the one or more intracellular signaling domains. In some embodiments, the CAR is an inhibitory CAR and includes a transmembrane domain and at least one intracellular inhibitory co-signaling domain each derived from a protein selected from PD-1, CTLA4, TIGIT, BTLA, and LIR1 (LILRB1), TIM3, KIR3DL1, NKG2A, LAG3, SLAP1, SLAP2, Dok-1, Dok-2, LAIR1, GRB-2, CD200R, SIRPa, HAVR, GITR, PD-L1, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, CD94, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, and SIGLEC-10.

[0301] In some embodiments, the transmembrane domain is derived from a first protein and the one or more intracellular signaling domains are derived from a second protein that are distinct from the first protein.

[0302] Natural Killer Cell Receptor (NKR) CARs

[0303] In some embodiments, a CAR of the present disclosure comprises one or more components of a natural killer cell receptor (NKR), thereby forming an NKR-CAR. The NKR component may be a transmembrane domain, a hinge domain, or a cytoplasmic domain from any suitable natural killer cell receptor, including without limitation, a killer cell immunoglobulin-like receptor (KIR), such as KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIRS DPI; a natural cytotoxicity receptor (NCR), such as NKp30, NKp44, NKp46; a signaling lymphocyte activation molecule (SLAM) family of immune cell receptor, such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10; an Fc receptor (FcR), such as CD16, and CD64; and an Ly49 receptor, such as LY49A and LY49C. In some embodiments, the NKR-CAR may interact with an adaptor molecule or intracellular signaling domain, such as DAP12. Exemplary configurations and sequences of CARs comprising NKR components are described in International Patent Publication WO2014 / 145252, published September 18, 2014.

[0304] Immunoresponsive cells

[0305] Certain aspects of the present disclosure relate to a cell, such as an immunoresponsive cell, that has been genetically engineered to comprise one or more chimeric receptors of the present disclosure or one or more nucleic acids encoding such chimeric receptors, and to methods of using such cells for treating myeloid malignancies (e.g., AML).

[0306] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a primary cell. In some embodiments, the mammalian cell is a cell line. In some embodiments, the mammalian cell, a bone marrow cell, a blood cell, a skin cell, bone cell, a muscle cell, a neuronal cell, a fat cell, a liver cell, or a heart cell. In some embodiments, the cell is a stem cell. Exemplary stem cells include, without limitation embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), adult stem cells, and tissue- specific stem cells, such as hematopoietic stem cells (blood stem cells), mesenchymal stem cells (MSC), neural stem cells, epithelial stem cells, or skin stem cells. In some embodiments, the cell is a cell that is derived or differentiated from a stem cell of the present disclosure. In some embodiments, the cell is an immune cell. Immune cells of the present disclosure may be isolated or differentiated from a stem cell of the present disclosure (e.g., from an ESC or iPSC). Exemplary immune cells include, without limitation, T cells (e.g., helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, alpha beta T cells, and gamma delta T cells), B cells, natural killer (NK) cells, dendritic cells, myeloid cells, macrophages, and monocytes. In some embodiments, the cell is a neuronal cell. Neuronal cells of the present disclosure may be isolated or differentiated from a stem cell of the present disclosure (e.g., from an ESC or iPSC). Exemplary neuronal cells include, without limitation, neural progenitor cells, neurons (e.g., sensory neurons, motor neurons, cholinergic neurons, GABAergic neurons, glutamatergic neurons, dopaminergic neurons, or serotonergic neurons), astrocytes, oligodendrocytes, and microglia.

[0307] In some embodiments, the cell is an immunoresponsive cell. Immunoresponsive cells of the present disclosure may be isolated or differentiated from a stem cell of the present disclosure (e.g., from an ESC or iPSC). Exemplary immunoresponsive cells of the present disclosure include, without limitation, cells of the lymphoid lineage. The lymphoid lineage, comprising B cells, T cells, and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. Examples of immunoresponsive cells of the lymphoid lineage include, without limitation, T cells, Natural Killer (NK) cells, embryonic stem cells, pluripotent stem cells, and induced pluripotent stem cells (e.g., those from which lymphoid cells may be derived or differentiated). T cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. In some embodiments, T cells of the present disclosure can be any type of T cells, including, without limitation, T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells, regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosal associated invariant T cells, and y5 T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. A patient's own T cells may be genetically modified to target specific antigens through the introduction of one or more chimeric receptors, such as a chimeric TCRs or CARs.

[0308] Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation in order to perform their cytotoxic effect on target cells.

[0309] In some embodiments, an immunoresponsive cell of the present disclosure is a T cell. T cells of the present disclosure may be autologous, allogeneic, or derived in vitro from engineered progenitor or stem cells.

[0310] In some embodiments, an immunoresponsive cell of the present disclosure is a universal T cell with deficient TCR-ap. Methods of developing universal T cells are described in the art, for example, in Valton et al., Molecular Therapy (2015); 23 9, 1507-1518, and Torikai et al., Blood 2012 119:5697-5705.

[0311] In some embodiments, an immunoresponsive cell of the present disclosure is an isolated immunoresponsive cell comprising one or more chimeric receptors of the present disclosure. In some embodiments, the immunoresponsive cell comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more chimeric receptors of the present disclosure.

[0312] In some embodiments, an immunoresponsive cell is a T cell. In some embodiments, an immunoresponsive cell is a Natural Killer (NK) cell.

[0313] In some embodiments, an immunoresponsive cell expresses or is capable of expressing an immune receptor. Immune receptors generally are capable of inducing signal transduction or changes in protein expression in the immune receptor-expressing cell that results in the modulation of an immune response upon binding to a cognate ligand (e.g., regulate, activate, initiate, stimulate, increase, prevent, attenuate, inhibit, reduce, decrease, inhibit, or suppress an immune response). For example, when CD3 chains present in a TCR / CAR cluster in response to ligand binding, an immunoreceptor tyrosine-based activation motifs (ITAMs)-meditated signal transduction cascade is produced. Specifically, in certain embodiments, when an endogenous TCR, exogenous TCR, chimeric TCR, or a CAR (specifically an activating CAR) binds their respective antigen, a formation of an immunological synapse occurs that includes clustering of many molecules near the bound receptor (e.g., CD4 or CD8, CD3y / 8 / e / ^, etc.). This clustering of membrane bound signaling molecules allows for IT AM motifs contained within the CD3 chains to become phosphorylated that in turn can initiate a T cell activation pathway and ultimately activates transcription factors, such as NF-KB and AP-1. These transcription factors are capable of inducing global gene expression of the T cell to increase IL-2 production for proliferation and expression of master regulator T cell proteins in order to initiate a T cell mediated immune response, such as cytokine production and / or T cell mediated killing.

[0314] Cells Expressing Multiple Chimeric Receptors

[0315] In some embodiments, a cell of the present disclosure (e.g., an immunoresponsive cell) comprises two or more chimeric receptors of the present disclosure. In some embodiments, the cell comprises two or more chimeric receptors, wherein one of the two or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises three or more chimeric receptors, wherein at least one of the three or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises four or more chimeric receptors, wherein at least one of the four or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises five or more chimeric receptors, wherein at least one of the five or more chimeric receptors is a chimeric inhibitory receptor.

[0316] In some embodiments, each of the two or more chimeric receptors comprise a different antigen-binding domain, e.g., that binds to the same antigen or to a different antigen. In some embodiments each antigen bound by the two or more chimeric receptors are expressed on the same cell, such as a myeloid cell type (e.g., same AML cell type). In some embodiments each antigen bound by the two or more chimeric receptors is an AML-associated antigen (e.g., FLT3, CD33, CD123, CLEC12A, CXCR4, EphA3, etc.).

[0317] In embodiments where a cell of the present disclosure (e.g., an immunoresponsive cell) expresses two or more distinct chimeric receptors, the antigen-binding domain of each of the different chimeric receptors may be designed such that the antigen-binding domains do not interact with one another. For example, a cell of the present disclosure (e.g., an immunoresponsive cell) expressing a first chimeric receptor (e.g., an EMCN-specific chimeric receptor) and a second chimeric receptor may comprise a first chimeric receptor that comprises an antigen-binding domain that does not form an association with the antigen-binding domain of the second chimeric receptor. For example, the antigen-binding domain of the first chimeric receptor may comprise an antibody fragment, such as an scFv, while the antigen-binding domain of the second chimeric receptor may comprise a VHH.

[0318] Without wishing to be bound by theory, it is believed that in cells having a plurality of chimeric membrane embedded receptors that each comprise an antigen-binding domain, interactions between the antigen-binding domains of each of the receptors can be undesirable, because such interactions may inhibit the ability of one or more of the antigen-binding domains to bind their cognate antigens. Accordingly, in embodiments where cells of the present disclosure (e.g., immunoresponsive cells) express two or more chimeric receptors, the chimeric receptors comprise antigen-binding domains that minimize such inhibitory interactions. In one embodiment, the antigen-binding domain of one chimeric receptor comprises an scFv and the antigen-binding domain of the second chimeric receptor comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence.

[0319] In some embodiments, when present on the surface of a cell, binding of the antigen- binding domain of the first chimeric receptor to its cognate antigen is not substantially reduced by the presence of the second chimeric receptor. In some embodiments, binding of the antigen- binding domain of the first chimeric receptor to its cognate antigen in the presence of the second chimeric receptor is 85%, 90%, 95%, 96%, 97%, 98%, or 99% of binding of the antigen-binding domain of the first chimeric receptor to its cognate antigen in the absence of the second chimeric receptor. In some embodiments, when present on the surface of a cell, the antigen-binding domains of the first chimeric receptor and the second chimeric receptor associate with one another less than if both were scFv antigen-binding domains. In some embodiments, the antigen- binding domains of the first chimeric receptor and the second chimeric receptor associate with one another 85%, 90%, 95%, 96%, 97%, 98%, or 99% less than if both were scFv antigen- binding domains.

[0320] Chimeric Inhibitory Receptors

[0321] In some embodiments, a cell of the present disclosure (e.g., an immunoresponsive cell) comprises one or more chimeric inhibitory receptors of the present disclosure. In some embodiments, each of the one or more chimeric inhibitory receptors comprises an antigen- binding domain that binds an antigen generally expressed on normal cells (e.g., cells generally considered to be healthy) but not on tumor cells, such as AML cells. In some embodiments, a chimeric inhibitory receptor includes an antigen-binding domain that binds EMCN (e.g., an EMCN-specific antigen-binding domain having one or more of the amino acid sequences listed in Table 11).

[0322] In some embodiments, the one or more chimeric inhibitory receptors bind antigens that are expressed on a non-tumor cell derived from a tissue selected from the group consisting of brain, neuronal tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, urinary bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

[0323] In some embodiments, a chimeric inhibitory receptor (e.g., an EMCN-specific chimeric inhibitory receptor) may be used, for example, with one or more activating chimeric receptors (e.g., activating chimeric TCRs or CARs) expressed on a cell of the present disclosure (e.g., an immunoresponsive cell) as NOT-logic gates to control, modulate, or otherwise inhibit one or more activities of the one or more activating chimeric receptors. In some embodiments, a chimeric inhibitory receptor of the present disclosure may inhibit one or more activities of a cell of the present disclosure (e.g., an immunoresponsive cell).

[0324] In some embodiments, a cell of the present disclosure comprises one or more chimeric inhibitory receptors of the present disclosure and further comprises a tumor-targeting chimeric receptor that binds to one or more tumor-associated antigens. In some embodiments, the one or more tumor-associated antigens include an AML- associated antigen. In some embodiments, the one or more tumor-associated antigens include CD33. In some embodiments, the one or more tumor-associated antigens include FLT3. In some embodiments, the one or more tumor- associated antigens include CD33 and FLT3.

