Polycistron chimeric protein expression system

Abstract

Described herein are polycistron expression systems encoding chimeric proteins, in particular membrane cleavable chimeric systems and chimeric antigen receptors. Also described herein are nucleic acids, cells, and methods involving them.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of each of U.S. Provisional Application No. 63 / 333,483, filed on April 21, 2022; U.S. Provisional Application No. 63 / 370,219, filed on August 2, 2022; and U.S. Provisional Application No. 63 / 440,668, filed on January 23, 2023, each of which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on XXXX, 20XX, is named XXX-XXX_sequencelisting.txt and is ###,### bytes in size. Background Art

[0005] Cell-based therapy platforms provide a promising approach for treating a variety of diseases. One such promising platform is CAR-T-based therapy in cancer treatment. Given their prospects, there is a need to improve cell-based therapies. An active area of ​​exploration is cell-based engineered therapy to produce and / or secrete effector molecules that can enhance cell-based therapies, such as cytokines, a process known as arming. For example, unarmed CAR-T therapy has poor efficacy against solid tumors, and arming can affect the entire cancer immunity cycle and enhance the activity of CAR-T. However, uncontrolled or unregulated arming strategies can have a negative impact on treatment, such as off-target effects and toxicity to subjects. Therefore, there is a need for additional methods to control and regulate the arming of cell-based therapies (such as regulating the production and / or secretion of payload effector molecules). Summary of the Invention

[0006] In some embodiments, there is provided herein a cell-based therapy platform, which relates to the regulated arming of cell-based therapy (including therapy (e.g., NK CAR) based on chimeric antigen receptor (CAR), such as the regulated secretion of payload effector molecules. In some embodiments, there is also provided herein a cell-based combination immunotherapy, which relates to the regulated arming of the targeted therapy of cancer (such as ovarian cancer, breast cancer, colon cancer, lung cancer and pancreatic cancer).

[0007] However, the therapy provided herein can limit the systemic toxicity of armament. For example, the immunotherapy provided herein can be tumor-specific and effective, while limiting the systemic toxicity and / or other off-target effects caused by armament. These therapies deliver proteins of interest in a regulated manner, such as immunomodulatory effector molecules, especially IL-15, including regulating secretion dynamics, cell state specificity and cell or tissue specificity. The design of delivery vehicles is optimized to improve the overall function of cell-based therapies (such as cancer therapy), including but not limited to membrane cleavage sites, promoters, linkers, signal peptides, delivery methods, combinations of immunomodulatory effector molecules, regulation and order optimization.

[0008] The design of the delivery vehicle was also optimized to express multiple components of the CAR-based therapy in a polycistronic system, including the chimeric antigen receptors (activating aCAR and inhibitory iCAR) and the membrane-cleavable chimeric protein.

[0009] Provided herein is a polycistronic expression system comprising an engineered nucleic acid comprising: (A) an exogenous polynucleotide encoding a membrane cleavable chimeric protein having the following formula from N-terminus to C-terminus: S–C–MT or MT–C–S, wherein S comprises a secretable effector molecule, wherein the secretable effector molecule comprises IL-15, C comprises a protease cleavage site, and MT comprises a cell membrane tethering domain, wherein S–C–MT or MT–C–S is constructed to be expressed as a single polypeptide; and (B) an exogenous polynucleotide encoding an inhibitory chimeric antigen receptor (iCAR), wherein the inhibitory chimeric antigen receptor comprises: (i) an antigen binding domain specific for endothelial mucin (EMCN); and (ii) an immunosuppressive agent. and (iv) one or more polypeptides selected from the group consisting of a signal peptide, a transmembrane domain, a hinge domain, a spacer, one or more peptide linkers, and combinations thereof.

[0010] In some aspects, the exogenous polynucleotides encoding each of the membrane-cleavable chimeric protein, the iCAR, and the bivalent aCAR are connected together by a polynucleotide linker encoding a 2A ribosomal skipping element. In some aspects, the 2A ribosomal skipping element is selected from the group consisting of a T2A ribosomal skipping element, an E2A ribosomal skipping element, a P2A ribosomal skipping element, an F2A ribosomal skipping element, a ribosomal skipping element fusion thereof, and a combination thereof. In some aspects, the ribosomal skipping element fusion comprises an E2A / T2A ribosomal skipping element. In some aspects, the E2A / T2A ribosomal skipping element comprises the amino acid sequence QCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 221). In some aspects, the E2A / T2A ribosomal skipping element is encoded by a polynucleotide sequence comprising the sequence CAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATCTAATCCTGGACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACGTGGAGGAAAACCCTGGACCT (SEQ ID NO: 222) or the sequence CAGTGCACAAATTATGCACTGCTGAAGCTCGCCGGGGATGTCGAGAGTAACCCAGGACCTGGAAGCGGAGAAGGTCGTGGTAGTCTACTAACGTGTGGTGATGTAGAAGAAAATCCTGGACCT (SEQ ID NO: 223).

[0011] In some aspects, the membrane cleavable chimeric protein, the iCAR, and the bivalent aCAR are encoded in the following order from 5' to 3': (i) membrane cleavable chimeric protein; (ii) bivalent aCAR; and (iii) iCAR. In some aspects, the membrane cleavable chimeric protein, the iCAR, and the bivalent aCAR are encoded in the following order from 5' to 3': (i) membrane cleavable chimeric protein; (ii) iCAR; and (iii) bivalent aCAR.

[0012] In some aspects, the secretory effector molecule comprises a signal peptide or a signal anchor sequence. In some aspects, the signal peptide comprises a natural signal peptide for the secretory effector molecule. In some aspects, the signal peptide comprises a non-natural signal peptide, or the signal anchor sequence comprises a non-natural signal anchor sequence for the secretory effector molecule. In some aspects, the non-natural signal peptide or the non-natural signal anchor sequence are selected from the group consisting of: IgE, IL-12, IL-2, optimization IL-2, trypsinogen-2, Gaussia luciferase, CD5, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin proprotein, azurocidin proprotein, osteonectin, CD33, IL-6, IL-8, CCL2, TIMP2, VEGFB, osteoprotegerin, serine protease inhibitor E1, GRO α, CXCL12, IL-21, CD8, NKG2D, TNFR2, GMCSF and GM-CSFRa. In some aspects, the non-natural signal peptide comprises an IgE signal peptide. In some aspects, the IgE signal peptide comprises the amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 228). In some aspects, the IgE signal peptide is encoded by a polynucleotide sequence comprising the sequence ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCT (SEQ ID NO: 229).

[0013] In some aspects, the IL-15 comprises the amino acid sequence NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 224). In some aspects, IL-15 is encoded by a polynucleotide sequence comprising the sequence AATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAACAGCCTGTCCAGCAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACACCTCA (SEQ ID NO: 225).

[0014] In some aspects, the protease cleavage site further comprises an N-terminal peptide linker, optionally selected from the group consisting of: SGGGGSGGGGSG; GGGSGGGGSGGGSLQ; GGS; GGSGGS; GGSGGSGGS; GGSGGSGGSGGS; GGSGGSGGSGGS; GGGS; GGGSGGGSGGGS; GGGSGGGSGGGS; GGGSGGGSGGGSGGGS; GGGGS; GGGGSGGGGSGGGS; GGGGSGGGGSGGGS; GGGGSGGGGSGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGSGGGGS; GSTSGSGKPGSGEGSTKG; and EAAAKEAAAKEAAAKEAAAK. In some aspects, the protease cleavage site further comprises an N-terminal peptide linker SGGGGSGGGGSG (SEQ ID NO: 230).

[0015] In some aspects, the protease cleavage site further comprises a C-terminal peptide linker, optionally selected from the group consisting of: SGGGGSGGGGSG; GGGSGGGGSGGGSLQ; GGS; GGSGGS; GGSGGSGGS; GGSGGSGGSGGS; GGSGGSGGSGGS; GGGS; GGGSGGGSGGGS; GGGSGGGSGGGS; GGGSGGGSGGGSGGGS; GGGGS; GGGGSGGGGSGGGS; GGGGSGGGGSGGGS; GGGGSGGGGSGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGSGGGGS; GSTSGSGKPGSGEGSTKG; and EAAAKEAAAKEAAAKEAAAK. In some aspects, the protease cleavage site further comprises a C-terminal linker GGGSGGGGSGGGSLQ (SEQ ID NO: 231).

[0016] In some aspects, the protease cleavage site comprises a tumor necrosis factor-alpha converting enzyme (TACE) specific cleavage site. In some aspects, the TACE specific cleavage site comprises the amino acid sequence VTPEPIFSLI (SEQ ID NO: 191). In some aspects, the TACE specific cleavage site comprises the amino acid sequence SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ (SEQ ID NO: 250). In some aspects, the TACE specific cleavage site is encoded by a polynucleotide sequence comprising the sequence TCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGTGACCCCTGAGCCTATCTTCAGCCTGATCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAA (SEQ ID NO: 251).

[0017] In some aspects, the cell membrane tethered domain comprises a transmembrane domain selected from the group consisting of PDGFR-β, CD8, CD28, CD3ζ-chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, LIR1, B7-1, and BTLA. In some aspects, the cell membrane tethered domain comprises a B7-1 transmembrane domain. In some aspects, the B7-1 transmembrane domain comprises the amino acid sequence LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 204). In some aspects, the B7-1 transmembrane domain is encoded by a polynucleotide sequence comprising the sequence TTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 252).

[0018] In some aspects, the membrane cleavable chimeric protein has the formula S-C-MT from N-terminus to C-terminus. In some aspects, the membrane cleavable chimeric protein comprises the amino acid sequence MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 226). In some aspects, the membrane cleavable chimeric protein is encoded by a polynucleotide sequence comprising the sequence (SEQ ID NO: 227).

[0019] In some aspects, an antigen binding domain that is 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 complementary determining region 1 (CDR-H1) having an amino acid sequence of RYDMH (SEQ ID NO: 291); a heavy chain complementary determining region 2 (CDR-H2) having an amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 296); and a heavy chain complementary determining region 3 (CDR-H3) having an amino acid sequence of RIKD (SEQ ID NO: 298), and the EMCN-VL comprises: a light chain complementary determining region 1 (CDR-L1) having an amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 299); a light chain complementary determining region 2 (CDR-L2) having an amino acid sequence of QVSKLDS (SEQ ID NO: 291). and a light chain complementary 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. In some aspects, the EMCN-VH comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQGTMVTVSS (SEQ ID NO: 302); and the EMCN-VL comprises the amino acid sequence DVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQGTKLEIK (SEQ ID NO: 310).

[0020] In some aspects, the EMCN-VH and the EMCN-VL are separated by a peptide linker. In some aspects, the antigen binding domain specific for EMCN comprises the structure VH-L-VL or VL-L-VH, wherein L is a peptide linker. In some aspects, the peptide linker comprises the amino acid sequence SGGGGSGGGGSG; GGGSGGGGSGGGSLQ; GGS; GGSGGS; GGSGGSGGS; GGSGGSGGSGGS; GGSGGSGGSGGS; GGGS; GGGSGGGS; GGGSGGGSGGGS; GGGSGGGSGGGS; GGGSGGGSGGGSGGGS; GGGGS; GGGGSGGGGSGGGS; GGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGS; GSTSGSGKPGSGEGSTKG; or EAAAKEAAAKEAAAKEAAAK. In some aspects, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 244). In some aspects, the peptide linker is encoded by a polynucleotide sequence comprising the sequence GGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCT (SEQ ID NO: 253).