[0325] Co-stimulatory ligands

[0326] In some embodiments, a cell of the present disclosure (e.g., an immunoresponsive cell) can further include one or more recombinant or exogenous co-stimulatory ligands. For example, the cell can be further transduced with one or more co-stimulatory ligands, such that the cell co- expresses or is induced to co-express one or more chimeric receptors of the present disclosure (e.g., the EMCN-specific, FLT3- specific, and / or CD33-specific CARs described herein) and one or more co-stimulatory ligands. Without wishing to be bound by theory, it is believed that the interaction between the one or more chimeric receptors and the one or more co-stimulatory ligands may provide a non- antigen- specific signal important for full activation of the cell. Examples of suitable co-stimulatory ligands include, without limitation, members of the tumor necrosis factor (TNF) superfamily, and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates the acute phase reaction. Its primary role is in the regulation of immune cells. Members of TNF superfamily share a number of common features. The majority of TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. Examples of suitable TNF superfamily members include, without limitation, nerve growth factor (NGF), CD40L (CD40L) / CD 154, CD137L / 4-1BBL, TNF-a, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor beta (TNFP) / lymphotoxin- alpha (LTa), lymphotoxin-beta (LTP), CD257 / B cell- activating factor (B AFF) / Bly s / THANK / Tall- 1, glucocorticoid- induced TNF Receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNESE 14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins that are involved in the recognition, binding, or adhesion processes of cells. These proteins share structural features with immunoglobulins and possess an immunoglobulin domain (fold). Examples of suitable immunoglobulin superfamily ligands include, without limitation, CD80 and CD86, both ligands for CD28, PD-L1 / (B7-H1) that ligands for PD-1. In certain embodiments, the one or more co-stimulatory ligands are selected from 4-1 BBL, CD80, CD86, CD70, OX40L, CD48, TNERSE14, PD-L1, and combinations thereof.

[0327] Chemokine receptor

[0328] In some embodiments, a cell of the present disclosure (e.g., an immunoresponsive cell) comprises one or more chimeric receptors (e.g., the EMCN- specific, ELT3-specific, and / or CD33-specific CARs described herein) and may further include one or more chemokine receptors. Lor example, transgenic expression of chemokine receptor CCR2b or CXCR2 in cells, such as T cells, enhances trafficking to CCL2- secreting or CXCL1 -secreting solid tumors (Craddock et al, J Immunother. 2010 Oct; 33(8):780-8 and Kershaw et al. Hum Gene Ther. 2002 Nov 1; 13(16): 1971 -80). Without wishing to be bound by theory, it is believed that chemokine receptors expressed on chimeric receptor-expressing cells of the present disclosure may recognize chemokines secreted by tumors and improve targeting of the cell to the tumor, which may facilitate the infiltration of the cell to the tumor and enhance the antitumor efficacy of the cell. Chemokine receptors of the present disclosure may include a naturally occurring chemokine receptor, a recombinant chemokine receptor, or a chemokine-binding fragment thereof. Examples of suitable chemokine receptors that may expressed on a cell of the present disclosure include, without limitation, a CXC chemokine receptor, such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7; a CC chemokine receptor, such as CCR1 , CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11; a CX3C chemokine receptor, such as CX3CR1; an XC chemokine receptor, such as XCR1; and chemokine-binding fragments thereof. In some embodiments, the chemokine receptor to be expressed on the cell is chosen based on the chemokines secreted by the tumor.

[0329] Chimeric Receptor Regulation

[0330] Some embodiments of the present disclosure relate to regulating one or more chimeric receptor activities of chimeric receptor-expressing cells of the present disclosure (e.g., the EMCN-specific CARs described herein). There are several ways chimeric receptor activities can be regulated. In some embodiments, a regulatable chimeric receptor, wherein one or more chimeric receptor activities can be controlled, may be desirable to optimize the safety and / or efficacy of the chimeric receptor therapy. For example, inducing apoptosis using a caspase fused to a dimerization domain (see, e.g., Di et al., N Engl. J. Med. 2011 Nov. 3; 365(18): 1673-1683) can be used as a safety switch in the chimeric receptor therapy. In some embodiments, a chimeric receptor-expressing cell of the present disclosure can also express an inducible Caspase-9 (iCaspase-9) that, upon administration of a dimerizer drug, such as rimiducid (IUPAC name: [(lR)-3-(3,4-dimethoxyphenyl)-l-[3-[2-[2-[[2-[3-[(lR)-3-(3,4-dimethoxyphenyl)-l-[(2S)- l-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2- carbonyl] oxypropyl]phenoxy] acetyl] amino] ethylamino] -2-oxoethoxy]phenyl]propyl] (2S)-1- [(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate), induces activation of the Caspase-9 and results in apoptosis of the cells. In some embodiments, the iCaspase-9 contains a binding domain that comprises a chemical inducer of dimerization (CID) that mediates dimerization in the presence of the CID, which results in inducible and selective depletion of the chimeric receptor-expressing cells.

[0331] Alternatively, in some embodiments a chimeric receptor of the present disclosure may be regulated by utilizing a small molecule or an antibody that deactivates or otherwise inhibits chimeric receptor activity. For example, an antibody may delete the chimeric receptor- expressing cells by inducing antibody dependent cell-mediated cytotoxicity (ADCC). In some embodiments, a chimeric receptor-expressing cell of the present disclosure may further express an antigen that is recognized by a molecule that is capable of inducing cell death by ADCC or complement-induced cell death. For example, a chimeric receptor-expressing cell of the present disclosure may further express a receptor capable of being targeted by an antibody or antibody fragment. Examples of suitable receptors that may be targeted by an antibody or antibody fragment include, without limitation, EpCAM, VEGFR, integrins (e.g., avP3, a4, al%p3, a4p7, a5pi, avP3, av), members of the TNF receptor superfamily (e.g., TRAIE-R1 and TRAIE-R2), PDGF receptor, interferon receptor, folate receptor, GPNMB, ICAM-1 , HEA-DR, CEA, CA- 125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11, CDlla / LFA-1, CD15, CD18 / ITGB2, CD19, CD20, CD22, CD23 / IgE Receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41 , CD44, CD51, CD52, CD62L, CD74, CD80, CD125, CD147 / basigin, CD152 / CTLA-4, CD154 / CD40L, CD195 / CCR5, CD319 / SLAMF7, and EGFR, and truncated versions thereof.

[0332] In some embodiments, a chimeric receptor-expressing cell of the present disclosure may also express a truncated epidermal growth factor receptor (EGFR) that lacks signaling capacity but retains an epitope that is recognized by molecules capable of inducing ADCC (e.g., WO201 1 / 056894).

[0333] In some embodiments, a chimeric receptor-expressing cell of the present disclosure further includes a highly expressing compact marker / suicide gene that combines target epitopes from both CD32 and CD20 antigens in the chimeric receptor-expressing cell, which binds an anti-CD20 antibody (e.g., rituximab) resulting in selective depletion of the chimeric receptor- expressing cell by ADCC. Other methods for depleting chimeric receptor-expressing cells of the present disclosure my include, without limitation, administration of a monoclonal anti-CD52 antibody that selectively binds and targets the chimeric receptor-expressing cell for destruction by inducing ADCC. In some embodiments, the chimeric receptor-expressing cell can be selectively targeted using a chimeric receptor ligand, such as an anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody can cause effector cell activity, such as ADCC or ADC activity. In some embodiments, the chimeric receptor ligand can be further coupled to an agent that induces cell killing, such as a toxin. In some embodiments, a chimeric receptor-expressing cell of the present disclosure may further express a target protein recognized by a cell depleting agent of the present disclosure. In some embodiments, the target protein is CD20 and the cell depleting agent is an anti-CD20 antibody. In such embodiments, the cell depleting agent is administered once it is desirable to reduce or eliminate the chimeric receptor-expressing cell. In some embodiments, the cell depleting agent is an anti-CD52 antibody.

[0334] In some embodiments, a regulated chimeric receptor comprises a set of polypeptides, in which the components of a chimeric receptor of the present disclosure are partitioned on separate polypeptides or members. For example, the set of polypeptides may include a dimerization switch that, when in the presence of a dimerization molecule, can couple the polypeptides to one another to form a functional chimeric receptor.

[0335] Endomucin-Specific Antigen-binding Domains

[0336] The present disclosure provides antigen-binding domains (e.g., single-chain variable fragments) that bind to endomucin (EMCN), chimeric proteins including antigen-binding domains that bind to EMCN (e.g., any of the iCARs described herein), and nucleic acids encoding such antigen -binding domains and chimeric proteins. Without wishing to be bound by theory, EMCN is a sialoglycoprotein that interferes with assembly of focal adhesion complexes and inhibits interaction between cells and extracellular matrix. EMCN-specific antigen-binding domains bind to human EMCN (e.g., Uniprot Q9ULC0, herein incorporated by reference for all purposes) or an epitope fragment thereof. EMCN can be expressed on cells generally considered to be healthy, such as healthy hematopoietic stem cells (HSCs), HSPCs, healthy multipotent progenitors (MPPs), healthy lympho-myeloid primed progenitor (LMPPs), and healthy hematopoietic progenitor cells (HPCs). EMCN can be expressed on hematopoietic stem and progenitor cells (HSPCs). EMCN can be expressed on HSCs. EMCN can be expressed on MPPs. EMCN can be expressed on LMPPs. EMCN can be expressed on HPCs. EMCN- specific antibodies have been previously described, including CBFYE-0213, V.7.C7.1, L4B1, L5F12, L10B5, L3F12, L6H3, , L9H8, and L10F12, as described in Samulowitz U. et al., Am. J. Path., 2002 May, 160(5): 1669-1681, herein incorporated by reference for all purposes.

[0337] The present disclosure provides an EMCN-specific antigen-binding domain including one or more of the amino acid sequences listed in Table 11.

[0338] Table 11 - EMCN-specific antigen-binding domains

[0339] In some embodiments, the antigen-binding domain specific for EMCN includes a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the EMCN-VH includes a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 291), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 296), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 298), and the EMCN-VL includes a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 299), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 300), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 301), and where the amino acid sequences of the CDR-H1, the CDR-H2, the CDR-H3, the CDR-L1, the CDR-L2, and the CDR-L3 of the reference antibody are defined based on the Kabat numbering scheme.

[0340] In some embodiments, the antigen-binding domain specific for EMCN includes a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the EMCN-VH includes the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNT HYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQGTMVTVSS (SEQ ID NO: 302); and the EMCN-VL includes the amino acid sequence DVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKL DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQGTKLEIK (SEQ ID NO: 310).

[0341] In some embodiments, the antigen-binding domain specific for EMCN includes the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNT HYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQGTMVTVSSGGG GSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPG QSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQ GTKLEIK (SEQ ID NO: 311). In some embodiments, the antigen-binding domain specific for EMCN is encoded by a polynucleotide sequence comprising the sequence GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAG ACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGATACGATATGCACTGGGTCCG ACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGTGATCTGGGGCAACGGCAACA CACACTACCACAGCGCCCTGAAGTCCCGGTTCACCATCTCCAGAGACAACAGCAAG AACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTA CTGCACCCTGAGAATCAAGGATTGGGGCCAGGGCACCATGGTCACCGTTTCTTCTG GAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCTGACGTGGTCAT GACACAGAGCCCTCTGAGCCTGCCTGTGACACTGGGACAGCCTGCCAGCATCAGCT GCAAGTCTAGCCAGTCTCTGGTGGCCAGCGACGAGAACACCTACCTGAACTGGTTC CAGCAGAGGCCCGGACAGTCTCCTAGACGGCTGATCTACCAGGTGTCCAAGCTGGA TAGCGGCGTGCCCGATAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGAA GATCAGCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTCTGCAAGGCATCC ATCTGCCTTGGACCTTTGGCCAGGGCACAAAGCTGGAAATCAAG (SEQ ID NO: 312). In some embodiments, the antigen-binding domain specific for EMCN is encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAG ACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGATACGATATGCACTGGGTCCG ACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGTGATCTGGGGCAACGGCAACA CACACTACCACAGCGCCCTGAAGTCCCGGTTCACCATCTCCAGAGACAACAGCAAG AACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTA CTGCACCCTGAGAATCAAGGATTGGGGCCAGGGCACCATGGTCACCGTTTCTTCTG GAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCTGACGTGGTCAT GACACAGAGCCCTCTGAGCCTGCCTGTGACACTGGGACAGCCTGCCAGCATCAGCT GCAAGTCTAGCCAGTCTCTGGTGGCCAGCGACGAGAACACCTACCTGAACTGGTTC CAGCAGAGGCCCGGACAGTCTCCTAGACGGCTGATCTACCAGGTGTCCAAGCTGGA TAGCGGCGTGCCCGATAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGAA GATCAGCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTCTGCAAGGCATCC ATCTGCCTTGGACCTTTGGCCAGGGCACAAAGCTGGAAATCAAG (SEQ ID NO: 312).

[0342] FLT3 and CD33-Specific Antigen-binding Domains

[0343] The present disclosure provides antigen-binding domains (e.g., single-chain variable fragments) that bind to FLT3 and CD33, chimeric proteins including antigen-binding domains that bind to FLT3 and CD33 (e.g., any of the aCARs described herein), and nucleic acids encoding such antigen -binding domains and chimeric proteins.

[0344] In some embodiments, chimeric receptors comprise one or more of the amino acid sequences listed in Table Al or Table A2. In some embodiments, activating chimeric receptors (aCARS) comprise one or more of the amino acid sequences listed in Table Al or Table A2. In some embodiments, bispecific-bivalent aCARS comprise one or more of the amino acid sequences listed in Table Al or Table A2. Table Al provides the variable domains of an antibody heavy chain or light chain. The CDRs were determined using the Kabat method and are in bold italic in Table Al for each variable heavy chain or variable light chain and shown in Table A2. In some embodiments, nucleic acids encoding any of the chimeric receptors of the present disclosure comprise one or more of the nucleic acid sequences listed in Table B.

[0345] In some embodiments, an antigen-binding domain specific for CD33 includes a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the CD33-VH includes: a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of DYNMH (SEQ ID NO: 402), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of YIYPYNGGTGYNQKFKSKA (SEQ ID NO: 403), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of GRPAMDYWGQ (SEQ ID NO: 404), and the CD33-VL includes: a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASES VDNYGISFMN (SEQ ID NO: 405), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of AASNQGS (SEQ ID NO: 406), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQSKEVPWT (SEQ ID NO: 407), and wherein the amino acid sequences of the CDR-H1, the CDR-H2, the CDR-H3, the CDR-L1, the CDR-L2, and the CDR-L3 of the reference antibody are defined based on the Kabat numbering scheme.

[0346] In some embodiments, an antigen-binding domain specific for CD33 includes a CD33- VH having the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGG TGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTV SS (SEQ ID NO: 329), and a CD33-VL having the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQG SGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 330).

[0347] In some embodiments, an antigen-binding domain specific for FLT3 includes a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the FLT3-VH includes a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GGTFSSYAIS (SEQ ID NO: 360), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of GIIPIFGTANYAQKFQG (SEQ ID NO: 361), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of FALFGFREQAFDI (SEQ ID NO: 362), and the FLT3-VL includes a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASQSISSYLN (SEQ ID NO: 363), a light chain complementarity determining region 2 (CDR- L2) having the amino acid sequence of AASSLQS (SEQ ID NO: 364), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQSYSTPFT (SEQ ID NO: 365), and wherein the amino acid sequences of the CDR-H1, the CDR-H2, the CDR-H3, the CDR-L1, the CDR-L2, and the CDR-L3 of the reference antibody are defined based on the Kabat numbering scheme.

[0348] In some embodiments, an antigen-binding domain specific for FLT3 includes a FLT3- VH having the amino acid sequence EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTAN YAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCATFALFGFREQAFDIWGQGTTV TVSS (SEQ ID NO: 315), and a FLT3-VL having the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDLATYYCQQSYSTPFTFGPGTKVDIK (SEQ ID NO: 316).

[0349] In some embodiments, a bivalent-bispecific aCAR includes an antigen-binding domain specific for FLT3 and an antigen-binding domain specific for CD33.

[0350] In some embodiments, a bivalent-bispecific aCAR specific for FLT3 and CD33 includes the amino acid sequence MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVR QAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCA TFALFGFREQAFDIWGQGTTVTVSSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYT FTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSL RSEDTAVYYCARGRPAMDYWGQGTLVTVSSGSTSGSGKPGSGEGSTKGDIQMTQSPSS LSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGS GSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKGGGGSDIQMTQSPSSLSA SVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL TISSLQPEDLATYYCQQSYSTPFTFGPGTKVDIKALSNSIMYFSHFVPVFLPAKPTTTPAP RPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YCNHRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA YKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 414).

[0351] In some embodiments, a bivalent-bispecific aCAR specific for FLT3 and CD33 includes the amino acid sequence MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVR QAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCA TFALFGFREQAFDIWGQGTTVTVSSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCK ASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAY MELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSGGGGSGGGGSDIQMTQSPSSLS ASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGS GTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKGGGGSGGGGSDIQMTQSPS SLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGT DFTLTISSLQPEDLATYYCQQSYSTPFTFGPGTKVDIKALSNSIMYFSHFVPVFLPAKPTT TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS LVITLYCNHRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSA DAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 415).

[0352] Engineered Nucleic Acids

[0353] Provided herein are multicistronic expression systems that include engineered nucleic acids encoding (i) a membrane-cleavable chimeric protein; (ii) a bivalent aCAR; and (iii) an iCAR.

[0354] In certain embodiments described herein, the multicistronic expression systems included engineered nucleic acids that encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding each of (i) a membrane-cleavable chimeric protein; (ii) a bivalent aCAR; and (iii) an iCAR.

[0355] The promoter is operably linked to expression cassette of the multicistronic system such that the exogenous polynucleotide sequence encoding each of the membrane-cleavable chimeric protein, the bivalent aCAR, and the iCAR is configured to be expressed as a single polypeptide.

[0356] An “engineered nucleic acid” is a nucleic acid that does not occur in nature. It should be understood, however, that while an engineered nucleic acid as a whole is not naturally- occurring, it may include nucleotide sequences that occur in nature. In some embodiments, an engineered nucleic acid comprises nucleotide sequences from different organisms (e.g., from different species). For example, in some embodiments, an engineered nucleic acid includes a murine nucleotide sequence, a bacterial nucleotide sequence, a human nucleotide sequence, and / or a viral nucleotide sequence. The term “engineered nucleic acids” includes recombinant nucleic acids and synthetic nucleic acids. A “recombinant nucleic acid” refers to a molecule that is constructed by joining nucleic acid molecules and, in some embodiments, can replicate in a live cell. A “synthetic nucleic acid” refers to a molecule that is amplified or chemically, or by other means, synthesized. Synthetic nucleic acids include those that are chemically modified, or otherwise modified, but can base pair with naturally- occurring nucleic acid molecules. Modifications include, but are not limited to, one or more modified intemucleotide linkages and non-natural nucleic acids. Modifications are described in further detail in U.S. Pat. No.

[0357] 6,673,611 and U.S. Application Publication 2004 / 0019001 and, each of which is incorporated by reference in their entirety. Modified intemucleotide linkages can be a phosphorodithioate or phosphorothioate linkage. Non-natural nucleic acids can be a locked nucleic acid (LNA), a peptide nucleic acid (PNA), glycol nucleic acid (GNA), a phosphorodiamidate morpholino oligomer (PMO or “morpholino”), and threose nucleic acid (TNA). Non-natural nucleic acids are described in further detail in International Application WO 1998 / 039352, U.S. Application Pub. No. 2013 / 0156849, and U.S. Pat. Nos. 6,670,461; 5,539,082; 5,185,444, each herein incorporated by reference in their entirety. Recombinant nucleic acids and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing. Engineered nucleic acid of the present disclosure may be encoded by a single molecule (e.g., included in the same plasmid or other vector) or by multiple different molecules (e.g., multiple different independently-replicating molecules). Engineered nucleic acids can be an isolated nucleic acid. Isolated nucleic acids include, but are not limited to a cDNA polynucleotide, an RNA polynucleotide, an RNAi oligonucleotide (e.g., siRNAs, miRNAs, antisense oligonucleotides, shRNAs, etc.), an mRNA polynucleotide, a circular plasmid, a linear DNA fragment, a vector, a minicircle, a ssDNA, a bacterial artificial chromosome (BAC), and yeast artificial chromosome (YAC), and an oligonucleotide.

[0358] Engineered nucleic acid of the present disclosure may be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, engineered nucleic acid constructs are produced using GIBSON ASSEMBLY® Cloning (see, e.g., Gibson, D.G. et al. Nature Methods, 343-345, 2009; and Gibson, D.G. et al. Nature Methods, 901-903, 2010, each of which is incorporated by reference herein). GIBSON ASSEMBLY® typically uses three enzymatic activities in a single-tube reaction: 5' exonuclease, the Y extension activity of a DNA polymerase and DNA ligase activity. The 5 ' exonuclease activity chews back the 5 ' end sequences and exposes the complementary sequence for annealing. The polymerase activity then fills in the gaps on the annealed regions. A DNA ligase then seals the nick and covalently links the DNA fragments together. The overlapping sequence of adjoining fragments is much longer than those used in Golden Gate Assembly, and therefore results in a higher percentage of correct assemblies. In some embodiments, engineered nucleic acid constructs are produced using IN- FUSION® cloning (Clontech). Promoters

[0359] In general, in all embodiments described herein, the engineered nucleic acids encoding the membrane-cleavable chimeric protein, the bivalent aCAR, and the iCAR encode an expression cassette containing a promoter. In some embodiments, an engineered nucleic acid (e.g., an engineered nucleic acid comprising an expression cassette) comprises a promoter operably linked to a nucleotide sequence (e.g., an exogenous polynucleotide sequence) encoding at least 2 distinct proteins. For example, the engineered nucleic acid may comprise a promoter operably linked to a nucleotide sequence encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 distinct proteins. In some embodiments, an engineered nucleic acid comprises a promoter operably linked to a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more distinct proteins. In some embodiments, an engineered nucleic acid (e.g., an engineered nucleic acid comprising an expression cassette) comprises a promoter operably linked to a nucleotide sequence (e.g., an exogenous polynucleotide sequence) encoding at least 2 membrane-cleavable chimeric proteins. For example, the engineered nucleic acid may comprise a promoter operably linked to a nucleotide sequence encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 membrane-cleavable chimeric proteins. In some embodiments, an engineered nucleic acid comprises a promoter operably linked to a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more membrane-cleavable chimeric proteins.

[0360] A “promoter” refers to a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter may also contain sub-regions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive, inducible, repressible, tissue-specific or any combination thereof. A promoter drives expression or drives transcription of the nucleic acid sequence that it regulates. Herein, a promoter is considered to be “operably linked” when it is in a correct functional location and orientation in relation to a nucleic acid sequence it regulates to control (“drive”) transcriptional initiation and / or expression of that sequence.

[0361] A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter can be referred to as “endogenous.” In some embodiments, a coding nucleic acid sequence may be positioned under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the encoded sequence in its natural environment. Such promoters may include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not "naturally occurring" such as, for example, those that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression through methods of genetic engineering that are known in the art. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including polymerase chain reaction (PCR) (see, e.g., U.S. Pat. No. 4,683,202 and U.S. Pat. No. 5,928,906).

[0362] Promoters of an engineered nucleic acid may be “inducible promoters,” which refer to promoters that are characterized by regulating (e.g., initiating or activating) transcriptional activity when in the presence of, influenced by or contacted by a signal. The signal may be endogenous or a normally exogenous condition (e.g., light), compound (e.g., chemical or non- chemical compound) or protein (e.g., cytokine) that contacts an inducible promoter in such a way as to be active in regulating transcriptional activity from the inducible promoter. Activation of transcription may involve directly acting on a promoter to drive transcription or indirectly acting on a promoter by inactivation a repressor that is preventing the promoter from driving transcription. Conversely, deactivation of transcription may involve directly acting on a promoter to prevent transcription or indirectly acting on a promoter by activating a repressor that then acts on the promoter.