[0021] In some aspects, an antigen binding domain specific for EMCN comprises the structure VH-L-VL and comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQGTMVTVSSGGGGSGGGGSGGGSDVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQGTKLEIK (SEQ ID NO: 311).In some aspects, the antigen-binding domain specific for EMCN is encoded by a polynucleotide sequence comprising the sequence GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGATACGATATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGTGATCTGGGGCAACGGCAACACACACTACCACAGCGCCCTGAAGTCCCGGTTCACCATCTCCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCACCCTGAGAATCAAGGATTGGGGCCAGGGCACCATGGTCACCGTTTCTTCTGGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCTGACGTGGTCATGACACAGAGCCCTCTGAGCCTGCCTGTGACACTGGGACAGCCTGCCAGCATCAGCTGCAAGTCTAGCCAGTCTCTGGTGGCCAGCGACGAGAACACCTACCTGAACTGGTTCCAGCAGAGGCCCGGACAGTCTCCTAGACGGCTGATCTACCAGGTGTCCAAGCTGGATAGCGGCGTGCCCGATAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGAAGATCAGCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTCTGCAAGGCATCCATCTGCCTTGGACCTTTGGCCAGGGCACAAAGCTGGAAATCAAG (SEQ ID NO:312).

[0022] In some aspects, the cell intracellular inhibitory domain comprises a LIR1 cell intracellular inhibitory domain. In some aspects, the LIR1 cell intracellular inhibitory domain comprises the amino acid sequence LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 285). In some aspects, the LIR1 intracellular inhibitory domain comprises a motif comprising the sequence CTGCGGCACAGAAGGCAGGGCAAGCACTGGACAAGCACCCAGAGAAAGGCCGACTTTCAGCATCCTGCTGGCGCCGTTGGACCTGAGCCTACAGATAGAGGACTGCAGTGGCGGTCTAGCCCTGCCGCTGATGCCCAAGAGGAAAATCTTTACGCCGCCGTGAAGCACACCCAGCCTGAGGATGGCGTGGAAATGGACACCAGATCTCCCCACGATGAGGACCCTCAGGCCGTGACATACG CAGAAGTGAAGCACTCCAGACCTCGGAGAGAGATGGCAAGCCCTCCATCTCCTCTGAGCGGCGAGTTCCTGGACACCAAAGACAGACAGGCCGAAGAGGACAGACAGATGGATACCGAAGCCGCCGCTTCTGA AGCCCCACAGGATGTGACATATGCCCAGCTGCATAGCCTGACACTGCGGAGAGAAGCCACAGAGCCTCCACCTTCTCAAGAAGGCCCATCTCCTGCCGTGCCTTCCATCTATGCCACTCTGGCCATTCAC(SEQ ID NO:286) polynucleotide sequence encoding.

[0023] In some aspects, the signal peptide is present in the iCAR.In some aspects, the signal peptide of the iCAR includes natural signal peptide, natural or non-natural signal peptide, optionally wherein the non-natural signal peptide or the non-natural signal anchor sequence are selected from the group consisting of the following: IgE, IL-12, IL-2, optimized IL-2, trypsinogen-2, Gaussia luciferase, CD5, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL-6, IL-8, CCL2, TIMP2, VEGFB, osteoprotegerin, serine protease inhibitor E1, GRO α, CXCL12, IL-21, CD8, NKG2D, TNFR2 and GMCSF.In some aspects, the signal peptide of the iCAR includes CD8 signal peptide.In some aspects, the CD8 signal peptide includes amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO:137). In some aspects, the CD8 signal peptide is encoded by a polynucleotide sequence comprising the sequence ATGGCTCTGCCCGTGACAGCTTTGCTGCTGCCTTTGGCACTGCTGCTGCATGCTGCTAGACCA (SEQ ID NO: 416).

[0024] In some aspects, the hinge domain is present in the iCAR. In some aspects, the hinge domain of the iCAR is selected from human Ig (immunoglobulin) hinge, IgG4 hinge, IgG2 hinge, CD8a hinge or IgD hinge, KIR2DS2 hinge, LNGFR hinge, LIR1 hinge, PDGFR-β extracellular joint and a combination thereof. In some aspects, the hinge domain of the iCAR includes LIR1 hinge. In some aspects, the LIR1 hinge includes the amino acid sequence HPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV (SEQ ID NO: 417). In some aspects, the LIR1 hinge is encoded by a polynucleotide sequence comprising the sequence CACCCATCCGATCCTCTCGAGCTGGTGGTTTCTGGACCTTCTGGCGGCCCTAGCAGCCCTACAACAGGACCTACAAGCACAAGCGGCCCTGAGGACCAACCTCTGACACCAACAGGCAGCGATCCTCAGTCTGGACTGGGGAGACATCTGGGCGTT (SEQ ID NO: 418). In some aspects, the hinge domain of the iCAR comprises a CD8 hinge. In some aspects, the CD8 hinge comprises the amino acid sequence TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 271). In some aspects, the CD8 hinge is encoded by a polynucleotide sequence comprising the sequence ACAACAACACCCGCACCTCGGCCTCCAACTCCAGCTCCAACAATTGCACTGCAACCCCTGAGTCTGAGGCCCGAGGCCTGTAGGCCAGCAGCTGGCGGAGCTGTTCACACTAGAGGCCTGGACTTTGCCTGTGAC (SEQ ID NO: 283).

[0025] In some aspects, there is a transmembrane domain of the iCAR. In some aspects, the transmembrane domain of the iCAR includes a transmembrane domain selected from the group consisting of: PDGFR-β, CD8, CD28, CD3ζ-chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, LIR1 and BTLA. In some aspects, the transmembrane domain of the iCAR includes the LIR1 transmembrane domain. In some aspects, the LIR1 transmembrane domain includes the amino acid sequence VIGILVAVILLLLLLLLLFLI (SEQ ID NO: 259). In some aspects, the LIR1 transmembrane domain is encoded by a polynucleotide sequence comprising the sequence GTGATCGGCATTCTGGTCGCCGTGATCCTGCTCCTGTTGCTCCTGCTGCTTCTGTTCCTGATC (SEQ ID NO: 260).

[0026] In some aspects, the iCAR comprises the amino acid sequence

[0027] MALPPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQG TMVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFG QGTKLEIKHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADA QEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH(SEQ ID NO:430); or

[0028] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQGTMVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQGTKLEIKngaaHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH(SEQ ID NO:419).

[0029] In some aspects, the iCAR is composed of sequences containing

[0030]

[0031] In some aspects, the iCAR comprises an amino acid sequence

[0032] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQGTMVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQGTKLEIKTTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACDVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 431); or

[0033] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQGTMVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQGTKLEIKngaaTTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACDVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH(SEQ ID NO:421).

[0034] In some aspects, the iCAR is composed of sequences containing

[0035]

[0036] In some aspects, an 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 complementary determining region 1 (CDR-H1) having an amino acid sequence of GGTFSSYAIS (SEQ ID NO: 360); a heavy chain complementary determining region 2 (CDR-H2) having an amino acid sequence of GIIPIFGTANYAQKFQG (SEQ ID NO: 361); and a heavy chain complementary determining region 3 (CDR-H3) having an amino acid sequence of FALFGFREQAFDI (SEQ ID NO: 362), and the FLT3-VL comprises: a light chain complementary determining region 1 (CDR-L1) having an amino acid sequence of RASQSISSYLN (SEQ ID NO: 363). NO: 363); a light chain complementary determining region 2 (CDR-L2) having the amino acid sequence of AASSLQS (SEQ ID NO: 364); and a light chain complementary 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. In some aspects, the FLT3-VH comprises the amino acid sequence of EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCATFALFGFREQAFDIWGQGTTVTVSS (SEQ ID NO: 315); and the FLT3-VL comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDLATYYCQQSYSTPFTFGPGTKVDIK (SEQ ID NO: 316).

[0037] In some aspects, the antigen binding domain that is 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 complementary determining region 1 (CDR-H1) having an amino acid sequence of DYNMH (SEQ ID NO: 402); a heavy chain complementary determining region 2 (CDR-H2) having an amino acid sequence of YIYPYNGGTGYNQKFKSKA (SEQ ID NO: 403); and a heavy chain complementary determining region 3 (CDR-H3) having an amino acid sequence of GRPAMDYWGQ (SEQ ID NO: 404), and the CD33-VL comprises: a light chain complementary determining region 1 (CDR-L1) having an amino acid sequence of RASESVDNYGISFMN (SEQ ID NO: 405). NO:405); a light chain complementary determining region 2 (CDR-L2), the CDR-L2 having the amino acid sequence of AASNQGS (SEQ ID NO:406); and a light chain complementary determining region 3 (CDR-L3), the 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. In some aspects, the CD33-VH comprises the amino acid sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS (SEQ ID NO: 329); and the CD33-VL comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 330).

[0038] In some aspects, FLT3-VH, FLT3-VL, CD33-VH, and CD33-VL are separated by a peptide linker. In some aspects, the aCAR antigen binding domain comprises the structure (FLT3-VH)–L1–(CD33-VH)–L2–(CD33-VL)–L3–(FLT3-VL), wherein L1, L2, and L3 are the first, second, and third peptide linkers, respectively. In some aspects, L1, L2, and / or L3 are each independently selected from the group consisting of: SGGGGSGGGGSG; GGGSGGGGSGGGSLQ; GGS; GGSGGS; GGSGGSGGS; GGSGGSGGSGGS; GGSGGSGGSGGSGGS; GGGS; GGGSGGGS; GGGSGGGSGGGS; GGGSGGGSGG GSGGGS; GGGSGGGSGGGSGGGSGGGS; GGGGS; GGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGSGGGGSGGGGS; GGGGSGGGGSGGGGSGGGGSGGGGS; GSTGSGKPGSGEGSTKG; and EAAAKEAAAKEAAAKEAAAK. In some aspects, the L1 peptide linker is the amino acid sequence GGGGS (SEQ ID NO: 242) or GGGGSGGGGS (SEQ ID NO: 243). In some aspects, the L1 peptide linker GGGGS (SEQ ID NO: 242) is encoded by a polynucleotide sequence comprising the sequence GGCGGCGGTGGCTCT (SEQ ID NO: 254), or the L1 peptide linker GGGGSGGGGS (SEQ ID NO: 243) is encoded by a polynucleotide sequence comprising the sequence GGAGGCGGAGGATCTGGTGGTGGTGGATCT (SEQ ID NO: 256). In some aspects, the L2 peptide linker is the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 247). In some aspects, the L2 peptide linker is encoded by a polynucleotide sequence comprising the sequence GGCTCTACATCTGGCTCTGGCAAACCTGGAAGCGGCGAGGGATCTACCAAGGGC (SEQ ID NO: 249). In some aspects, the L2 peptide linker is the amino acid sequence GGGGSGGGGS (SEQ ID NO: 243). In some aspects, the L2 peptide linker is encoded by a polynucleotide sequence comprising the sequence GGTGGCGGAGGAAGTGGCGGCGGAGGCTCT (SEQ ID NO: 257). In some aspects, the L3 peptide linker is the amino acid sequence GGGGS (SEQ ID NO: 242) or GGGGSGGGGS (SEQ ID NO: 243).In some aspects, 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).

[0039] In some aspects, the intracellular signaling domain of the aCAR that stimulates an immune response is selected from the group consisting of: CD3-ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d, CD97, CD2, ICOS, CD27, CD154, CD8, OX40, 4-1BB, CD28, ZAP40, CD30, GITR, HVEM, DAP10, DAP12, MyD88, 2B4, CD40, PD-1, LFA-1, CD7, LIGHT, NKG2C, B7-H3, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, SLAM protein, activating NK cell receptor, BTLA, Toll ligand receptor, CDS, ICAM-1, (CD11a / CD18), BAFFR, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile type), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a and combinations thereof. In some aspects, the intracellular signaling domain of the aCAR that stimulates an immune response comprises a CD28 co-stimulatory domain and a CD3ζ signaling domain. In some aspects, the CD28 co-stimulatory domain comprises the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 287).In some aspects, the CD28 costimulatory domain is encoded by a polynucleotide sequence comprising the sequence AGAAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCCGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGATTTCGCCGCCTACCGGTCC (SEQ ID NO: 288). In some aspects, the CD3 zeta signaling domain comprises the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 289). In some aspects, the CD3 zeta signaling domain is encoded by a polynucleotide sequence comprising the sequence AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 290).

[0040] In some aspects, there is a hinge domain of the aCAR. In some aspects, the hinge domain of the aCAR is selected from human Ig (immunoglobulin) hinge, IgG4 hinge, IgG2 hinge, CD8a hinge or IgD hinge, KIR2DS2 hinge, LNGFR hinge, LIR1 hinge, PDGFR-β extracellular joint and a combination thereof. In some aspects, the hinge domain of the aCAR includes a CD8 hinge. In some aspects, the CD8 hinge includes the amino acid sequence ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 272). In some aspects, the CD8 hinge is encoded by a polynucleotide sequence comprising the sequence GCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTGCCCGCCAAGCCTACAACAACCCCTGCTCCTAGACCACCTACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCCGAAGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCATACAAGAGGACTGGATTTTGCCTGCGAC (SEQ ID NO: 284).

[0041] In some aspects, there is a transmembrane domain of the aCAR. In some aspects, the transmembrane domain of the aCAR is selected from human Ig (immunoglobulin) hinge, IgG4 hinge, IgG2 hinge, CD8a hinge or IgD hinge, KIR2DS2 hinge, LNGFR hinge, LIR1 hinge, PDGFR-β extracellular linker and a combination thereof. In some aspects, the transmembrane domain of the aCAR includes a CD8 hinge. In some aspects, the CD8 transmembrane includes the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 206). In some aspects, the CD8 transmembrane is encoded by a polynucleotide sequence comprising the sequence ATCTATATCTGGGCCCCTCTGGCTGGCACATGCGGAGTTCTGCTGCTCAGCCTGGTCATCACCCTGTACTGCAACCACAGA (SEQ ID NO: 261).

[0042] In some aspects, there is the aCAR signal peptide of the aCAR. In some aspects, the signal peptide of the aCAR is selected from the group consisting of: IgE, IL-12, IL-2, optimization IL-2, trypsinogen-2, Gaussia luciferase, CD5, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL-6, IL-8, CCL2, TIMP2, VEGFB, osteoprotegerin, serine protease inhibitor E1, GRO α, CXCL12, IL-21, CD8, NKG2D, TNFR2, GMCSF and GM-CSFRa. In some aspects, the signal peptide of the aCAR includes GM-CSFRa signal peptide. In some aspects, the GM-CSFRa signal peptide includes amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 423). In some aspects, the GM-CSFRa signal peptide is encoded by a polynucleotide sequence comprising the sequence ATGCTGCTGCTGGTTACATCTCTGCTGCTGTGCGAGCTGCCCCATCCTGCCTTTCTGCTTATTCCT (SEQ ID NO: 424).

[0043] In some aspects, the aCAR comprises the amino acid sequence

[0044] MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCATFALFGFREQAFDIWGQGTTVTVSSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSGSTSGSGKPGSGEGSTKGDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQSYSTPFTFGPGTKVDIKALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:414).

[0045] In some aspects, the aCAR comprises the amino acid sequence

[0046] (SEQ ID NO:415).

[0047] Also provided herein is an expression vector comprising any one of the polycistronic expression systems provided herein. In some aspects, the expression vector comprises the polynucleotide of SEQ ID NO: 425, SEQ ID NO: 426, SEQ ID NO: 427, SEQ ID NO: 428, SEQ ID NO: 433, SEQ ID NO: 432, or SEQ ID NO: 429.

[0048] Also provided herein is a cell of separation, the cell of the separation comprising any one of the polycistronic expression system provided herein or any one of the expression vector provided herein. In some aspects, the cell includes an immune cell. In some aspects, the cell is selected from the group consisting of: T cell, natural killer (NK) cell, cytotoxic T lymphocyte (CTL), regulatory T cell, natural killer T (NKT) cell, myeloid cell, macrophage, human embryonic stem cell (ESC), ESC derived cell, pluripotent stem cell and induced pluripotent stem cell (iPSC) and iPSC derived cell. In some aspects, the cell is a NK cell.

[0049] Also provided herein is a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:425, SEQ ID NO:426, SEQ ID NO:427, SEQ ID NO:428, SEQ ID NO:433, SEQ ID NO:432, or SEQ ID NO:429.

[0050] Also provided herein is a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 425.

[0051] Also provided herein is a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 426.

[0052] Also provided herein is a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 427.

[0053] Also provided herein is a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 428.

[0054] Also provided herein is a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 429.

[0055] Also provided herein is a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 432.

[0056] Also provided herein is a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 433.

[0057] Also provided herein is a method of treating a subject in need thereof, the method comprising administering a therapeutically effective dose of any of the isolated cells provided herein or any of the polynucleotides provided herein.

[0058] Also provided herein is a method of treating a subject having cancer, the method comprising administering to the subject an immunotherapy comprising any of the isolated cells provided herein or any of the polynucleotides provided herein.

[0059] A method of stimulating a cell-mediated immune response against tumor cells in a subject, the method comprising administering to a subject having a tumor a therapeutically effective dose of any of the isolated cells provided herein or any of the polynucleotides provided herein. In some aspects, the isolated cells are derived from the subject. In some aspects, the isolated cells are allogeneic with respect to the subject.

[0060] Also provided herein is a method of making an engineered cell, the method comprising transducing an isolated cell with any of the polycistronic expression systems provided herein, any of the expression vectors provided herein, or any of the polynucleotides provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 A schematic diagram illustrating the components and organizational structure of the entire FLT3 OR CD33 NOT EMCN logic-gated CAR-NK cell system is provided.

[0062] Figure 2 A schematic diagram illustrating the components evaluated in optimizing the FLT3 OR CD33 NOT EMCN logic-gated CAR-NK cell system is provided.

[0063] Figure 3 Shown is the gating strategy for evaluating iCAR and aCAR expression by flow cytometry.

[0064] Figure 4 Shown are aCAR and iCAR expression at day 15 for the indicated series of RetroVec constructs.

[0065] Figure 5 Shown are aCAR and iCAR expression at day 15 for the indicated series of self-inactivating (SIN) γ-retroviral SINvec constructs.

[0066] Figure 6 Representative flow cytometric plots of membrane-bound IL-15 expression at day 15 for the indicated series of RetroVec constructs are shown.

[0067] Figure 7 Representative flow cytometric plots of membrane-bound IL-15 expression at day 15 for the indicated series of SINvec constructs are shown.

[0068] Figure 8 Shown is the first series of comparisons of aCAR, iCAR expression, and membrane-bound IL-15 for the indicated series of RetroVec and SINvec constructs.

[0069] Figure 9A second series of comparisons of aCAR, iCAR expression, and membrane-bound IL-15 for the indicated series of RetroVec and SINvec constructs is shown.

[0070] Figure 10 Shown is a third series of comparisons of aCAR, iCAR expression, and membrane-bound IL-15 for the indicated series of RetroVec and SINvec constructs.

[0071] Figure 11 Shown is a summary of aCAR and iCAR expression assessed by MFI at day 15 for the indicated series of RetroVec and SINvec constructs.

[0072] Figure 12 A general summary of expression considerations for the various components and tissue structures evaluated is shown.

[0073] Figure 13 Shown are the total cytotoxicity of constructs using the LIR1 ICD against a target cell line expressing FLT3 / CD33 (AML cell line MV4-11) and a target cell line engineered to also express the NOT gate target antigen EMCN (MV4-11+EMCN), both ordered in an aCAR to iCAR organization (top) and an iCAR to aCAR organization (bottom).

[0074] Figure 14 Shown are normalized cytotoxicity of constructs using LIR1 ICD against a target cell line expressing FLT3 / CD33 (AML cell line MV4-11) and a target cell line engineered to also express the NOT gate target antigen EMCN (MV4-11+EMCN).

[0075] Figure 15 Shown are the total cytotoxicity of constructs using the LIR1 ICD at the indicated effector:target ratios on a target cell line expressing FLT3 / CD33 (AML cell line MV4-11) and a target cell line engineered to also express the NOT gate target antigen EMCN (MV4-11+EMCN) at day 17 for constructs ordered in an aCAR to iCAR organizational structure.

[0076] Figure 16 Shown are normalized cytotoxicity at day 10 for constructs using the LIR1 ICD at the indicated effector:target ratios against a target cell line expressing FLT3 / CD33 (AML cell line MV4-11) and a target cell line engineered to also express the NOT gate target antigen EMCN (MV4-11+EMCN), both ordered in an aCAR to iCAR organization (top) and an iCAR to aCAR organization (bottom).

[0077] Figure 17 Shown is a schematic diagram of a membrane cleavable system as described herein, wherein the desired payload is expressed as a chimeric protein in which a protease cleavage site is inserted between the payload and the membrane tethered domain. The left figure shows a schematic diagram of a membrane cleavable system using a transmembrane structure (e.g., a chimeric protein comprises a transmembrane domain). The right figure shows a schematic diagram of a membrane cleavable system using a membrane-connected structure (e.g., a chimeric protein comprises a post-translational modification tag that allows the cell membrane to be connected).

[0078] Figure 18 Representative membrane-cleavable systems for regulated secretion of IL-15 are shown, in which IL-15 is expressed as a chimeric protein with a cleavage site that can be cleaved by TACE inserted between the IL-15 payload and the membrane tethering domain. The left panel illustrates type I transmembrane structures (e.g., S–C–MT) using type I transmembrane domains (such as PDGFR-β and CD8). The right panel illustrates type II transmembrane structures (e.g., MT–C–S) using type II transmembrane domains (such as NKG2D and TNFR2).

[0079] Figure 19 The components of the indicated constructs encoding a bivalent FLT3 OR CD33 aCAR or a monospecific aCAR and their order are shown.

[0080] Figure 20 Shown are the survival curves of bivalent FLT3 OR CD33 CAR-NK cells and monospecific CAR-NK cells in the MV4-11 mouse tumor model.

[0081] Figure 21 Shown are representative tumor imaging of bivalent FLT3 OR CD33 CAR-NK cells and monospecific CAR-NK cells in the MV4-11 murine tumor model.

[0082] Figure 22 Various labeled and label-free combinations of aCAR and iCAR are illustrated.

[0083] Figure 23 Representative flow cytometric plots of “no virus” controls used to evaluate tagged and untagged CAR are shown.

[0084] Figure 24 Representative flow cytometric plots of the untagged constructs SB07401 and SB07405 at day 8 are shown.

[0085] Figure 25 Representative flow cytometric plots of only the iCAR-tagged construct SB06467 at day 8 are shown.

[0086] Figure 26 Shown is a summary of the percentage of CD33 / EMCN double-positive cells for the indicated constructs with either the loop 6 (upper panel) or loop 4 (lower panel) linker.

[0087] Figure 27 Shown are the summaries of expression of each of CD33, FLT3, and EMCN CARs for the indicated constructs with a loop 4 linker.

[0088] Figure 28 Shown are the summaries of expression of each of CD33, FLT3, and EMCN CARs for the indicated constructs with a loop 6 linker.

[0089] Figure 29 Shown are the total cytotoxicity of constructs using the LIR1 ICD at a 1:2 effector:target ratio against a target cell line expressing FLT3 / CD33 (AML cell line MV4-11) and a target cell line engineered to also express the NOT gate target antigen EMCN (MV4-11+EMCN).