[0363] A promoter is “responsive to” or “modulated by” a local tumor state (e.g., inflammation or hypoxia) or signal if in the presence of that state or signal, transcription from the promoter is activated, deactivated, increased, or decreased. In some embodiments, the promoter comprises a response element. A “response element” is a short sequence of DNA within a promoter region that binds specific molecules (e.g., transcription factors) that modulate (regulate) gene expression from the promoter. Response elements that may be used in accordance with the present disclosure include, without limitation, a phloretin-adjustable control element (PEACE), a zinc-finger DNA-binding domain (DBD), an interferon-gamma-activated sequence (GAS) (Decker, T. et al. J Interferon Cytokine Res. 1997 Mar; 17(3): 121-34, incorporated herein by reference), an interferon- stimulated response element (ISRE) (Han, K. J. et al. J Biol Chem. 2004 Apr 9;279(15): 15652-61, incorporated herein by reference), a NF-kappaB response element (Wang, V. et al. Cell Reports. 2012; 2(4): 824-839, incorporated herein by reference), and a STAT3 response element (Zhang, D. et al. J of Biol Chem. 1996; 271: 9503-9509, incorporated herein by reference). Other response elements are encompassed herein. Response elements can also contain tandem repeats (e.g., consecutive repeats of the same nucleotide sequence encoding the response element) to generally increase sensitivity of the response element to its cognate binding molecule. Tandem repeats can be labeled 2X, 3X, 4X, 5X, etc. to denote the number of repeats present.

[0364] Non-limiting examples of responsive promoters (also referred to as “inducible promoters”) (e.g., TGF-beta responsive promoters) are listed in Table 12A, which shows the design of the promoter and transcription factor, as well as the effect of the inducer molecule towards the transcription factor (TF) and transgene transcription (T) is shown (B, binding; D, dissociation; n.d., not determined) (A, activation; DA, deactivation; DR, derepression) (see Homer, M. & Weber, W. FEBS Letters 586 (2012) 20784-2096m, and references cited therein).

[0365] Non-limiting examples of components of inducible promoters include those presented in Table 12B.

[0366] Table 12A. Examples of Responsive Promoters Table 12B. Exemplary Components of Inducible Promoters

[0367] Non-limiting examples of promoters include the cytomegalovirus (CMV) promoter, the elongation factor 1-alpha (EFla) promoter, the elongation factor (EFS) promoter, the MND promoter (a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer), the phosphoglycerate kinase (PGK) promoter, the spleen focus-forming virus (SFFV) promoter, the simian virus 40 (SV40) promoter, and the ubiquitin C (UbC) promoter (see Table 12C). Table 12C. Exemplary Constitutive Promoters

[0368] The promoter can be a tissue-specific promoter. In general, a tissue- specific promoter directs transcription of a nucleic acid, (e.g. , the engineered nucleic acids encoding the chimeric proteins, such as membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S) such that expression is limited to a specific cell type, organelle, or tissue. Tissue- specific promoters include, but are not limited to, albumin (liver specific, Pinkert et al., (1987)), lymphoid specific promoters (Calame and Eaton, 1988), particular promoters of T-cell receptors (Winoto and Baltimore, (1989)) and immunoglobulins; Banerji et al., (1983); Queen and Baltimore, 1983), neuron specific promoters (e.g. the neurofilament promoter; Byrne and Ruddle, 1989), pancreas specific promoters (Edlund et al., (1985)) or mammary gland specific promoters (milk whey promoter, U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166) as well as developmentally regulated promoters such as the murine hox promoters (Kessel and Gruss, Science 249:374-379 (1990)) or the a-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3:537-546 (1989)), the contents of each of which are fully incorporated by reference herein. The promoter can be constitutive in the respective specific cell type, organelle, or tissue. Tissue-specific promoters and / or regulatory elements can also include promoters from the liver fatty acid binding (FAB) protein gene, specific for colon epithelial cells; the insulin gene, specific for pancreatic cells; the transphyretin, .alpha.1- antitrypsin, plasminogen activator inhibitor type 1 (PAI- 1), apolipoprotein Al and LDL receptor genes, specific for liver cells; the myelin basic protein (MBP) gene, specific for oligodendrocytes; the glial fibrillary acidic protein (GFAP) gene, specific for glial cells; OPSIN, specific for targeting to the eye; and the neural-specific enolase (NSE) promoter that is specific for nerve cells. Examples of tissue- specific promoters include, but are not limited to, the promoter for creatine kinase, which has been used to direct expression in muscle and cardiac tissue and immunoglobulin heavy or light chain promoters for expression in B cells. Other tissue specific promoters include the human smooth muscle alpha- actin promoter. Exemplary tissue- specific expression elements for the liver include but are not limited to HMG-COA reductase promoter, sterol regulatory element 1, phosphoenol pyruvate carboxy kinase (PEPCK) promoter, human C- reactive protein (CRP) promoter, human glucokinase promoter, cholesterol L 7-alpha hydroylase (CYP-7) promoter, beta- galactosidase alpha-2,6 sialylkansferase promoter, insulin-like growth factor binding protein (IGFBP-I) promoter, aldolase B promoter, human transferrin promoter, and collagen type I promoter. Exemplary tissue-specific expression elements for the prostate include but are not limited to the prostatic acid phosphatase (PAP) promoter, prostatic secretory protein of 94 (PSP 94) promoter, prostate specific antigen complex promoter, and human glandular kallikrein gene promoter (hgt-1). Exemplary tissue- specific expression elements for gastric tissue include but are not limited to the human H+ / K+-ATPase alpha subunit promoter. Exemplary tissue- specific expression elements for the pancreas include but are not limited to pancreatitis associated protein promoter (PAP), elastase 1 transcriptional enhancer, pancreas specific amylase and elastase enhancer promoter, and pancreatic cholesterol esterase gene promoter. Exemplary tissue-specific expression elements for the endometrium include, but are not limited to, the uteroglobin promoter. Exemplary tissue-specific expression elements for adrenal cells include, but are not limited to, cholesterol side-chain cleavage (SCC) promoter. Exemplary tissue- specific expression elements for the general nervous system include, but are not limited to, gamma-gamman enolase (neuron- specific enolase, NSE) promoter. Exemplary tissue-specific expression elements for the brain include, but are not limited to, the neurofilament heavy chain (NF-H) promoter. Exemplary tissue-specific expression elements for lymphocytes include, but are not limited to, the human CGL-l / granzyme B promoter, the terminal deoxy transferase (TdT), lambda 5, VpreB, and lek (lymphocyte specific tyrosine protein kinase p561ck) promoter, the humans CD2 promoter and its 3 ' transcriptional enhancer, and the human NK and T cell specific activation (NKG5) promoter. Exemplary tissue-specific expression elements for the colon include, but are not limited to, pp60c-src tyrosine kinase promoter, organ-specific neoantigens (OSNs) promoter, and colon specific antigen-P promoter. Tissue- specific expression elements for breast cells are for example, but are not limited to, the human alpha-lactalbumin promoter. Exemplary tissue- specific expression elements for the lung include, but are not limited to, the cystic fibrosis transmembrane conductance regulator (CFTR) gene promoter.

[0369] In some embodiments, a promoter of the present disclosure is modulated by signals within a tumor microenvironment. A tumor microenvironment is considered to modulate a promoter if, in the presence of the tumor microenvironment, the activity of the promoter is increased or decreased by at least 10%, relative to activity of the promoter in the absence of the tumor microenvironment. In some embodiments, the activity of the promoter is increased or decreased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, relative to activity of the promoter in the absence of the tumor microenvironment. For example, the activity of the promoter is increased or decreased by 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20-200%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100%, or 50-200%, relative to activity of the promoter in the absence of the tumor microenvironment.

[0370] In some embodiments, the activity of the promoter is increased or decreased by at least 2 fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100 fold), relative to activity of the promoter in the absence of the tumor microenvironment. For example, the activity of the promoter is increased or decreased by at least 3 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, or at least 100 fold, relative to activity of the promoter in the absence of the tumor microenvironment. In some embodiments, the activity of the promoter is increased or decreased by 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100 fold, relative to activity of the promoter in the absence of the tumor microenvironment.

[0371] In some embodiments, a promoter of the present disclosure is activated under a hypoxic condition. A “hypoxic condition” is a condition where the body or a region of the body is deprived of adequate oxygen supply at the tissue level. Hypoxic conditions can cause inflammation (e.g., the level of inflammatory cytokines increase under hypoxic conditions). In some embodiments, the promoter that is activated under hypoxic condition is operably linked to a nucleotide encoding a chimeric proteins that decreases the expression of activity of inflammatory cytokines, thus reducing the inflammation caused by the hypoxic condition. In some embodiments, the promoter that is activated under hypoxic conditions comprises a hypoxia responsive element (HRE). A “hypoxia responsive element (HRE)” is a response element that responds to hypoxia-inducible factor (HIF). The HRE, in some embodiments, comprises a consensus motif NCGTG (where N is either A or G).

[0372] Activation-Conditional Control Polypeptide (ACP) Promoter Systems

[0373] In some embodiments, a synthetic promoter is a promoter system including an activation-conditional control polypeptide- (ACP-) binding domain sequence and a promoter sequence. Such a system is also referred to herein as an “ACP-responsive promoter.” In general, an ACP promoter system includes a first expression cassette encoding an activation-conditional control polypeptide (ACP) and a second expression cassette encoding an ACP-responsive promoter operably linked to an exogenous polynucleotide sequence, such as the exogenous polynucleotide sequence encoding the membrane-cleavable chimeric proteins described herein or any other protein of interest (e.g., a protease). In some embodiments, the first expression cassette and second expression cassette are each encoded by a separate engineered nucleic acid. In other embodiments, the first expression cassette and the second expression cassette are encoded by the same engineered nucleic acid. The ACP-responsive promoter can be operably linked to a nucleotide sequence encoding a single protein of interest or multiple proteins of interest.

[0374] The promoters of the ACP promoter system, e.g., either a promoter driving expression of the ACP or the promoter sequence of the ACP-responsive promoter, can include any of the promoter sequences described herein (see “Promoters” above). The ACP-responsive promoter can be derived from minP, NFkB response element, CREB response element, NF AT response element, SRF response element 1, SRF response element 2, API response element, TCF-LEF response element promoter fusion, Hypoxia responsive element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducer molecule responsive promoters, and tandem repeats thereof. In some embodiments, the ACP-responsive promoter includes a minimal promoter.

[0375] In some embodiments, the ACP-binding domain includes one or more zinc finger binding sites. In some embodiments, the ACP-responsive promoter includes a minimal promoter and the ACP-binding domain includes one or more zinc finger binding sites. The ACP-binding domain can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more zinc finger binding sites. In some embodiments, the transcription factor is a zinc -finger-containing transcription factor. In some embodiments, the zinc-finger-containing transcription factor is a synthetic transcription factor. In some embodiments, the ACP-binding domain includes one or more zinc finger binding sites and the ACP has a DNA-binding zinc finger protein domain (ZF protein domain). In some embodiments, the ACP has a DNA-binding zinc finger protein domain (ZF protein domain) and an effector domain. In some embodiments, the ACP-binding domain includes one or more zinc finger binding sites and the ACP has a DNA-binding zinc finger protein domain (ZF protein domain) and an effector domain. In some embodiments, the ZF protein domain is modular in design and is composed of zinc finger arrays (ZFA). A zinc finger array comprises multiple zinc finger protein motifs that are linked together. Each zinc finger motif binds to a different nucleic acid motif. This results in a ZFA with specificity to any desired nucleic acid sequence, e.g., a ZFA with desired specificity to an ACP-binding domain having a specific zinc finger binding site composition and / or configuration. The ZF motifs can be directly adjacent to each other, or separated by a flexible linker sequence. In some embodiments, a ZFA is an array, string, or chain of ZF motifs arranged in tandem. A ZFA can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,1 3,

[0376] 14, or 15 zinc finger motifs. The ZFA can have from 1-10, 1-15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4- 9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, or 5-15 zinc finger motifs. The ZF protein domain can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more ZFAs. The ZF domain can have from 1-10, 1-

[0377] 15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, or 5-15 ZFAs. In some embodiments, the ZF protein domain comprises one to ten ZFA(s). In some embodiments, the ZF protein domain comprises at least one ZFA. In some embodiments, the ZF protein domain comprises at least two ZFAs. In some embodiments, the ZF protein domain comprises at least three ZFAs. In some embodiments, the ZF protein domain comprises at least four ZFAs. In some embodiments, the ZF protein domain comprises at least five ZFAs. In some embodiments, the ZF protein domain comprises at least ten ZFAs. In some embodiments, the ACP is a transcriptional modulator. In some embodiments, the ACP is a transcriptional repressor. In some embodiments, the ACP is a transcriptional activator. In some embodiments, the ACP is a transcription factor. In some embodiments, the ACP comprises a DNA-binding domain and a transcriptional effector domain. In some embodiments, the DNA-binding domain comprises a tetracycline (or derivative thereof) repressor (TetR) domain. In some embodiments, the ACP is an antigen recognizing receptor of the present disclosure.