[0090] Figure 30 Shown are normalized cytotoxicity (normalized to a no virus control) of constructs using the LIR1 ICD at a 1:2 effector:target ratio against a target cell line expressing FLT3 / CD33 (AML cell line MV4-11) and a target cell line engineered to also express the NOT gate target antigen EMCN (MV4-11+EMCN).

[0091] Figure 31 Shown are normalized cytotoxicity (normalized to a no virus control) of constructs using the LIR1 ICD at a 1:4 effector:target ratio against a target cell line expressing FLT3 / CD33 (AML cell line MV4-11) and a target cell line engineered to also express the NOT gate target antigen EMCN (MV4-11+EMCN).

[0092] Figure 32 Shown are specific cytotoxicity of target cell lines expressing CD33 (SEM) and target cell lines that also express the NOT gate target antigen (SEM+EMCN) at a 1:2 effector:target ratio. The graph provides SEM EMCN- in the left column and EMCN+ in the right column, respectively.

[0093] Figure 33A Shown are specific cytotoxicity of target cell lines expressing CD33 and target cell lines that also express the NOT gate target antigen (SEM+EMCN) at a 1:2 effector:target ratio in a serial killing assay at 24 hours. Figure 33BShown are specific cytotoxicity of target cell lines expressing CD33 and target cell lines that also express the NOT gate target antigen (SEM+EMCN) at a 1:2 effector:target ratio in a serial killing assay at 48 hours. Figure 33A Shown are specific cytotoxicity of target cell lines expressing CD33 and those also expressing the NOT gate target antigen (SEM+EMCN) at a 1:2 effector:target ratio in a serial killing assay at 96 hours, EMCN+ right column, respectively.

[0094] Figure 34A A general schematic diagram of the in vivo experiments evaluating NOT logic-gated CAR-NK cells is shown. Figure 34B Shown Figure 34A Characterization of SEM CD33+ and a 50:50 mixture of SEM CD33+ / EMCN+ cells shown in .

[0095] Figure 35 Shown are the results of an in vivo experiment in which a 1:1 mixture of SEM+ / -EMCN was injected into mice and treated with NK cells transduced with the corresponding vectors. The top row shows BLI images on day 11, while the bottom panel shows BLI images on day 19 after tumor injection.

[0096] Figure 36 Shown Figure 35 Figure 5 shows the results of quantified regions of interest (ROIs) at days 11 and 19.

[0097] Figure 37A The proportion of healthy cells in mice on day 14 of an in vivo study in which mice were injected with a 1:1 mixture of SEM+ / -EMCN and treated with NK cells transduced with the corresponding constructs is shown. The circle compares the percentage of EMCN+SEM cells between OR / NOT and OR-gated CAR-NK cell treatments, showing that the percentage of EMCN+SEM cells was significantly higher in animals treated with OR / NOT-gated CAR-NK cells, demonstrating non-gated protection of EMCN+ cells. Figure 37B Shown in Figure 37A Proportion of EMCN+SEM cells at day 21 of the indicated studies. Figure 37C Shown in Figure 37A The proportion of EMCN+SEM cells at day 27 of the indicated studies.

[0098] Figure 38A Shown are the mean BLI measurements of mice on the corresponding days of the in vivo study, in which mice were injected with MV4-11 cells and treated with NK cells transduced with the corresponding constructs. Figure 38B Shown in Figure 38A During the in vivo studies shown, Figure 38A Quantified BLI signals of the indicated mice.

[0099] Figure 39A Shown are BLI measurements of mice on the corresponding days of the in vivo study, in which mice were injected with MV4-11 cells and treated with NK cells transduced with the corresponding constructs.

[0100] Figure 39B Shown are representative images at day 55 after tumor transplantation of mice treated with PBS, untransduced NK cells, or NK cells transduced with SB07412 or SB07418.

[0101] Figure 39C Shown are the survival curves and median survival (in days) for the treatment groups tested.

[0102] Figure 39D Shown Figure 39C Continuation of the indicated study, in which data have been collected through Day 120.

[0103] Figure 40 Shown are the testing of NK cells expressing aCAR and different iCAR intracellular domains in a cytotoxicity assay.

[0104] Figure 41A Shown is a comparison of cell-mediated cytotoxicity against MOLM-13 using non-engineered or engineered NK cells.

[0105] Figure 41B Shown is a comparison of cell-mediated cytotoxicity of MV4-11 using non-engineered or engineered NK cells.

[0106] Figure 41C Shown is a comparison of cell-mediated cytotoxicity against SEM-CD33 using non-engineered or engineered NK cells.

[0107] Figure 41D NK cytotoxicity of MOLM-13 cells at various E:T ratios is shown.

[0108] Figure 41E NK cytotoxicity of MV4-11 at various E:T ratios is shown.

[0109] Figure 41F Shown are NK cytotoxicity of engineered and non-engineered NK cells from multiple experiments plotted against CD33 CAR expression.

[0110] Figure 42 Shown are heat maps visualizing fold expression analysis of cytokines produced by NK cells co-cultured with MV4-11 or MOLM-13.

[0111] Figure 43A Shown are NK cytotoxicity of engineered and non-engineered NK cells derived from donor A co-cultured with primary AML samples.

[0112] Figure 43B Shown are NK cytotoxicity of engineered and non-engineered NK cells derived from donor B co-cultured with primary AML samples.

[0113] Figure 44 Shown is a heat map analysis of NK activation markers analyzed by flow cytometry.

[0114] Figure 45A Shown is the expression of surface-associated IL 15 in transduced NK cells derived from different donors.

[0115] Figure 45B Shown is the expression of secreted IL15 in transduced NK cells derived from different donors.

[0116] Figure 45C Shown is the expression of CD33 / FLT3 bivalent aCAR in transduced NK cells derived from different donors.

[0117] Figure 45D Shown is the expression of EMCN iCAR in engineered NK cells derived from different donors.

[0118] Figure 46A Shown are the results of a cytotoxicity assay of engineered NK cells incubated with human stem cells expressing EMCN.

[0119] Figure 46B Shown are the results of a cytotoxicity assay of engineered NK cells incubated with AML (leukemia cells).

[0120] Figure 46C Shown are the results of a cytotoxicity assay of engineered NK cells incubated with human stem progenitor cells.

[0121] Figure 46D Results of cytotoxicity assays of engineered NK cells incubated with primary healthy MPPs are shown. Results for the entire healthy MPP population are shown.

[0122] Figure 46E Results of cytotoxicity assays of engineered NK cells incubated with primary healthy MPPs are shown. Results for a healthy EMCN+MPP population are shown.

[0123] Figure 47Shown is EMCN expression on various hematopoietic stem and progenitor cell (HSPC) subsets, including hematopoietic progenitors (HPCs), lympho-myeloid primed progenitors (LMPPs), and multipotent progenitors (MPPs), in addition to HSCs, in CD34-enriched primary human bone marrow cells from two representative donors.

[0124] Figure 48 Figure 2 shows the flow cytometry gating strategy for counting the number of viable remaining EMCN+HSCs after the killing assay. From left to right and top to bottom, progressive gating is performed for (1) cell size parameters, (2) single cells, (3) CD56-negative cells (i.e., non-NK cells), (4) viable CD34+ undifferentiated cells, (5) CD45RA–CD38– cells (i.e., HSCs and MPPs), (6) CD90+ cells (i.e., HSCs), and (7) EMCN+HSCs. FMO controls were used to set gates for CD90 and EMCN. DETAILED DESCRIPTION

[0125] The present disclosure provides multicistronic expression systems. These multicistronic systems encode (i) membrane-cleavable chimeric proteins; (ii) bivalent aCARs; and (iii) iCARs. These multicistronic systems include FLT3 OR CD33 NOT EMCN logic-gated CARs and membrane-cleavable chimeric proteins encoding IL-15.

[0126] Polycistronic expression systems are provided, comprising engineered nucleic acids encoding:

[0127] (A) An exogenous polynucleotide encoding a membrane-cleavable chimeric protein having the following formula from N-terminus to C-terminus: S–C–MT or MT–C–S, wherein S comprises a secretable effector molecule, wherein the secretable effector molecule comprises IL-15, C comprises a protease cleavage site, and MT comprises a cell membrane tethering domain. S–C–MT or MT–C–S is constructed to be expressed as a single polypeptide;

[0128] (B) an exogenous polynucleotide encoding an inhibitory chimeric antigen receptor (iCAR) comprising: (i) an antigen binding domain specific for endothelial mucin (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, one or more peptide linkers, and combinations thereof; and

[0129] (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, one or more peptide linkers, and combinations thereof.

[0130] The expression systems described herein are polycistronic, i.e., more than one individual polypeptide (e.g., multiple chimeric proteins) can be produced from a single mRNA transcript. By using various linkers, engineered nucleic acids can become polycistronic, for example, a polynucleotide sequence encoding a first chimeric protein can be linked to a nucleotide sequence encoding a second chimeric protein, such as in a first gene: linker: second gene 5' to 3' orientation. The linker can encode a 2A ribosomal skipping element, such as T2A. Other 2A ribosomal skipping elements include, but are not limited to, E2A, P2A, and F2A. The 2A ribosomal skipping element allows for the production of separate polypeptides encoded by the first and second genes during translation. The 2A ribosomal skipping element can include a fusion peptide of the 2A ribosomal skipping element, including but not limited to the E2A / T2A ribosomal skipping element. In certain embodiments, the E2A / T2A ribosomal skipping element comprises the amino acid sequence of GSGQCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 219). An exemplary nucleic acid encoding the E2A / T2A ribosomal skipping element is GGTAGCGGCCAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATCTAATCCTGGACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACGTGGAGGAAAACCCTGGACCT (SEQ ID NO: 220). In certain embodiments, the nucleic acid encoding the E2A / T2A ribosomal skipping element comprises 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, the engineered nucleic acids disclosed herein comprise the E2A / T2A ribosomal skipping element. In certain embodiments, the E2A / T2A ribosomal skipping element comprises the amino acid sequence of QCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 221). An exemplary nucleic acid encoding the E2A / T2A ribosomal skipping element is CAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATCTAATCCTGGACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACGTGGAGGAAAACCCTGGACCT (SEQ ID NO: 222).Another exemplary nucleic acid encoding the E2A / T2A ribosomal skipping element is CAGTGCACAAATTATGCACTGCTGAAGCTCGCCGGGGATGTCGAGAGTAACCCAGGACCTGGAAGCGGAGAAGGTCGTGGTAGTCTACTAACGTGTGGTGATGTAGAAGAAAATCCTGGACCT (SEQ ID NO: 223). In certain embodiments, the nucleic acid encoding the E2A / T2A ribosomal skipping element comprises 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.

[0131] The linker can encode a cleavable linker polypeptide sequence, such as a furin cleavage site or a TEV cleavage site, wherein upon expression, the cleavable linker polypeptide is cleaved, thereby producing separate polypeptides encoded by the first gene and the second gene. The cleavable linker can comprise a polypeptide sequence that further promotes cleavage, such as such a flexible linker (e.g., a Gly-Ser-Gly sequence).

[0132] The linker may encode an internal ribosome entry site (IRES) such that separate polypeptides encoded by the first and second genes are produced during translation. The linker may encode a splice acceptor, such as a viral splice acceptor.

[0133] In some embodiments, the linker is a furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, the linker is a furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, the linker is a furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, the linker is a furin-Gly-Ser-Gly-2A fusion polypeptide.

[0134] In general, polycistronic systems can use any number of linkers or combinations of linkers to express any number of genes or portions thereof (e.g., an engineered nucleic acid can encode a first chimeric protein, a second chimeric protein, and a third chimeric protein, each separated by a linker such that separate polypeptides encoded by the first chimeric protein, the second chimeric protein, and the third chimeric protein are produced).

[0135] In general, the exogenous polynucleotides encoding each of the membrane-cleavable chimeric protein, iCAR, and bivalent aCAR can be encoded in any order. For example, the membrane-cleavable chimeric protein, iCAR, and bivalent aCAR can be encoded by the polycistronic system in the following order from 5' to 3': (i) membrane-cleavable chimeric protein; (ii) bivalent aCAR; and (iii) iCAR. For another example, the membrane-cleavable chimeric protein, iCAR, and bivalent aCAR can be encoded by the polycistronic system in the following order from 5' to 3': i) membrane-cleavable chimeric protein; (ii) iCAR; and (iii) bivalent aCAR.

[0136] Membrane-cleavable chimeric protein

[0137] The polycistronic system herein encodes a membrane-cleavable chimeric protein having the formula S-C-MT or MT-C-S in an orientation from N-terminus to C-terminus and is expressed as a single polypeptide. S refers to the secretable effector molecule. C refers to the protease cleavage site. MT refers to the cell membrane tethering domain. The membrane-cleavable chimeric protein is engineered such that the secretion of the effector molecule can be regulated in a protease-dependent manner. Specifically, the membrane-cleavable chimeric protein is engineered such that the secretion of the effector molecule can be regulated as part of a "membrane-cleavable" system, wherein the incorporation of the protease cleavage site ("C") and the cell membrane tethering domain ("MT") allows for the secretion of the effector molecule to be regulated 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 typically regulate secretion through the following cellular processes:

[0138] -MT: The cell membrane tethering domain comprises a transmembrane domain (or transmembrane-intracellular domain) that directs the cellular trafficking of the chimeric protein, such that the protein is inserted into or otherwise associated with the cell membrane ("tethered")

[0139] -C: After the chimeric protein is expressed and localized to the cell membrane, the protease cleavage site directs the cleavage of the chimeric protein, allowing the effector molecule to be 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 payload with desired kinetics (e.g., the ratio of membrane-bound chimeric protein levels to secreted chimeric protein levels).

[0140] Figure 17Shown is a schematic diagram of a membrane cleavable system as described herein, wherein the desired payload is expressed as a chimeric protein in which a protease cleavage site is inserted between the payload and the membrane tethered domain. The left figure shows a schematic diagram of a membrane cleavable system using a transmembrane structure (e.g., a chimeric protein comprises a transmembrane domain). The right figure shows a schematic diagram of a membrane cleavable system using a membrane-connected structure (e.g., a chimeric protein comprises a post-translational modification tag that allows the cell membrane to be connected).

[0141] Figure 18 Representative membrane-cleavable systems for regulated secretion of IL-15 are shown, in which IL-15 is expressed as a chimeric protein with a cleavage site that can be cleaved by TACE inserted between the IL-15 payload and the membrane tethering domain. The left panel illustrates type I transmembrane structures (e.g., S–C–MT) using type I transmembrane domains (such as PDGFR-β and CD8). The right panel illustrates type II transmembrane structures (e.g., MT–C–S) using type II transmembrane domains (such as NKG2D and TNFR2).

[0142] In some aspects, provided herein are membrane-cleavable chimeric proteins (or engineered nucleic acids encoding membrane-cleavable chimeric proteins) having a protein of interest (e.g., any effector molecule described herein), a protease cleavage site, and a cell membrane tethering domain.

[0143] 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 the bound molecule. For example, an effector molecule can act as a ligand for increasing or decreasing enzymatic activity, gene expression, or cell signaling. Thus, in some embodiments, effector molecules modulate (activate or inhibit) different immunoregulatory mechanisms. By directly binding to and modulating a molecule, an effector molecule can also indirectly modulate a second downstream molecule.

[0144] In certain embodiments described herein (e.g., generally for all membrane-cleavable chimeric proteins described herein), the effector molecule is a secretable effector molecule (e.g., referred to as "S" in the formula S-C-MT or MT-C-S of the membrane-cleavable chimeric proteins described herein). Non-limiting examples of effector molecules include cytokines, chemokines, enzymes that modulate metabolite levels, growth factors, coactivator molecules, tumor microenvironment modifiers, ligands, peptides, enzymes, antibodies, antibodies that modulate cytokines, homing molecules, and / or integrins, or decoy molecules.

[0145] The term "modulate" encompasses maintaining biological activity, inhibiting (partially or completely) biological activity, and stimulating / activating (partially or completely) biological activity. The term also encompasses reducing or increasing (e.g., enhancing) biological activity. Two different effector molecules are considered to "modulate different tumor-mediated immunosuppressive mechanisms" when the tumor-mediated immunosuppressive mechanism modulated by one effector molecule (e.g., stimulating T cell signaling) is different from the tumor-mediated immunosuppressive mechanism modulated by another effector molecule (e.g., stimulating antigen presentation and / or processing).

[0146] Regulation by effector molecules can be direct or indirect. Direct regulation occurs when an effector molecule binds to another molecule and modulates the activity of that molecule. Indirect regulation occurs when an effector molecule binds to another molecule, modulates the activity of that molecule, and as a result of this modulation, modulates the activity of yet another molecule (to which the effector molecule is not bound).

[0147] In some embodiments, at least one effector molecule modulates tumor-mediated immunosuppressive mechanisms such that immunostimulation and / or anti-tumor immune responses (e.g., systemically or in the tumor microenvironment) are increased by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). For example, modulation of tumor-mediated immunosuppressive mechanisms can increase immunostimulation and / or anti-tumor immune responses 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%, or at least 100%. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in an increase in immunostimulation and / or anti-tumor immune response 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%. It will be understood that an "increase" in immunostimulatory and / or anti-tumor immune response, eg, systemically or in the tumor microenvironment, is relative to the immunostimulatory and / or anti-tumor immune response that would otherwise occur in the absence of the effector molecule.

[0148] In some embodiments, at least one effector molecule modulates tumor-mediated immunosuppressive mechanisms such that immunostimulation and / or anti-tumor immune responses (e.g., systemically or in the tumor microenvironment) are increased by at least 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 20-fold, 25-fold, 50-fold, or 100-fold). For example, modulating tumor-mediated immunosuppressive mechanisms can increase immunostimulation and / or anti-tumor immune responses 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, modulating tumor-mediated immunosuppressive mechanisms increases immunostimulation and / or anti-tumor immune responses by 2-10-fold, 2-20-fold, 2-30-fold, 2-40-fold, 2-50-fold, 2-60-fold, 2-70-fold, 2-80-fold, 2-90-fold, or 2-100-fold.

[0149] Non-limiting examples of immune stimulation and / or anti-tumor immune mechanisms include T cell signaling, activity and / or recruitment, antigen presentation and / or processing, natural killer cell-mediated cytotoxicity signaling, activity and / or recruitment, dendritic cell differentiation and / or maturation, immune cell recruitment, proinflammatory macrophage signaling, activity and / or recruitment, matrix degradation, immunostimulatory metabolite production, interferon gene stimulator (STING) signaling (which increases IFN secretion and Th1 polarization, promoting anti-tumor immune response) and / or type I interferon signaling. Effector molecules can stimulate at least one (one or more) of the aforementioned immune stimulation mechanisms, thereby increasing the immune stimulation response. Changes in the aforementioned immune stimulation and / or anti-tumor immune mechanisms can be assessed, for example, using in vitro assays for T cell proliferation or cytotoxicity, in vitro antigen presentation assays, (e.g., specific marker) expression assays and / or (e.g., cytokine) cell secretion assays.

[0150] In some embodiments, at least one effector molecule modulates tumor-mediated immunosuppressive mechanisms such that an immunosuppressive response (e.g., systemically or in the tumor microenvironment) is reduced by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). For example, modulating tumor-mediated immunosuppressive mechanisms can reduce 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%, or at least 100%. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in a reduction of the immunosuppressive response 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%. It will be understood that a "reduction" of an immunosuppressive response, e.g., systemically or in the tumor microenvironment, is relative to the immunosuppressive response that would have occurred in the absence of the effector molecule.

[0151] In some embodiments, at least one effector molecule modulates a tumor-mediated immunosuppressive mechanism such that an immunosuppressive response (e.g., systemically or in the tumor microenvironment) is reduced by at least 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 20-fold, 25-fold, 50-fold, or 100-fold). For example, modulating a tumor-mediated immunosuppressive mechanism can reduce the 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, modulating a tumor-mediated immunosuppressive mechanism reduces the immunosuppressive response by 2-10-fold, 2-20-fold, 2-30-fold, 2-40-fold, 2-50-fold, 2-60-fold, 2-70-fold, 2-80-fold, 2-90-fold, or 2-100-fold.

[0152] 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. The effector molecule can inhibit at least one (one or more) of the aforementioned immunosuppressive mechanisms, thereby reducing the immunosuppressive response. The changes in the aforementioned immunosuppressive mechanisms can be assessed, for example, by measuring an increase in T cell proliferation and / or an increase in IFNγ production (negative costimulatory signaling, T reg cell signaling and / or MDSC); Annexin V / PI flow cytometry (pro-apoptotic signaling); flow cytometry for expression, e.g., PDL1 expression (tumor checkpoint molecule production / maintenance); ELISA, RNA was assayed by qPCR and enzymatic assays, e.g., IDO tryptophan catabolism (immunosuppressive factor / metabolite production); and PI3K, Akt, p38 phosphorylation (VEGF signaling).

[0153] In some embodiments, the effector molecules act additively: the effect of the two effector molecules can be equal to the sum of the effects of the two effector molecules acting separately. In other embodiments, the effector molecules act synergistically: the effect of the two effector molecules can be greater than the combined function of the two effector molecules.

[0154] Effector molecules that modulate tumor-mediated immunosuppression mechanisms and / or modify the tumor microenvironment can 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, ligands TRAP, and / or small blocking peptides), intracellular factors that control cell state (e.g., microRNAs and / or transcription factors that modulate cell state to enhance pro-inflammatory properties), factors packaged into exosomes (e.g., microRNAs, cytoplasmic factors, and / or extracellular factors), surface displayed factors (e.g., checkpoint inhibitors, TRAIL), and / or metabolic genes (e.g., enzymes that produce / regulate or degrade metabolites or amino acids).

[0155] In some embodiments, at least one effector molecule stimulates immunostimulatory mechanisms in the tumor microenvironment and / or suppresses immunosuppressive mechanisms in the tumor microenvironment.

[0156] In some embodiments, at least one effector molecule (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 matrix degradation, (h) stimulates immunostimulatory metabolite production, (i) stimulates type I interferon signaling, (j) inhibits negative costimulatory signaling, (k) inhibits pro-apoptotic signaling in anti-tumor immune cells, (l) inhibits T regulatory (T) reg ) cell signaling, activity, and / or recruitment, (m) inhibit tumor checkpoint molecules, (n) stimulate stimulator of interferon genes (STING) signaling, (o) inhibit myeloid-derived suppressor cell signaling, activity, and / or recruitment, (p) degrade immunosuppressive factors / metabolites, (q) inhibit vascular endothelial growth factor signaling, and / or (r) directly kill tumor cells.

[0157] In some embodiments, the effector molecule can be selected from the following non-limiting molecular classes: cytokines, antibodies, chemokines, nucleotides, peptides, and enzymes. Non-limiting examples of the aforementioned effector molecule classes are listed in Table 1, and specific sequences encoding exemplary effector molecules are listed in Table 2. The effector molecules can be human, such as those listed in Table 1 or Table 2, or human equivalents of the murine effector molecules listed in Table 1 or Table 2. The effector molecules can be human-derived, such as endogenous human effector molecules or effector molecules that have been functionally modified and / or optimized (e.g., codon optimized to improve expression, modified to improve stability, or modified at their 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 desired improvement, such as codon optimization for a specific species (e.g., human, mouse, bacteria, etc.).