[0378] The ACP can also further include an effector domain, such as a transcriptional effector domain. For instance, a transcriptional effector domain can be the effector or activator domain of a transcription factor. Transcription factor activation domains are also known as transactivation domains, and act as scaffold domains for proteins such as transcription coregulators that act to activate or repress transcription of genes. Any suitable transcriptional effector domains can be used in the ACP including, but not limited to, a Herpes Simplex Virus Protein 16 (VP 16) activation domain; an activation domain consisting of four tandem copies of VP 16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains, the tripartite activator is known as a VPR activation domain; a histone acetyltransferase (HAT) core domain of the human El A- associated protein p300, known as a p300 HAT core activation domain; a Kriippel associated box (KRAB) repression domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain, or any combination thereof.

[0379] In some embodiments, the effector domain is s transcription effector domain selected from: a Herpes Simplex Virus Protein 16 (VP 16) activation domain; an activation domain consisting of four tandem copies of VP 16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains, the tripartite activator is known as a VPR activation domain; a histone acetyltransferase (HAT) core domain of the human E1A- associated protein p300, known as a p300 HAT core activation domain; a Kriippel associated box (KRAB) repression domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain. In some embodiments, the ACP is a small molecule (e.g., drug) inducible polypeptide. For example, in some embodiments, the ACP may be induced by tetracycline (or derivative thereof), and comprises a TetR domain and a VP 16 effector domain. In some embodiments, the ACP includes an estrogen receptor variant, such as ERT2, and may be regulated by tamoxifen, or a metabolite thereof (such as 4-hydroxy-tamoxifen [4-OHT], N-desmethyltamoxifen, tamoxifen-N-oxide, or endoxifen), through tamoxifen-controlled nuclear localization.

[0380] In some embodiments, the ACP is a small molecule (e.g., drug) inducible polypeptide that includes a repressible protease and one or more cognate cleavage sites of the repressible protease. In some embodiments, a repressible protease is active (cleaves a cognate cleavage site) in the absence of the specific agent and is inactive (does not cleave a cognate cleavage site) in the presence of the specific agent. In some embodiments, the specific agent is a protease inhibitor. In some embodiments, the protease inhibitor specifically inhibits a given repressible protease of the present disclosure. The repressible protease can be any of the proteases described herein that is capable of inactivation by the presence or absence of a specific agent (see “Protease Cleavage Site” above for exemplary repressible proteases, cognate cleavage sites, and protease inhibitors).

[0381] In some embodiments, the ACP has a degron domain (see “Degron Systems and Domains” above for exemplary degron sequences). The degron domain can be in any order or position relative to the individual domains of the ACP. For example, the degron domain can be N-terminal of the repressible protease, C-terminal of the repressible protease, N-terminal of the ZF protein domain, C-terminal of the ZF protein domain, N-terminal of the effector domain, or C-terminal of the effector domain.

[0382] Multicistronic and Multiple Promoter Systems

[0383] In some embodiments, engineered nucleic acids (e.g., an engineered nucleic acid comprising an expression cassette) are configured to produce multiple chimeric proteins. For example, nucleic acids may be configured to produce 2-20 different chimeric proteins. In some embodiments, nucleic acids are configured to produce 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14,

[0384] 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14,

[0385] 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13,

[0386] 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5- 11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8- 18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12- 16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17- 19, 17-18, 18-20, 18-19, or 19-20 chimeric proteins. In some embodiments, nucleic acids are configured to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chimeric proteins.

[0387] In general, the engineered nucleic acids described herein are multicistronic, i.e., as described above.

[0388] Engineered nucleic acids can also use multiple promoters to express genes from multiple ORFs, i.e., more than one separate mRNA transcripts can be produced from a single engineered nucleic acid. For example, a first promoter can be operably linked to a polynucleotide sequence encoding a first chimeric protein, and a second promoter can be operably linked to a polynucleotide sequence encoding a second chimeric protein. In general, any number of promoters can be used to express any number of chimeric proteins. In some embodiments, at least one of the ORFs expressed from the multiple promoters can be multicistronic.

[0389] “Einkers,” as used herein can refer to polypeptides that link a first polypeptide sequence and a second polypeptide sequence, the multicistronic linkers described above, or the additional promoters that are operably linked to additional ORFs described above.

[0390] Engineered Cells

[0391] Provided herein are engineered cells, and methods of producing the engineered cells, that produce each of (i) a membrane-cleavable chimeric protein; (ii) a bivalent aCAR; and (iii) an iCAR.. In general, engineered cells of the present disclosure may be engineered to express the chimeric proteins provided for herein, such as each of the membrane-cleavable chimeric proteins, the bivalent aCARs, and the iCARsdescribed herein. These cells are referred to herein as “engineered cells.” These cells, which typically contain engineered nucleic acid, do not occur in nature. In some embodiments, the cells are engineered to include a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding a chimeric protein, for example, a membrane-cleavable chimeric protein. An engineered cell can comprise an engineered nucleic acid integrated into the cell’s genome. An engineered cell can comprise an engineered nucleic acid capable of expression without integrating into the cell’s genome, for example, engineered with a transient expression system such as a plasmid or mRNA.

[0392] The present disclosure also encompasses additivity and synergy between a chimeric protein(s) and the engineered cell from which they are produced. In some embodiments, cells are engineered to produce at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) chimeric proteins, for example ate least two membrane-cleavable chimeric proteins. In other embodiments, cells are engineered to produce at least one chimeric proteins having an effector molecule that is not natively produced by the cells. Such an effector molecule may, for example, complement the function of effector molecules natively produced by the cells.

[0393] In some embodiments, cells are engineered to express membrane-tethered anti-CD3 and / or anti-CD28 agonist extracellular domains.

[0394] In some embodiments, a cell (e.g., an immune cell or a stem cell) is engineered to produce multiple chimeric proteins. For example, cells may be engineered to produce 2-20 different chimeric proteins, such as 2-20 different membrane-cleavable chimeric proteins. In some embodiments, cells engineered to produce 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13,

[0395] 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13,

[0396] 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12,

[0397] 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5- 10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9- 13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11- 20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14- 17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, or 19-20 chimeric proteins. In some embodiments, cells are engineered to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chimeric proteins.

[0398] In some embodiments, engineered cells comprise one or more engineered nucleic acids encoding a promoter operably linked to a nucleotide sequence encoding a chimeric protein. In some embodiments, cells are engineered to include a plurality of engineered nucleic acids, e.g., at least two engineered nucleic acids, each encoding a promoter operably linked to a nucleotide sequence encoding at least one (e.g., 1, 2 or 3) chimeric protein. For example, cells may be engineered to comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10, engineered nucleic acids, each encoding a promoter operably linked to a nucleotide sequence encoding at least one (e.g., 1, 2 or 3) chimeric protein. In some embodiments, the cells are engineered to comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more engineered nucleic acids, each encoding a promoter operably linked to a nucleotide sequence encoding at least one (e.g., 1, 2 or 3) chimeric protein. Engineered cells can comprise an engineered nucleic acid encoding at least one of the linkers described above, such as polypeptides that link a first polypeptide sequence and a second polypeptide sequence, one or more multicistronic linker described above, one or more additional promoters operably linked to additional ORFs, or a combination thereof.

[0399] In some embodiments, a cell (e.g., an immune cell or a stem cell) is engineered to express a protease. In some embodiments, a cell is engineered to express a protease heterologous to a cell. In some embodiments, a cell is engineered to express a protease heterologous to a cell expressing a chimeric protein, such as a heterologous protease that cleaves the protease cleavage site of a membrane-cleavable chimeric protein. In some embodiments, engineered cells comprise one or more engineered nucleic acids encoding a promoter operably linked to a nucleotide sequence encoding a protease, such as a heterologous protease. Protease and protease cleavage sites are described in greater detail in the Section herein titled “Protease Cleavage site.”

[0400] In some embodiments, a cell (e.g., an immune cell or a stem cell) is engineered to produce at least one homing molecule. “Homing,” refers to active navigation (migration) of a cell to a target site (e.g., a cell, tissue (e.g., tumor), or organ). A “homing molecule” refers to a molecule that directs cells to a target site. In some embodiments, a homing molecule functions to recognize and / or initiate interaction of an engineered cell to a target site. Non-limiting examples of homing molecules include CXCR1, CCR9, CXCR2, CXCR3, CXCR4, CCR2, CCR4, FPR2, VEGFR, IL6R, CXCR1, CSCR7, and PDGFR.

[0401] In some embodiments, a homing molecule is a chemokine receptor (cell surface molecule that binds to a chemokine). Non-limiting examples of chemokine receptors that may be produced by the engineered cells of the present disclosure include: CXC chemokine receptors (e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7), CC chemokine receptors (CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, and CCR11), CX3C chemokine receptors (e.g., CX3CR1, which binds to CX3CL1), and XC chemokine receptors (e.g., XCR1). In some embodiments, a chemokine receptor is a G protein- linked transmembrane receptor, or a member of the tumor necrosis factor (TNF) receptor superfamily (including but not limited to TNFRSF1A, TNFRSF1B). In some embodiments, cells are engineered to produce CXCL8, CXCL9, and / or CXCL10 (promote T-cell recruitment), CCL3 and / or CXCL5, CCL21 (Thl recruitment and polarization). In some embodiments, cells are engineered to produce CXCR4.

[0402] In some embodiments, a cell (e.g., an immune cell or a stem cell) is engineered to produce G-protein coupled receptors (GPCRs) that detect N-formylated-containing oligopeptides (including but not limited to FPR2 and FPRL1).

[0403] In some embodiments, a cell (e.g., an immune cell or a stem cell) is engineered to produce receptors that detect interleukins (including but not limited to IL6R). In some embodiments, a cell (e.g., an immune cell or a stem cell) is engineered to produce receptors that detect growth factors secreted from other cells, tissues, or tumors (including but not limited to FGFR, PDGFR, EGFR, and receptors of the VEGF family, including but not limited to VEGF-C and VEGF-D).

[0404] In some embodiments, a cell (e.g., an immune cell or a stem cell) is engineered to produce one or more integrins. Cells of the present disclosure may be engineered to produce any combination of integrin a and P subunits. The a subunit of an integrin may be, without limitation: ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, IGTA7, ITGA8, ITGA9, IGTA10, IGTA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGA2B, ITGAX. The p subunit of an integrin may be, without limitation: ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, and ITGB8.

[0405] In some embodiments, a cell (e.g., an immune cell or a stem cell) is engineered to produce one or more matrix metalloproteinases (MMP). Non-limiting examples of MMPs include MMP-2, MMP-9, and MMP. In some embodiments, cells are engineered to produce an inhibitor of a molecule (e.g., protein) that inhibits MMPs. For example, cells may be engineered to express an inhibitor (e.g., an RNAi molecule) of membrane type 1 MMP (MT1-MMP) or TIMP metallopeptidase inhibitor 1 (TIMP-1).

[0406] In some embodiments, a cell (e.g., an immune cell or a stem cell) is engineered to produce a ligand that binds to selectin (e.g., hematopoietic cell E- / L-selectin ligand (HCELL), Dykstran et al., Stem Cells. 2016 Oct;34(10):2501-2511) on the endothelium of a target tissue, for example.

[0407] The term “homing molecule” also encompasses transcription factors that regulate the production of molecules that improve / enhance homing of cells.