[0158] In some embodiments, the effector molecule comprises interleukin 12 (IL-12), such as a dimer of p35 and p40, commonly 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 set forth in SEQ ID NO: 203.

[0159] 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 comprising IL-15 and the extracellular portion of the IL-15 receptor alpha (IL-15Rα), such as the sushi domain shown in SEQ ID NO: 201. An exemplary IL-15 / IL-15Rα sushi domain fusion is provided as SEQ ID NO: 202. In some embodiments, the effector molecule comprises IL-15 having the amino acid sequence NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENL IILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 224). In some embodiments, IL-15 is encoded by a polynucleotide sequence comprising the sequence AATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAACAGCCTGTCCAGCAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACACCTCA (SEQ ID NO: 225).In some embodiments, IL-15 is encoded by a polynucleotide sequence comprising a sequence 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 AATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAACAGCCTGTCCAGCAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACACCTCA (SEQ ID NO: 225).

[0160] In some embodiments, the membrane cleavable chimeric protein comprises the amino acid sequence MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 226). In some embodiments, the membrane cleavable chimeric protein is encoded by a polynucleotide sequence comprising the sequence (SEQ ID NO: 227).In some embodiments, the membrane-cleavable chimeric protein is encoded by a polynucleotide sequence comprising 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: 227).

[0161] Table 1: Exemplary effector molecules

[0162]

[0163]

[0164] Table 2: Exemplary effector molecule sequences

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179] Secretion signals and signal anchors

[0180] One or more effector molecules of the chimeric proteins provided herein can be secretable effector molecules having a secretory signal peptide (also referred to as a signal peptide or signal sequence) at the N-terminus of the chimeric protein (e.g., the N-terminus of the effector molecule of S-C-MT), which directs newly synthesized proteins destined for secretion or membrane localization (also referred to as membrane insertion) to the appropriate protein processing pathway. For chimeric proteins having the formula MT-C-S, the membrane tethering domain typically has a signal anchor sequence (e.g., a signal anchor sequence of a type II transmembrane protein) that directs newly synthesized proteins destined for membrane localization to the appropriate protein processing pathway. For chimeric proteins having the formula S-C-MT, a membrane tethering domain (usually without a separate secretory signal peptide) having an inverted signal anchor sequence (e.g., a signal anchor sequence of certain type III transmembrane proteins) can be used that directs newly synthesized proteins destined for membrane localization to the appropriate protein processing pathway.

[0181] In general, for all membrane-cleavable chimeric proteins described herein, 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 having two or more chimeric proteins, each chimeric protein can comprise a secretion signal. In embodiments having two or more chimeric proteins, each chimeric protein can comprise a secretion signal, enabling each effector molecule to be secreted from the engineered cell following cleavage at a protease cleavage site.

[0182] The secretion signal peptide operably linked to the effector molecule can be a natural secretion signal peptide (e.g., a secretion signal peptide is typically endogenously linked to a given effector molecule). The secretion signal peptide operably linked to the effector molecule can be a non-natural secretion signal peptide. Non-natural secretion signal peptides can promote improved expression and function, such as maintaining secretion in a specific environment (such as a tumor microenvironment). Non-limiting examples of non-natural secretion signal peptides are shown in Table 3.

[0183] The secretion signal peptide can be an IgE signal peptide. The IgE signal peptide can include the amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 228). The IgE signal peptide can be encoded by a polynucleotide sequence including the sequence ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCT (SEQ ID NO: 229). The IgE signal peptide can be encoded by a polynucleotide sequence comprising a sequence 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).

[0184] The secretion signal peptide of the membrane-cleavable chimeric protein (e.g., IL-15) can be an IgE signal peptide. The IgE signal peptide of the membrane-cleavable chimeric protein (e.g., IL-15) can include the amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 228). The IgE signal peptide of the membrane-cleavable chimeric protein (e.g., IL-15) can be encoded by a polynucleotide sequence including the sequence ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCT (SEQ ID NO: 229). The IgE signal peptide of the membrane-cleavable chimeric protein (e.g., IL-15) can be encoded by a polynucleotide sequence comprising 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).

[0185] Table 3. Exemplary signal secretion peptides

[0186]

[0187]

[0188]

[0189] Protease cleavage site

[0190] In certain embodiments, the chimeric proteins provided herein (e.g., generally, for all membrane-cleavable chimeric proteins described herein) comprise a protease cleavage site (e.g., referred to as "C" in the formula S-C-MT or MT-C-S of the membrane-cleavable chimeric proteins described herein). Generally, the protease cleavage site can be any amino acid sequence motif that is 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 ADAM15 protease cleavage site, an ADAM17 protease cleavage site, an ADAM19 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 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.

[0191] An example of a protease cleavage site is the hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease cleavage site, including but not limited to NS3 / NS4A, NS4A / NS4B, NS4B / NS5A or NS5A / NS5B cleavage sites. For a description of representative sequences of NS3 proteases and cleavage sites thereof for various HCV strains, see, for example, Hepatitis C Viruses: Genomes and Molecular Biology (SLTan, ed., Taylor & Francis, 2006), Chapter 6, pages 163-206; the entire document is incorporated herein by reference. For example, sequences of the HCV NS4A / 4B protease cleavage site, the HCV NS5A / 5B protease cleavage site, the C-terminal degron with the NS4A / 4B protease cleavage site, and the N-terminal degron with the HCV NS5A / 5B protease cleavage site are provided. Representative NS3 sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, e.g., 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, CB F60817, AHH29575, AIZ00747, AIZ00744, ABI36969, ABN05226, KF516075, KF516074, KF516056, AB826684, AB826683, JX171009, JX171008, JX171000, EU847455, EF154714, GU085487, JX171065, JX171063; all of the sequences entered as of the filing date of this application are incorporated herein by reference.

[0192] Another example of a protease cleavage site is an ADAM17 specific protease (also known as tumor necrosis factor-α converting enzyme [TACE]) cleavage site. The ADAM17 specific protease cleavage site can be an endogenous sequence of a substrate that is naturally cleaved by ADAM17. The ADAM17 specific protease cleavage site can be an engineered sequence that can be cleaved by ADAM17. The engineered ADAM17 specific protease cleavage site can be engineered to obtain specific desired properties, including but not limited to optimal expression of the chimeric protein, specificity for ADAM17, the cleavage rate of ADAM17, the ratio of the level of secreted chimeric protein to the level of membrane-bound chimeric protein, and cleavage under different cell states. The protease cleavage site can be selected for the specific cleavage of ADAM17. For example, certain protease cleavage sites that can be cleaved by ADAM17 can also be cleaved by other ADAM family proteases such as ADAM10. Therefore, the ADAM17 specific protease cleavage site can be selected and / or engineered to reduce or eliminate the cleavage of other proteases such as ADAM10. The protease cleavage site can be selected for the cleavage rate of ADAM17. For example, it may be desirable to select a protease cleavage site showing the specific cleavage rate of ADAM17 (such as the cleavage kinetics reduced relative to the endogenous sequence of the substrate naturally cut by ADAM17). In such cases, generally speaking, a specific cleavage rate can be selected to regulate the processing rate of chimeric protein, then the release / secretion rate of the payload effector molecule is regulated. Therefore, the ADAM17 specific protease cleavage site can be selected and / or engineered so that the sequence exhibits the ADAM17 cleavage rate of expectation. The protease cleavage site can be selected for the specific cutting of ADAM17 and the cleavage rate of ADAM17. Exemplary ADAM17 specific protease cleavage sites (including those sites that exhibit specific specificity and cleavage rate kinetics) are shown in Table 4A below, wherein cleavage site (P5-P1: N-terminal; P1 '-P5 ': C-terminal) is mentioned. Further details of ADAM17 and ADAM10, including expression and protease cleavage sites, are described in Sharma et al. (J Immunol 2017 Oct 15, 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 of which is incorporated herein by reference for all purposes.

[0193] Table 4A - Sequences of various ADAM17 protease cleavage sites

[0194] P5 P4 P3 P2 P1 P1' P2' P3' P4' P5' full sequence SEQ ID NO P R A E A V K G G PRAEAVKGG 179 P R A E A L K G G PRAEALKGG 180 P R A E Y S K G G PRAEYSKGG 181 P R A E P I K G G PRAEPIKGG 182 P R A E A Y K G G PRAEAYKGG 183 P R A E S S K G G PRAESSKGG 184 P R A E F T K G G PRAEFTKGG 185 D E P H Y S Q R R DEPHYSQRR 187 P P L G P I F N P G PPLGPIFNPG 188 P L A Q A Y R S S PLAQAYRSS 189 T P I D S S F N P D TPIDSSFNPD 190 V T P E P I F S L I VTPEPIFSLI 191

[0195] In some embodiments, the protease cleavage site comprises a first region having an amino acid sequence of PRAE (SEQ ID NO: 176). In some embodiments, the protease cleavage site comprises a second region having an amino acid sequence of KGG (SEQ ID NO: 177). In some embodiments, the first region is located at the N-terminus of the second region. In some embodiments, the protease cleavage site comprises an amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 178), wherein X1 is A, Y, P, S or F, and wherein X2 is V, L, S, I, Y, T or A. In some embodiments, the protease cleavage site comprises an amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 178), wherein X1 is A, Y, P, S or F, and wherein X2 is V, L, S, I, Y or T. In some embodiments, the protease cleavage site comprises an amino acid sequence of PRAEAVKGG (SEQ ID NO: 179). In some embodiments, the protease cleavage site comprises an 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).

[0196] 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 ADAM17.

[0197] In some embodiments, the protease cleavage site may include an N-terminal peptide linker, such as SGGGGSGGGGSG (SEQ ID NO: 230). In some embodiments, the protease cleavage site may include a C-terminal peptide linker, such as GGGSGGGGSGGGSLQ (SEQ ID NO: 231). In some embodiments, the protease cleavage site may include an N-terminal peptide linker and a C-terminal peptide linker, such as GGGSGGGGSGGGSLQ (SEQ ID NO: 230) and GGGSGGGGSGGGSLQ (SEQ ID NO: 231).

[0198] In some embodiments, the protease cleavage site may include a tumor necrosis factor-alpha converting enzyme (TACE) specific cleavage site (also known as ADAM17), an N-terminal peptide linker, and a C-terminal peptide linker. In some embodiments, the protease cleavage site may include a 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 may include a TACE specific cleavage site, an N-terminal peptide linker, and a C-terminal peptide linker, and has the amino acid sequence SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ (SEQ ID NO: 250). In some embodiments, the protease cleavage site may include a TACE specific cleavage site, an N-terminal peptide linker, and a C-terminal peptide linker, consisting of a protein comprising the sequence

[0199] TCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGTGACCCCTGAGCCTATCTTCAGCCTGATC GGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAA (SEQ ID NO: 251). In some embodiments, the protease cleavage site may include a TACE specific cleavage site, an N-terminal peptide linker and a C-terminal peptide linker,

[0200] TCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGTGACCCCTGAGCCTATCTTCAGCCTGATC GGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAA (SEQ ID NO: 251) 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.

[0201] In some embodiments, the protease cleavage site comprises the amino acid sequence of ITQGLAVSTISSFF (SEQ ID NO: 198), which is a cleavage site native to CD16 and cleavable by ADAM17.