[0408] Also provided herein are engineered cells that are engineered to produce multiple chimeric proteins, at least two of which include effector molecules that modulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or more) chimeric protein includes an effector molecule that stimulates at least one immuno stimulatory mechanism in the tumor microenvironment, or inhibits at least one immunosuppressive mechanism in the tumor microenvironment. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or more) chimeric protein includes an effector molecule that inhibits at least one immunosuppressive mechanism in the tumor microenvironment, and at least one chimeric protein (e.g., 1, 2, 3, 4, 5, or more) inhibits at least one immunosuppressive mechanism in the tumor microenvironment. In yet other embodiments, at least two (e.g., 2, 3, 4, 5, or more) chimeric proteins includes an effector molecule that stimulate at least one immuno stimulatory mechanism in the tumor microenvironment. In still other embodiments, at least two (e.g., 1, 2, 3, 4, 5, or more) chimeric proteins includes an effector molecule that inhibit at least one immunosuppressive mechanism in the tumor microenvironment.

[0409] In some embodiments, a cell (e.g., an immune cell or a stem cell) is engineered to produce at least one chimeric protein including an effector molecule that stimulates T cell signaling, activity and / or recruitment. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that stimulates antigen presentation and / or processing. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that stimulates natural killer cell-mediated cytotoxic signaling, activity and / or recruitment. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that stimulates dendritic cell differentiation and / or maturation. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that stimulates immune cell recruitment. In some embodiments, a cell is engineered to produce at least one chimeric protein includes an effector molecule that that stimulates Ml macrophage signaling, activity and / or recruitment. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that stimulates Thl polarization. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that stimulates stroma degradation. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that stimulates immuno stimulatory metabolite production. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that stimulates Type I interferon signaling. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that inhibits negative costimulatory signaling. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that inhibits pro-apoptotic signaling (e.g., via TRAIL) of anti-tumor immune cells. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that inhibits T regulatory (Treg) cell signaling, activity and / or recruitment. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that inhibits tumor checkpoint molecules. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that activates stimulator of interferon genes (STING) signaling. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that inhibits myeloid-derived suppressor cell signaling, activity and / or recruitment. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that degrades immunosuppressive factors / metabolites. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that inhibits vascular endothelial growth factor signaling. In some embodiments, a cell is engineered to produce at least one chimeric protein that includes an effector molecule that directly kills tumor cells (e.g., granzyme, perforin, oncolytic viruses, cytolytic peptides and enzymes, anti-tumor antibodies, e.g., that trigger ADCC).

[0410] In some embodiments, at least one chimeric protein including an effector molecule that: stimulates T cell signaling, activity and / or recruitment, stimulates antigen presentation and / or processing, stimulates natural killer cell-mediated cytotoxic signaling, activity and / or recruitment, stimulates dendritic cell differentiation and / or maturation, stimulates immune cell recruitment, stimulates macrophage signaling, stimulates stroma degradation, stimulates immuno stimulatory metabolite production, or stimulates Type I interferon signaling; and at least one chimeric protein...

Claims

CLAIMSWhat is claimed is:

1. A method of treating a subject known or suspected of having cancer, the method comprising:(A) administering a cell therapy to the subject, wherein the cell therapy comprises an immunoresponsive cell engineered to express a recombinant cytokine, optionally wherein the immunoresponsive cell engineered to express the recombinant cytokine comprises an exogenous polynucleotide encoding a controlled release cytokine, oriented from N-terminal to C-terminal, having the formula:S - C - MT or MT - C - S whereinS comprises a secretable effector molecule, wherein the secretable effector molecule comprises the recombinant cytokine,C comprises a protease cleavage site, andMT comprises a cell membrane tethering domain, wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide; and(B) administering a pharmaceutically effective amount of IL-2, optionally wherein the immunoresponsive cell is engineered to express the recombinant cytokine is from a multicistronic expression system, optionally wherein the recombinant cytokine comprises IL- 15 or a functional fragment thereof, optionally wherein the recombinant cytokine comprises crIL15.

2. The method of claim 1, wherein the immunoresponsive cell comprises a. an exogenous polynucleotide encoding an inhibitory chimeric antigen receptor (iCAR) comprising: (i) an antigen-binding domain specific for endomucin (EMCN); (ii) one or more intracellular inhibitory domains that inhibit an immune response; and (iii) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof, and / or b. an exogenous polynucleotide encoding a bivalent activating chimeric antigen receptor (aCAR) comprising: (i) an antigen-binding domain specific for FLT3;(ii) an antigen-binding domain specific for CD33; (iii) one or more intracellular signaling domains that stimulate an immune response; and (iv) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

3. The method of any one of claims 1-2, wherein the administration of IL-2 comprises multiple administrations, optionally wherein the multiple administrations of IL-2 is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 administrations, optionally wherein IL-2 is administered within 4, within 5, within 7, or within 8 hours following the start of the administration of the cell therapy, optionally wherein IL-2 is administered within 4 to 8 hours following the administration of the cell therapy, optionally wherein IL-2 is administered within 6 to 8 hours following the administration of the cell therapy, optionally wherein IL-2 is administered within 4 hour, 5 hours, 6 hours, 7 hours or 8 hours, following the administration of the cell therapy, optionally wherein IL-2 is administered between 4 to 8 hours following the administration of the cell therapy, optionally wherein IL-2 is administered between 6 to 8 hours following the administration of the cell therapy, optionally wherein IL-2 is administered within 24 or 48 hours after the administering of the cell therapy, optionally wherein the pharmaceutically effective amount of of IL-2 is between 1-10 MIU, optionally wherein the pharmaceutically effective amount of IL-2 is a dose of 3 MIU.

4. The method of any one of claims 1-3, wherein the administration of the cell therapy comprises multiple administrations, optionally wherein IL-2 is administered at least 1, at least 2, a least 3 times a week following each administration of the cell therapy, optionally wherein the cell therapy is administered again at least on day 7 and / or day 14 following the initial administration of the cell therapy, optionally wherein the cell therapy is administered again at least on day 7 following the initial administration of the cell therapy, optionally wherein the cell therapy is administered again at least on day 7 and day 14 following the initial administration of the cell therapy, optionally wherein the cell therapy and the pharmaceutically effective amount of IL-2 are administered again at least on day 7 and / or day 14 following the initial administration of the cell therapy, optionally wherein the cell therapy and the pharmaceutically effective amount of IL-2 are administered again at least on day 7 following the initial administration of the cell therapy, optionally wherein the cell therapy and the pharmaceutically effective amount of IL-2 are administered again at least on day 7 and day 14 following the initialadministration of the cell therapy, optionally wherein the pharmaceutically effective amount of of IL-2 is between 1-10 MIU, optionally wherein the pharmaceutically effective amount of IL-2 is a dose of 3 MIU.

5. The method of any one of claims 1-4, wherein the subject has, prior to administration of the cell therapy, been administered one or more lymphodepletion agents, optionally wherein the one or more lymphodepletion agents is administered 5 days prior to administration of the cell therapy, optionally wherein the one or more lymphodepletion agents is administered daily until administration of the cell therapy, optionally wherein the one or more lympodepletion agents comprises fludarabine, cytarabine (Ara-C), or combinations thereof, optionally wherein the one or more lymphodepletion agents are administered intravenously, optionally wherein fludarabine is administered at a dose of 30 mg / m2 and cytarabine (Ara-C) is administered at a dose of 2 g / m2.

6. The method of any one of claims 1-5, wherein the method of treatment comprises i) a reduction in tumor volume in the subject; ii) treatment of a solid tumor in the subject; and / or iii) inducing an immune response in the subject.

7. The method of any one of claims 1-6, wherein the immunoresponsive cell comprises an engineered nucleic acid comprising at least one selected from the group consisting of:(A) an exogenous polynucleotide encoding a controlled release cytokine, oriented from N-terminal to C-terminal, having the formula:S - C - MT or MT - C - S whereinS comprises a secretable effector molecule, wherein the secretable effector molecule comprises the recombinant cytokine,C comprises a protease cleavage site, andMT comprises a cell membrane tethering domain, wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide;(B) an exogenous polynucleotide encoding an inhibitory chimeric antigen receptor(iCAR) comprising:(i) an antigen-binding domain specific for endomucin (EMCN);(ii) one or more intracellular inhibitory domains that inhibit an immune response; and(iii) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof; and(C) an exogenous polynucleotide encoding a bivalent activating chimeric antigen receptor (aCAR) comprising:(i) an antigen-binding domain specific for FLT3;(ii) an antigen-binding domain specific for CD33;(iii) one or more intracellular signaling domains that stimulate an immune response; and(iv) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof; optionally wherein the membrane-cleavable chimeric protein, the iCAR, and the bivalent aCAR are encoded in order from 5’ to 3’: (i) membrane-cleavable chimeric protein; (ii) bivalent aCAR; and (iii) iCAR; or from 5’ to 3’: (i) membrane-cleavable chimeric protein; (ii) iCAR; and (iii) bivalent aCAR.

8. The multicistronic expression system of any one of claims 1-7, wherein the exogenous polynucleotides encoding each of the membrane-cleavable chimeric protein, the iCAR, and the bivalent aCAR are linked together by a polynucleotide linker encoding a 2A ribosome skipping element, optionally wherein the 2A ribosome skipping element is selected from the group consisting of: a T2A ribosome skipping element, a E2A ribosome skipping element, a P2A ribosome skipping element, a F2A ribosome skipping element, ribosome skipping element fusions thereof, and combinations thereof, optionally wherein the ribosome skipping element fusion comprises an E2A / T2A ribosome skipping element, optionally wherein the E2A / T2A ribosome skipping element comprises the amino acid sequence QCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 221), optionally wherein the E2A / T2A ribosome skipping element is encoded by a polynucleotide sequence comprising the sequence CAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATCTAATC CTGGACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACGT GGAGGAAAACCCTGGACCT (SEQ ID NO: 222) or the sequence CAGTGCACAAATTATGCACTGCTGAAGCTCGCCGGGGATGTCGAGAGTAACC CAGGACCTGGAAGCGGAGAAGGTCGTGGTAGTCTACTAACGTGTGGTGATGT AGAAGAAAATCCTGGACCT (SEQ ID NO: 223).

9. The multicistronic expression system of any one of claims 1-8, wherein the protease cleavage site further comprises the N-terminal peptide linker, optionally selected from the group consisting of: SGGGGSGGGGSG (SEQ ID NO: 230); GGGSGGGGSGGGSLQ (SEQ ID NO: 231); GGS (SEQ ID NO: 232); GGSGGS (SEQ ID NO: 233); GGSGGSGGS (SEQ ID NO: 234); GGSGGSGGSGGS (SEQ ID NO: 235); GGSGGSGGSGGSGGS (SEQ ID NO: 236); GGGS (SEQ ID NO: 237); GGGSGGGS (SEQ ID NO: 238); GGGSGGGSGGGS (SEQ ID NO: 239); GGGSGGGSGGGSGGGS (SEQ ID NO: 240); GGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 241); GGGGS (SEQ ID NO: 242); GGGGSGGGGS (SEQ ID NO: 243); GGGGSGGGGSGGGGS (SEQ ID NO: 244); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 245); GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 246);GSTSGSGKPGSGEGSTKG (SEQ ID NO: 247); and EAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 248), optionally wherein the protease cleavage site further comprises the N- terminal peptide linker SGGGGSGGGGSG (SEQ ID NO: 230), optionally wherein the protease cleavage site further comprises a C-terminal peptide linker, optionally selectedfrom the group consisting of: SGGGGSGGGGSG (SEQ ID NO: 230); GGGSGGGGSGGGSLQ (SEQ ID NO: 231); GGS (SEQ ID NO: 232); GGSGGS (SEQ ID NO: 233); GGSGGSGGS (SEQ ID NO: 234); GGSGGSGGSGGS (SEQ ID NO: 235); GGSGGSGGSGGSGGS (SEQ ID NO: 236); GGGS (SEQ ID NO: 237); GGGSGGGS (SEQ ID NO: 238); GGGSGGGSGGGS (SEQ ID NO: 239); GGGSGGGSGGGSGGGS (SEQ ID NO: 240); GGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 241); GGGGS (SEQ ID NO: 242); GGGGSGGGGS (SEQ ID NO: 243); GGGGSGGGGSGGGGS (SEQ ID NO: 244); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 245);GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 246); GSTSGSGKPGSGEGSTKG (SEQ ID NO: 247); and EAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 248), optionally wherein the protease cleavage site further comprises the C- terminal linker GGGSGGGGSGGGSLQ (SEQ ID NO: 231), optionally wherein the protease cleavage site comprises a Tumor Necrosis Factor-a Converting Enzyme (TACE)-specific cleavage site, optionally wherein the TACE-specific cleavage site comprises the amino acid sequence VTPEPIFSLI (SEQ ID NO: 191), optionally, wherein the TACE-specific cleavage site comprises the amino acid sequence SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ (SEQ ID NO: 250), optionally wherein the TACE-specific cleavage site is encoded by a polynucleotide sequence comprising the sequenceTCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGTGACCCCTGAGCCTA TCTTCAGCCTGATCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATC TCTTCAA (SEQ ID NO: 251).

10. The multicistronic expression system of any one of claims 1-9, wherein the cell membrane tethering domain comprises a transmembrane domain selected from the group consisting of: PDGFR-beta, CD8, CD28, CD3zeta-chain, CD4, 4- IBB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, LIR1, B7-1, and BTLA, optionally wherein the cell membrane tethering domain comprises a B7-1 transmembrane domain, optionally wherein the B7-1 transmembrane domain comprises the amino acid sequence LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 204), optionally wherein the B7-1 transmembrane domain is encoded by a polynucleotide sequence comprising the sequence TTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGAT CTGCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 252), optionally wherein the membrane-cleavable chimeric protein, oriented from N-terminal to C- terminal, has the formula S - C - MT, optionally wherein the membrane-cleavable chimeric protein comprises the amino acid sequence MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCK VTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEE KNIKEFLQSFVHIVQMFINTSSGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSL QLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 226), optionally wherein the membrane-cleavable chimeric protein is encoded by a polynucleotide sequence comprising the sequence ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTC TAATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAG AGCATGCACATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCT GCAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCT GGAAAGCGGCGACGCCAGCATCCACGATACCGTGGAAAATCTGATCATCCTG GCCAACAACAGCCTGTCCAGCAACGGCAATGTGACCGAGAGCGGCTGCAAAG AGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGT CCACATCGTGCAGATGTTCATCAACACCTCATCAGGCGGCGGTGGTAGTGGA GGCGGAGGCTCAGGCGTGACCCCTGAGCCTATCTTCAGCCTGATCGGCGGAG GTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAATTGCTGCCTAGCTGG GCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGACCTA CTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAG AGAAAGCGTGCGGCCTGTG (SEQ ID NO: 227).

11. The multicistronic expression system of any one of claims 1-10, wherein the antigen- binding domain specific for EMCN comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the EMCN-VH comprises: a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 291), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 296), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 298), andthe EMCN-VL comprises: a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 299), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 300), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 301), and wherein the amino acid sequences of the CDR-H1, the CDR-H2, the CDR-H3, the CDR- Ll, the CDR-L2, and the CDR-L3 of the reference antibody are defined based on the Kabat numbering scheme; optionally wherein the EMCN-VH and the EMCN-VL are separated by a peptide linker, optionally wherein the antigen-binding domain specific for EMCN comprises the structure VH-L-VL or VL-L-VH, wherein L is the peptide linker, optionally wherein the peptide linker comprises the amino acid sequence SGGGGSGGGGSG (SEQ ID NO: 230); GGGSGGGGSGGGSLQ (SEQ ID NO: 231); GGS (SEQ ID NO: 232); GGSGGS (SEQ ID NO: 233); GGSGGSGGS (SEQ ID NO: 234); GGSGGSGGSGGS (SEQ ID NO: 235); GGSGGSGGSGGSGGS (SEQ ID NO: 236); GGGS (SEQ ID NO: 237) ; GGGSGGGS (SEQ ID NO: 238); GGGSGGGSGGGS (SEQ ID NO: 239); GGGSGGGSGGGSGGGS (SEQ ID NO: 240);GGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 241); GGGGS (SEQ ID NO: 242); GGGGSGGGGS (SEQ ID NO: 243); GGGGSGGGGSGGGGS (SEQ ID NO: 244); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 245);GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 246);GSTSGSGKPGSGEGSTKG (SEQ ID NO: 247); or EAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 248), optionally wherein the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 244), optionally wherein the peptide linker is encoded by a polynucleotide sequence comprising the sequence GGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCT (SEQ ID NO: 253), optionally wherein the intracellular inhibitory domain comprises a LIR1 intracellular inhibitory domain, optionally wherein the LIR1 intracellular inhibitory domain comprises the amino acid sequence LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYA AVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSI YATLAIH (SEQ ID NO: 285), optionally wherein the LIR1 intracellular inhibitory domain is encoded by a polynucleotide sequence comprising the sequence CTGCGGCACAGAAGGCAGGGCAAGCACTGGACAAGCACCCAGAGAAAGGCC GACTTTCAGCATCCTGCTGGCGCCGTTGGACCTGAGCCTACAGATAGAGGAC TGCAGTGGCGGTCTAGCCCTGCCGCTGATGCCCAAGAGGAAAATCTTTACGC CGCCGTGAAGCACACCCAGCCTGAGGATGGCGTGGAAATGGACACCAGATC TCCCCACGATGAGGACCCTCAGGCCGTGACATACGCAGAAGTGAAGCACTCC AGACCTCGGAGAGAGATGGCAAGCCCTCCATCTCCTCTGAGCGGCGAGTTCC TGGACACCAAAGACAGACAGGCCGAAGAGGACAGACAGATGGATACCGAA GCCGCCGCTTCTGAAGCCCCACAGGATGTGACATATGCCCAGCTGCATAGCC TGACACTGCGGAGAGAAGCCACAGAGCCTCCACCTTCTCAAGAAGGCCCATC TCCTGCCGTGCCTTCCATCTATGCCACTCTGGCCATTCAC (SEQ ID NO: 286), optionally wherein (A) the signal peptide is present in the iCAR, optionally wherein the signal peptide of the iCAR comprises a native signal peptide native or a non-native signal peptide, optionally wherein the non-native signal peptide or the non-native signal- anchor sequence is selected from the group consisting of: IgE, IL- 12, IL-2, optimized IL- 2, trypsiongen-2, Gaussia luciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL-6, IL- 8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin El, GROalpha, CXCL12, IL-21, CD8, NKG2D, TNFR2, and GMCSF, optionally wherein the signal peptide of the iCAR comprises a CD8 signal peptide, optionally wherein the CD8 signal peptide comprises the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 137), optionally wherein the CD8 signal peptide is encoded by a polynucleotide sequence comprising the sequenceATGGCTCTGCCCGTGACAGCTTTGCTGCTGCCTTTGGCACTGCTGCTGCATGC TGCTAGACCA (SEQ ID NO: 416); and / or wherein (B) the hinge domain is present in the iCAR comprises, optionally wherein the hinge domain of the iCAR is selected from the group consisting of a human Ig (immunoglobulin) hinge, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge, a KIR2DS2 hinge, an LNGFR hinge, a LIR1 hinge, a PDGFR-beta extracellular linker, and combinations thereof, optionally wherein the hinge domain of the iCAR comprises a LIR1 hinge, optionally wherein the LIR1 hinge comprises the amino acid sequence HPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV (SEQID NO: 417), optionally wherein the LIR1 hinge comprises is encoded by a polynucleotide sequence comprising the sequence CACCCATCCGATCCTCTCGAGCTGGTGGTTTCTGGACCTTCTGGCGGCCCTAG CAGCCCTACAACAGGACCTACAAGCACAAGCGGCCCTGAGGACCAACCTCT GACACCAACAGGCAGCGATCCTCAGTCTGGACTGGGGAGACATCTGGGCGTT (SEQ ID NO: 418); optionally wherein the hinge domain of the iCAR comprises a CD8 hinge, optionally wherein the CD8 hinge comprises the amino acid sequence TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 271), optionally wherein the CD8 hinge comprises is encoded by a polynucleotide sequence comprising the sequenceACAACAACACCCGCACCTCGGCCTCCAACTCCAGCTCCAACAATTGCACTGC AACCCCTGAGTCTGAGGCCCGAGGCCTGTAGGCCAGCAGCTGGCGGAGCTGT TCACACTAGAGGCCTGGACTTTGCCTGTGAC (SEQ ID NO: 283); and / or wherein (C) the transmembrane domain of the iCAR is present, optionally wherein the transmembrane domain of the iCAR comprises a transmembrane domain selected from the group consisting of: PDGFR-beta, CD8, CD28, CD3zeta-chain, CD4, 4- IBB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, LIR1, and BTLA, optionally wherein the transmembrane domain of the iCAR comprises a LIR1 transmembrane domain, optionally wherein the LIR1 transmembrane domain comprises the amino acid sequence VIGILVAVILLLLLLLLLFLI (SEQ ID NO: 259), optionally wherein the LIR1 transmembrane domain is encoded by a polynucleotide sequence comprising the sequenceGTGATCGGCATTCTGGTCGCCGTGATCCTGCTCCTGTTGCTCCTGCTGCTTCT GTTCCTGATC (SEQ ID NO: 260), optionally wherein the iCAR comprises the amino acid sequence of MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYD MHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSL RAEDTAVYYCTLRIKDWGQGTMVTVSSGGGGSGGGGSGGGGSDVVMTQSPLS LPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPD RFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQGTKLEIKngaaHPSDP LELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVIL LLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAA DAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPP SPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 419), optionally wherein the iCAR is encoded by a polynucleotide sequence comprising the sequence ofATGGCTCTGCCCGTGACAGCTTTGCTGCTGCCTTTGGCACTGCTGCTGCATGCTGCTAGACCAGAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGATACGATATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGTGATCTGGGGCAACGGCAACACACACTACCACAGCGCCCTGAAGTCCCGGTTCACCATCTCCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCACCCTGAGAATCAAGGATTGGGGCCAGGGCACCATGGTCACCGTTTCTTCTGGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCTGACGTGGTCATGACACAGAGCCCTCTGAGCCTGCCTGTGACACTGGGACAGCCTGCCAGCATCAGCTGCAAGTCTAGCCAGTCTCTGGTGGCCAGCGACGAGAACACCTACCTGAACTGGTTCCAGCAGAGGCCCGGACAGTCTCCTAGACGGCTGATCTACCAGGTGTCCAAGCTGGATAGCGGCGTGCCCGATAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGAAGATCAGCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTCTGCAAGGCATCCATCTGCCTTGGACCTTTGGCCAGGGCACAAAGCTGGAAATCAAGAATGGCGCTGCACACCCATCCGATCCTCTCGAGCTGGTGGTTTCTGGACCTTCTGGCGGCCCTAGCAGCCCTACAACAGGACCTACAAGCACAAGCGGCCCTGAGGACCAACCTCTGACACCAACAGGCAGCGATCCTCAGTCTGGACTGGGGAGACATCTGGGCGTTGTGATCGGCATTCTGGTCGCCGTGATCCTGCTCCTGTTGCTCCTGCTGCTTCTGTTCCTGATCCTGCGGCACAGAAGGCAGGGCAAGCACTGGACAAGCACCCAGAGAAAGGCCGACTTTCAGCATCCTGCTGGCGCCGTTGGACCTGAGCCTACAGATAGAGGACTGCAGTGGCGGTCTAGCCCTGCCGCTGATGCCCAAGAGGAAAATCTTTACGCCGCCGTGAAGCACACCCAGCCTGAGGATGGCGTGGAAATGGACACCAGATCTCCCCACGATGAGGACCCTCAGGCCGTGACATACGCAGAAGTGAAGCACTCCAGACCTCGGAGAGAGATGGCAAGCCCTCCATCTCCTCTGAGCGGCGAGTTCCTGGACACCAAAGACAGACAGGCCGAAGAGGACAGACAGATGGATACCGAAGCCGCCGCTTCTGAAGCCCCACAGGATGTGACATATGCCCAGCTGCATAGCCTGACACTGCGGAGAGAAGCCACAGAGCCTCCACCTTCTCAAGAAGGCCCATCTCCTGCCGTGCCTTCCATCTATGCCAC TCTGGCCATTCAC (SEQ ID NO: 420).