[0202] The protease cleavage site can be the C-terminus of the secretable effector molecule. The protease cleavage site can be the N-terminus of the secretable effector molecule. In general, for all membrane-cleavable chimeric proteins described herein, the protease cleavage site is any of the following: (1) the C-terminus of the secretable effector molecule and the N-terminus of the cell membrane tethered domain (in other words, the protease cleavage site is between the secretable effector molecule and the cell membrane tethered domain); or (2) the N-terminus of the secretable effector molecule and the C-terminus of the cell membrane tethered domain (also between the secretable effector molecule and the cell membrane tethered domain, but the domain orientation is reversed). The protease cleavage site can be connected to the secretable effector molecule by a polypeptide linker (i.e., a polypeptide sequence that is not generally considered to be a part of the effector molecule or protease cleavage site). The protease cleavage site can be connected to the cell membrane tethered domain by a polypeptide linker (i.e., a polypeptide sequence that is not generally considered to be a part of the cell membrane tethered domain or protease cleavage site). The polypeptide linker can be any amino acid sequence connecting the first polypeptide sequence and the second polypeptide sequence. The polypeptide linker can be a flexible linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, a GSG linker (e.g., [GS]4GG [SEQ ID NO: 182]), A(EAAAK)3A (SEQ ID NO: 183), and a Whitlow linker (e.g., a "KEGS" 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, which is incorporated herein by reference). Other exemplary polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, and SEQ ID NO: 197. Other polypeptide linkers can be selected based on desired properties (e.g., length, flexibility, amino acid composition, etc.) and are known to those skilled in the art.

[0203] In a membrane cleavable system, after the chimeric protein is expressed and localized to the cell membrane, the protease cleavage site directs the cleavage of the chimeric protein, allowing the release ("secretion") of the effector molecule into the extracellular space of the cell.

[0204] In general, the protease that cuts the protease cleavage site is a protease that has specificity to this particular protease cleavage site. For example, in the case of a disintegrin-metalloproteinase ("ADAM") family protease, the protease that cuts a specific ADAM protease cleavage site is usually limited to the ADAM protease of the specific ADAM protease cleavage site motif of specific recognition. The protease cleavage site can be selected and / or engineered so as to reduce or eliminate the cutting of undesirable protease. Protease can be membrane-bound or membrane-connected. Protease can be secreted, for example, in a specific cellular environment such as a tumor microenvironment ("TME"), secreted.

[0205] The protease of the protease cleavage site of the cutting chimeric protein can be expressed in the same cells expressing the chimeric protein. The protease of the protease cleavage site of the cutting chimeric protein can be endogenous for the cells expressing the chimeric protein. In other words, the cells engineered to express the chimeric protein can endogenously express a protease that has specificity for the protease cleavage site present in the chimeric protein. The endogenous expression of protease refers to both expression under general steady-state conditions (for example, generally considered to be healthy cells) and differential expression (for example, expression upregulated in tumor cells) under non-steady-state conditions. The protease cleavage site can be selected based on known proteases endogenously expressed by the desired cell population. In such cases, in general, the cutting of the protease cleavage site (and therefore the release / secretion of the payload) can be limited to those cells of interest, because cell-restricted proteases need to contact the protease cleavage site of the chimeric protein expressed in the same cells. For example, without wishing to be bound by theory, ADAM17 is considered to be limited to NK cells and T cells. Therefore, the selection of ADAM17 specific protease cleavage site can limit the cutting of protease cleavage site to the NK cells and T cells of co-expressing chimeric protein.In other examples, protease cleavage site can be selected for the specific tumor-associated protease expressed in the specific tumor colony of interest (for example, in the specific tumor cells engineered to express chimeric protein).Protease and / or expression database can be used to select appropriate protease cleavage site, such as by consulting Oncomine (www.oncomine.org), European Bioinformatic Institute (European Bioinformatic Institute) (www.ebi.ac.uk) (particularly (www.ebi.ac.uk / gxa)), PMAP (www.proteolysis.org), ExPASyPeptide Cutter (ca.expasy.org / tools / peptide cutter) and PMAP.Cut DB (cutdb.burnham.org) to select the protease cleavage site cut by tumor-associated protease, each of these documents is incorporated by reference for all purposes.

[0206] The protease that cuts the protease cleavage site of the chimeric protein can be heterologous to the cell expressing the chimeric protein. For example, the cell engineered to express the chimeric protein can also be engineered to express a protease that is not usually expressed by the cell, and the protease has specificity for the protease cleavage site present in the chimeric protein. The cell engineered to express both the chimeric protein and the protease can be engineered to express each by a separate engineered nucleic acid or by a polycistronic system (polycistronic and multi-promoter systems are described in more detail in the section entitled "Polycistronic and Multi-Promoter Systems" herein). Heterologous proteases and their corresponding protease cleavage sites can be selected as described above with reference to endogenous proteases.

[0207] The protease that cuts the protease cleavage site of chimeric protein can be expressed on a separate cell different from the cell expressing the chimeric protein. For example, protease can be expressed in a specific cellular environment (such as a tumor microenvironment) generally. In such cases, generally speaking, the cutting of the protease cleavage site can be limited to those cellular environments of interest (e.g., tumor microenvironment) because environmentally restricted proteases need to contact the protease cleavage site. In embodiments with a membrane-cleavable chimeric protein, generally speaking, the secretion of the effector molecule can be limited to those cellular environments of interest (e.g., tumor microenvironment) because environmentally restricted proteases need to contact the protease cleavage site. The protease that cuts the protease cleavage site of chimeric protein can be endogenous for different separate cells. The protease that cuts the protease cleavage site of chimeric protein can be heterologous for different separate cells. For example, different separate cells can be engineered to express a protease that is not usually expressed by different separate cells.

[0208] Protease includes but is not limited to type 1 transmembrane protease, type II transmembrane protease, GPI anchor protease, ADAM8 protease, ADAM9 protease, ADAM10 protease, ADAM12 protease, ADAM15 protease, ADAM17 protease, ADAM19 protease, ADAM20 protease, ADAM21 protease, ADAM28 protease, ADAM30 protease, ADAM33 protease, BACE1 protease, BACE2 protease, SIP protease, MT1-MMP protease, MT3-MMP protease, MT5-MMP protease, furin, PCSK7 protease, matriptase protease, matriptase-2 protease and MMP9 protease. Protease can be NS3 protease. Protease can be ADAM17 protease.

[0209] Protease can be tumor-associated protease, such as cathepsin, cysteine ​​protease, aspartyl protease, serine protease or metalloprotease. Specific examples of tumor-associated protease 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, ADAMTS, CD10 (CALLA) or prostate specific antigen. Protease can also include but is not limited to the protease listed in Table 4B below. Exemplary homologous protease cleavage sites of some proteases are also listed in Table 4B.