12. The multicistronic expression system of any one of claims 1-11, wherein the antigen- binding domain specific for FLT3 comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the FLT3-VH comprises: a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GGTFSSYAIS (SEQ ID NO: 360), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of GIIPIFGTANYAQKFQG (SEQ ID NO: 361), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of FALFGFREQAFDI (SEQ ID NO: 362), and the FLT3-VL comprises: a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASQSISSYLN (SEQ ID NO: 363), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of AASSLQS (SEQ ID NO: 364), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQSYSTPFT (SEQ ID NO: 365), and wherein the amino acid sequences of the CDR-H1, the CDR-H2, the CDR-H3, the CDR- Ll, the CDR-L2, and the CDR-L3 of the reference antibody are defined based on the Kabat numbering scheme; optionally wherein: the FLT3-VH comprises the amino acid sequenceEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPI FGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCATFALFGFREQA FDIWGQGTTVTVSS (SEQ ID NO: 315); and the FLT3-VL comprises the amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQSYSTPFTFGPGTKVDIK (SEQ ID NO: 316), optionally wherein the FLT3-VH, and the FLT3-VL are separated by peptide linkers.

13. The multicistronic expression system of any one of claims 1-12, wherein the antigen- binding domain specific for CD33 comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein:the CD33-VH comprises: a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of DYNMH (SEQ ID NO: 402), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of YIYPYNGGTGYNQKFKSKA (SEQ ID NO: 403), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of GRPAMDYWGQ (SEQ ID NO: 404), and the CD33-VL comprises: a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASESVDNYGISFMN (SEQ ID NO: 405), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of AASNQGS (SEQ ID NO: 406), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQSKEVPWT (SEQ ID NO: 407), and wherein the amino acid sequences of the CDR-H1, the CDR-H2, the CDR-H3, the CDR- Ll, the CDR-L2, and the CDR-L3 of the reference antibody are defined based on the Kabat numbering scheme; optionally wherein: the CD33-VH comprises the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIY PYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDY WGQGTLVTVSS (SEQ ID NO: 329); and the CD33-VL comprises the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAA SNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEI K (SEQ ID NO: 330), optionally wherein the CD33-VH, and the CD33-VL are separated by peptide linkers.

14. The multicistronic expression system of any one of claims 1-13, wherein the aCAR antigen binding domains comprises the structure (FLT3-VH) - LI - (CD33-VH) - L2 - (CD33-VL) - L3 - (FLT3-VL), wherein LI, L2, and L3 are a first, a second, and a third peptide linker, respectively; optionally wherein the LI, L2, and / or L3 are each independently selected from the group consisting of: SEQ ID Nos 230 - 248, optionally wherein the LI peptide linker is the amino acid sequence GGGGS (SEQ ID NO: 242) orGGGGSGGGGS (SEQ ID NO: 243), optionally wherein the LI peptide linker GGGGS (SEQ ID NO: 242) is encoded by a polynucleotide sequence comprising the sequence GGCGGCGGTGGCTCT (SEQ ID NO: 254) or the LI peptide linker GGGGSGGGGS (SEQ ID NO: 243) is encoded by a polynucleotide sequence comprising the sequence GGAGGCGGAGGATCTGGTGGTGGTGGATCT (SEQ ID NO: 256), optionally wherein the L2 peptide linker is the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 247), optionally wherein the L2 peptide linker is encoded by a polynucleotide sequence comprising the sequence GGCTCTACATCTGGCTCTGGCAAACCTGGAAGCGGCGAGGGATCTACCAAGG GC (SEQ ID NO: 249), optionally wherein the L2 peptide linker is the amino acid sequence GGGGSGGGGS (SEQ ID NO: 243), optionally wherein the L2 peptide linker is encoded by a polynucleotide sequence comprising the sequence GGTGGCGGAGGAAGTGGCGGCGGAGGCTCT (SEQ ID NO: 257), optionally wherein the L3 peptide linker is the amino acid sequence GGGGS (SEQ ID NO: 242)or GGGGSGGGGS (SEQ ID NO: 243), optionally wherein the L3 peptide linker GGGGS (SEQ ID NO: 242) is encoded by a polynucleotide sequence comprising the sequence GGTGGCGGCGGATCC (SEQ ID NO: 255) or the L3 peptide linker GGGGSGGGGS (SEQ ID NO: 243) is encoded by a polynucleotide sequence comprising the sequence GGCGGTGGCGGATCTGGCGGAGGTGGCAGT (SEQ ID NO: 258), optionally wherein the aCAR intracellular signaling domains that stimulate an immune response is selected from the group consisting of: CD3-zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278, FceRI, DAP10, DAP12, CD66d, CD97, CD2, ICOS, CD27, CD154, CD8, 0X40, 4-1BB, CD28, ZAP40, CD30, GITR, HVEM, DAP10, DAP12, MyD88, 2B4, CD40, PD-1, LFA-1, CD7, LIGHT, NKG2C, B7-H3, an MHC class I molecule, a TNF receptor protein, an Immunoglobulin- like protein, a cytokine receptor, an integrin, a SLAM protein, an activating NK cell receptor, BTLA, a Toll ligand receptor, CDS, ICAM-1, (CDlla / CD18), BAFFR, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG(CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, and combinations thereof, optionally wherein the aCAR intracellular signaling domains that stimulate an immune response comprise a CD28 co-stimulatory domain and a CD3(^ signaling domain, optionally wherein the CD28 co-stimulatory domain comprises the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 287), optionally wherein the CD28 co-stimulatory domain is encoded by a polynucleotide sequence comprising the sequenceAGAAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCT AGACGGCCCGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAG ATTTCGCCGCCTACCGGTCC (SEQ ID NO: 288), optionally wherein the CD3^ signaling domain comprises the amino acid sequenceRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR (SEQ ID NO: 289), optionally wherein the CD3(^ signaling domain is encoded by a polynucleotide sequence comprising the sequenceAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAG AACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGG AAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCG GAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGAC GCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 290), optionally wherein(A) the hinge domain of the aCAR is present, optionally wherein the hinge domain of the aCAR is selected from the group consisting of a human Ig (immunoglobulin) hinge, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge, a KIR2DS2 hinge, an LNGFR hinge, a LIR1 hinge, a PDGFR-beta extracellular linker, and combinations thereof, optionally wherein the hinge domain of the aCAR comprises a CD8 hinge, optionally wherein the CD8 hinge comprises the amino acid sequenceALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACD (SEQ ID NO: 272), optionally wherein the CD8 hinge comprises is encoded by a polynucleotide sequence comprising the sequenceGCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTGCC CGCCAAGCCTACAACAACCCCTGCTCCTAGACCACCTACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCCGAAGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCATACAAGAGGACTGGATTTTGCCTGCGAC (SEQ ID NO: 284), and / or wherein(B) the transmembrane domain of the aCAR is present, optionally wherein the transmembrane domain of the aCAR is selected from the group consisting of a human Ig (immunoglobulin) hinge, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge, a KIR2DS2 hinge, an LNGFR hinge, a LIR1 hinge, a PDGFR-beta extracellular linker, and combinations thereof, optionally wherein the transmembrane domain of the aCAR comprises a CD8 hinge, optionally wherein the CD8 transmembrane comprises the amino acid sequence IYIWAPEAGTCGVEEESEVITEYCNHR (SEQ ID NO: 206), optionally wherein the CD8 transmembrane comprises is encoded by a polynucleotide sequence comprising the sequenceATCTATATCTGGGCCCCTCTGGCTGGCACATGCGGAGTTCTGCTGCTCAGCCT GGTCATCACCCTGTACTGCAACCACAGA (SEQ ID NO: 261); and / or wherein(C) the aCAR signal peptide of the aCAR is present, optionally wherein the signal peptide of the aCAR is selected from the group consisting of: IgE, IL- 12, IL-2, optimized IL-2, trypsiongen-2, Gaussia luciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL-6, IL- 8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin El, GROalpha, CXCL12, IL-21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa, optionally wherein the signal peptide of the aCAR comprises a GM-CSFRa signal peptide, optionally wherein the GM-CSFRa signal peptide comprises the amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 423), optionally wherein the GM-CSFRa signal peptide is encoded by a polynucleotide sequence comprising the sequenceATGCTGCTGCTGGTTACATCTCTGCTGCTGTGCGAGCTGCCCCATCCTGCCTT TCTGCTTATTCCT (SEQ ID NO: 424), optionally wherein the aCAR comprises the amino acid sequenceMLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAI SWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRS EDTAVYYCATFALFGFREQAFDIWGQGTTVTVSSGGGGSQVQLVQSGAEVKKP GSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKS KATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSGSTSGSGKPGSGEGSTKGDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWF QQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQS KEVPWTFGQGTKVEIKGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLN WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQ QSYSTPFTFGPGTKVDIKALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQ PLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRR SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAY KQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 414), optionally wherein the aCAR comprises the amino acid sequence MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAI SWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRS EDTAVYYCATFALFGFREQAFDIWGQGTTVTVSSGGGGSGGGGSQVQLVQSGA EVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYN QKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTV SSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQ KPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKE VPWTFGQGTKVEIKGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISS YLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDLATY YCQQSYSTPFTFGPGTKVDIKALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTI ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAP AYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 415).

15. An expression vector comprising the multicistronic expression system of any one of claims 1-14.

16. An isolated cell comprising the multicistronic expression system of any one of claims 1- 15 or the expression vector of claim 59, optionally wherein the cell comprises an immune cell, optionally wherein the cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a Natural Killer T (NKT) cell, a myeloid cell, a macrophage, a human embryonic stem cell(ESC), an ESC-derived cell, a pluripotent stem cell, and induced pluripotent stem cell (iPSC), and an iPSC-derived cell, optionally wherein the cell is an NK cell.

17. A method of treating a subject in need thereof or stimulating a cell-mediated immune response to a tumor cell in a subject, the method comprising administering a therapeutically effective dose of any of the isolated cells of claim 16, optionally wherein the subject has cancer, optionally wherein the isolated cell is derived from the subject or is allogeneic with reference to the subject.

18. A method of treating a subject known or suspected of having cancer, the method comprising:(A) administering a cell therapy to the subject, wherein the cell therapy comprises an immunoresponsive cell engineered to express a recombinant cytokine, wherein the immunoresponsive cell engineered to express the recombinant cytokine comprises an exogenous polynucleotide encoding a controlled release cytokine, oriented from N- terminal to C-terminal, having the formula:S - C - MT or MT - C - S whereinS comprises a secretable effector molecule, wherein the secretable effector molecule comprises the recombinant cytokine,C comprises a protease cleavage site, andMT comprises a cell membrane tethering domain, wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide; and(B) administering a pharmaceutically effective amount of an agent selected from the group consisting of:(i) cytarabine (Ara-C);(ii) decitabine;(iii) venetoclax;(iv) a combination of fludarabine and Ara-C; or(V) or a combination of decitabine and venetoclax;optionally wherein the immunoresponsive cell comprises an exogenous polynucleotide encoding an inhibitory chimeric antigen receptor (iCAR) and / or an a bivalent activating chimeric antigen receptor (aCAR); optionally wherein wherein the iCAR comprises: (i) an antigen-binding domain specific for endomucin (EMCN); (ii) one or more intracellular inhibitory domains that inhibit an immune response; and (iii) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof; optionally wherein b) wherein the aCAR comprises (i) an antigen-binding domain specific for FLT3; (ii) an antigen-binding domain specific for CD33; (iii) one or more intracellular signaling domains that stimulate an immune response; and (iv) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof, optionally wherein the agent comprises the combination of fludarabine and Ara-C, optionally wherein the fludarabine is administered at a dose of 30 mg / m2 and the cytarabine (Ara-C) is administered at a dose of 2 g / m2, optionally wherein when the Ara-C is administered, the Ara-C is administered at a dose of 2 g / m2, optionally wherein the agent is administered intravenously, optionally wherein the agent is administered 5 days prior to administration of the cell therapy.

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