[0210] Table 4B: Exemplary proteases with cognate cleavage sites and inhibitors

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220] The protease may be any of the following human proteases (MEROPS peptidase database numbers are provided in parentheses; Rawlings ND, Morton FR, Kok, CY, Kong, J. & Barrett AJ (2008) MEROPS: the peptidase database. Nucleic Acids Res. Res. 36 Database Special, D320-325; incorporated herein by reference for all purposes): Pepsin A (MER000885), Pepsin (MER000894), Membrane Aspartic Acid Proteinase-2 (MER005870), Renin (MER000917), Cathepsin D (MER000911), Cathepsin E (MER000944), Membrane Aspartic Acid Proteinase-1 (MER005534), Novo Aspartic Acid Proteinase A (MER004981), Mername-AA034 Peptidase (MER014038), Pepsin A4 (MER037290), Pepsin A5 (Homo sapiens) (MER037291), hCG1733572 (Homo sapiens) type putative peptidase (MER107386), Novo Aspartic Acid Proteinase B pseudogene (MER004982), CYMP gp (Homo sapiens) (MER002929), subfamily A1A unspecified peptidase (MER181559), mouse mammary tumor virus retropepsin (MER048030), rabbit endogenous retroviral endopeptidase (MER043650), S71-related human endogenous retropepsin (MER001812), RTVL-H type putative peptidase (MER047117), RTVL-H type putative peptidase (MER047133), RTVL-H type putative peptidase (MER047160), RTVL-H type putative peptidase (MER047206), RTVL-H type putative peptidase (MER047253), RTVL-H type putative peptidase (MER047260), RTV L-H type putative peptidase (MER047291), RTVL-H type putative peptidase (MER047418), RTVL-H type putative peptidase (MER047440), RTVL-H type putative peptidase (MER047479), RTVL-H type putative peptidase (MER047559), RTVL-H type putative peptidase (MER047583), RTVL-H type putative peptidase (MER015446), human endogenous retroviral retropepsin homolog 1 (MER015479), human endogenous retroviral retropepsin homolog 2 (MER015481), endogenous retroviral retropepsin pseudogene 1 (Homo sapiens chromosome 14) (MER029977),Endogenous retroviral retropepsin pseudogene 2 (Homo sapiens chromosome 8) (MER029665), endogenous retroviral retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER002660), endogenous retroviral retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER030286), endogenous retroviral retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER047144), endogenous retroviral retropepsin pseudogene 5 (Homo sapiens chromosome 12) (MER029664), endogenous retroviral retropepsin pseudogene 6 (Homo sapiens chromosome 7) (MER00209 ... pepsin pseudogene 7 (Homo sapiens chromosome 6) (MER029776), endogenous retroviral retropepsin pseudogene 8 (Homo sapiens chromosome Y) (MER030291), endogenous retroviral retropepsin pseudogene 9 (Homo sapiens chromosome 19) (MER029680), endogenous retroviral retropepsin pseudogene 10 (Homo sapiens chromosome 12) (MER002848), endogenous retroviral retropepsin pseudogene 11 (Homo sapiens chromosome 17) (MER004378), endogenous retroviral retropepsin pseudogene 12 (Homo sapiens chromosome 11) (MER003344), endogenous retroviral retropepsin pseudogene 13 (Homo sapiens chromosome 2 and similar) (MER029779), endogenous retroviral retropepsin pseudogene 14 (Homo sapiens chromosome 2) (MER029778), endogenous retroviral retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047158), endogenous retroviral retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047332), endogenous retroviral retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER003182), endogenous retroviral retropepsin pseudogene 16 (MER047165), endogenous retroviral retropepsin pseudogene 16 (MER047178), Endogenous retroviral retrovirus pepsin pseudogene 16 (MER047200), endogenous retroviral retrovirus pepsin pseudogene 16 (MER047315), endogenous retroviral retrovirus pepsin pseudogene 16 (MER047405), endogenous retroviral retrovirus pepsin pseudogene 16 (MER030292), endogenous retroviral retrovirus pepsin pseudogene 17 (Homo sapiens chromosome 8) (MER005305), endogenous retroviral retrovirus pepsin pseudogene 18 (Homo sapiens chromosome 4) (MER030288), endogenous retroviral retrovirus pepsin pseudogene 19 (Homo sapiens chromosome 16) (MER001740),Endogenous retroviral retropepsin pseudogene 21 (Homo sapiens) (MER047222), endogenous retroviral retropepsin pseudogene 21 (Homo sapiens) (MER047454), endogenous retroviral retropepsin pseudogene 21 (Homo sapiens) (MER047477), endogenous retroviral retropepsin pseudogene 21 (Homo sapiens) (MER004403), endogenous retroviral retropepsin pseudogene 22 (Homo sapiens chromosome X) (MER030287), subfamily A2A nonpeptidase homolog (MER047046), subfamily A2A nonpeptidase homolog (MER047052), subfamily A2A nonpeptidase homolog (MER047047). 7076), subfamily A2A non-peptidase homolog (MER047080), subfamily A2A non-peptidase homolog (MER047088), subfamily A2A non-peptidase homolog (MER047089), subfamily A2A non-peptidase homolog (MER047091), subfamily A2A non-peptidase homolog (MER047092), subfamily A2A non-peptidase homolog (MER047093), subfamily A2A non-peptidase homolog (MER047094), subfamily A2A non-peptidase homolog (MER047097), subfamily A2A non-peptidase homolog (MER047099), subfamily A2A non-peptidase homolog (MER047101), subfamily A 2A non-peptidase homolog (MER047102), subfamily A2A non-peptidase homolog (MER047107), subfamily A2A non-peptidase homolog (MER047108), subfamily A2A non-peptidase homolog (MER047109), subfamily A2A non-peptidase homolog (MER047110), subfamily A2A non-peptidase homolog (MER047111), subfamily A2A non-peptidase homolog (MER047114), subfamily A2A non-peptidase homolog (MER047118), subfamily A2A non-peptidase homolog (MER047121), subfamily A2A non-peptidase homolog (MER047122), subfamily A2A non-peptidase homolog (MER047123), subfamily A2A non-peptidase homolog (MER047124). ER047126), subfamily A2A non-peptidase homolog (MER047129), subfamily A2A non-peptidase homolog (MER047130), subfamily A2A non-peptidase homolog (MER047134), subfamily A2A non-peptidase homolog (MER047135), subfamily A2A non-peptidase homolog (MER047137), subfamily A2A non-peptidase homolog (MER047140), subfamily A2A non-peptidase homolog (MER047141), subfamily A2A non-peptidase homolog (MER047142), subfamily A2A non-peptidase homolog (MER047148), subfamily A2A non-peptidase homolog (MER047149),Subfamily A2A non-peptidase homolog (MER047151), subfamily A2A non-peptidase homolog (MER047154), subfamily A2A non-peptidase homolog (MER047155), subfamily A2A non-peptidase homolog (MER047156), subfamily A2A non-peptidase homolog (MER047157), subfamily A2A non-peptidase homolog (MER047159), subfamily A2A non-peptidase homolog (MER047161), subfamily A2A non-peptidase homolog (MER047163), subfamily A2A non-peptidase homolog (MER047166), subfamily A2A non-peptidase homolog (MER047171), subfamily A2A Non-peptidase homolog (MER047173), Subfamily A2A non-peptidase homolog (MER047174), Subfamily A2A non-peptidase homolog (MER047179), Subfamily A2A non-peptidase homolog (MER047183), Subfamily A2A non-peptidase homolog (MER047186), Subfamily A2A non-peptidase homolog (MER047190), Subfamily A2A non-peptidase homolog (MER047191), Subfamily A2A non-peptidase homolog (MER047196), Subfamily A2A non-peptidase homolog (MER047198), Subfamily A2A non-peptidase homolog (MER047199), Subfamily A2A non-peptidase homolog (MER047201), subfamily A2A non-peptidase homolog (MER047202), subfamily A2A non-peptidase homolog (MER047203), subfamily A2A non-peptidase homolog (MER047204), subfamily A2A non-peptidase homolog (MER047205), subfamily A2A non-peptidase homolog (MER047207), subfamily A2A non-peptidase homolog (MER047208), subfamily A2A non-peptidase homolog (MER047210), subfamily A2A non-peptidase homolog (MER047211), subfamily A2A non-peptidase homolog (MER047212), subfamily A2A non-peptidase homolog (MER047213). 7213), subfamily A2A non-peptidase homolog (MER047215), subfamily A2A non-peptidase homolog (MER047216), subfamily A2A non-peptidase homolog (MER047218), subfamily A2A non-peptidase homolog (MER047219), subfamily A2A non-peptidase homolog (MER047221), subfamily A2A non-peptidase homolog (MER047224), subfamily A2A non-peptidase homolog (MER047225), subfamily A2A non-peptidase homolog (MER047226), subfamily A2A non-peptidase homolog (MER047227), subfamily A2A non-peptidase homolog (MER047230),Subfamily A2A non-peptidase homolog (MER047232), subfamily A2A non-peptidase homolog (MER047233), subfamily A2A non-peptidase homolog (MER047234), subfamily A2A non-peptidase homolog (MER047236), subfamily A2A non-peptidase homolog (MER047238), subfamily A2A non-peptidase homolog (MER047239), subfamily A2A non-peptidase homolog (MER047240), subfamily A2A non-peptidase homolog (MER047242), subfamily A2A non-peptidase homolog (MER047243), subfamily A2A non-peptidase homolog (MER047249), subfamily A2A Non-peptidase homolog (MER047251), Subfamily A2A non-peptidase homolog (MER047252), Subfamily A2A non-peptidase homolog (MER047254), Subfamily A2A non-peptidase homolog (MER047255), Subfamily A2A non-peptidase homolog (MER047263), Subfamily A2A non-peptidase homolog (MER047265), Subfamily A2A non-peptidase homolog (MER047266), Subfamily A2A non-peptidase homolog (MER047267), Subfamily A2A non-peptidase homolog (MER047268), Subfamily A2A non-peptidase homolog (MER047269), Subfamily A2A non-peptidase homolog (MER047272), subfamily A2A nonpeptidase homolog (MER047273), subfamily A2A nonpeptidase homolog (MER047274), subfamily A2A nonpeptidase homolog (MER047275), subfamily A2A nonpeptidase homolog (MER047276), subfamily A2A nonpeptidase homolog (MER047279), subfamily A2A nonpeptidase homolog (MER047280), subfamily A2A nonpeptidase homolog (MER047281), subfamily A2A nonpeptidase homolog (MER047282), subfamily A2A nonpeptidase homolog (MER047284), subfamily A2A nonpeptidase homolog (MER047285). 7285), subfamily A2A non-peptidase homolog (MER047289), subfamily A2A non-peptidase homolog (MER047290), subfamily A2A non-peptidase homolog (MER047294), subfamily A2A non-peptidase homolog (MER047295), subfamily A2A non-peptidase homolog (MER047298), subfamily A2A non-peptidase homolog (MER047300), subfamily A2A non-peptidase homolog (MER047302), subfamily A2A non-peptidase homolog (MER047304), subfamily A2A non-peptidase homolog (MER047305), subfamily A2A non-peptidase homolog (MER047306),Subfamily A2A non-peptidase homolog (MER047307), subfamily A2A non-peptidase homolog (MER047310), subfamily A2A non-peptidase homolog (MER047311), subfamily A2A non-peptidase homolog (MER047314), subfamily A2A non-peptidase homolog (MER047318), subfamily A2A non-peptidase homolog (MER047320), subfamily A2A non-peptidase homolog (MER047321), subfamily A2A non-peptidase homolog (MER047322), subfamily A2A non-peptidase homolog (MER047326), subfamily A2A non-peptidase homolog (MER047327), subfamily A2A Non-peptidase homolog (MER047330), Subfamily A2A non-peptidase homolog (MER047333), Subfamily A2A non-peptidase homolog (MER047362), Subfamily A2A non-peptidase homolog (MER047366), Subfamily A2A non-peptidase homolog (MER047369), Subfamily A2A non-peptidase homolog (MER047370), Subfamily A2A non-peptidase homolog (MER047371), Subfamily A2A non-peptidase homolog (MER047375), Subfamily A2A non-peptidase homolog (MER047376), Subfamily A2A non-peptidase homolog (MER047381), Subfamily A2A non-peptidase homolog (MER047383), subfamily A2A nonpeptidase homolog (MER047384), subfamily A2A nonpeptidase homolog (MER047385), subfamily A2A nonpeptidase homolog (MER047388), subfamily A2A nonpeptidase homolog (MER047389), subfamily A2A nonpeptidase homolog (MER047391), subfamily A2A nonpeptidase homolog (MER047394), subfamily A2A nonpeptidase homolog (MER047396), subfamily A2A nonpeptidase homolog (MER047400), subfamily A2A nonpeptidase homolog (MER047401), subfamily A2A nonpeptidase homolog (MER047402). 7403), subfamily A2A non-peptidase homolog (MER047406), subfamily A2A non-peptidase homolog (MER047407), subfamily A2A non-peptidase homolog (MER047410), subfamily A2A non-peptidase homolog (MER047411), subfamily A2A non-peptidase homolog (MER047413), subfamily A2A non-peptidase homolog (MER047414), subfamily A2A non-peptidase homolog (MER047416), subfamily A2A non-peptidase homolog (MER047417), subfamily A2A non-peptidase homolog (MER047420), subfamily A2A non-peptidase homolog (MER047423),Subfamily A2A non-peptidase homolog (MER047424), subfamily A2A non-peptidase homolog (MER047428), subfamily A2A non-peptidase homolog (MER047429), subfamily A2A non-peptidase homolog (MER047431), subfamily A2A non-peptidase homolog (MER047434), subfamily A2A non-peptidase homolog (MER047439), subfamily A2A non-peptidase homolog (MER047442), subfamily A2A non-peptidase homolog (MER047445), subfamily A2A non-peptidase homolog (MER047449), subfamily A2A non-peptidase homolog (MER047450), subfamily A2A Non-peptidase homolog (MER047452), Subfamily A2A non-peptidase homolog (MER047455), Subfamily A2A non-peptidase homolog (MER047457), Subfamily A2A non-peptidase homolog (MER047458), Subfamily A2A non-peptidase homolog (MER047459), Subfamily A2A non-peptidase homolog (MER047463), Subfamily A2A non-peptidase homolog (MER047468), Subfamily A2A non-peptidase homolog (MER047469), Subfamily A2A non-peptidase homolog (MER047470), Subfamily A2A non-peptidase homolog (MER047476), Subfamily A2A non-peptidase homolog (MER047478), subfamily A2A nonpeptidase homolog (MER047483), subfamily A2A nonpeptidase homolog (MER047488), subfamily A2A nonpeptidase homolog (MER047489), subfamily A2A nonpeptidase homolog (MER047490), subfamily A2A nonpeptidase homolog (MER047493), subfamily A2A nonpeptidase homolog (MER047494), subfamily A2A nonpeptidase homolog (MER047495), subfamily A2A nonpeptidase homolog (MER047496), subfamily A2A nonpeptidase homolog (MER047497), subfamily A2A nonpeptidase homolog (MER047498). 7499), subfamily A2A non-peptidase homolog (MER047502), subfamily A2A non-peptidase homolog (MER047504), subfamily A2A non-peptidase homolog (MER047511), subfamily A2A non-peptidase homolog (MER047513), subfamily A2A non-peptidase homolog (MER047514), subfamily A2A non-peptidase homolog (MER047515), subfamily A2A non-peptidase homolog (MER047516), subfamily A2A non-peptidase homolog (MER047520), subfamily A2A non-peptidase homolog (MER047533), subfamily A2A non-peptidase homolog (MER047537),Subfamily A2A nonpeptidase homolog (MER047569), subfamily A2A nonpeptidase homolog (MER047570), subfamily A2A nonpeptidase homolog (MER047584), subfamily A2A nonpeptidase homolog (MER047603), subfamily A2A nonpeptidase homolog (MER047604), subfamily A2A nonpeptidase homolog (MER047606), subfamily A2A nonpeptidase homolog (MER047609), subfamily A2A nonpeptidase homolog (MER047616), subfamily A2A nonpeptidase homolog (MER047619), subfamily A2A nonpeptidase homolog (MER047648), subfamily A2A nonpeptidase homolog (MER047649), subfamily A2A non-peptidase homolog (MER047662), subfamily A2A non-peptidase homolog (MER048004), subfamily A2A non-peptidase homolog (MER048018), subfamily A2A non-peptidase homolog (MER048019), subfamily A2A non-peptidase homolog (MER048023), subfamily A2A non-peptidase homolog (MER048037), subfamily A2A unspecified peptidase (MER047164), subfamily A2A unspecified peptidase (MER047231), subfamily A2A unspecified peptidase (MER047386), skin aspartic proteinase (MER057097), presenilin 1 (MER005221), presenilin 2 (MER005223), impas impas 1 peptidase (MER019701), impas 1 peptidase (MER184722), impas 4 peptidase (MER019715), impas 2 peptidase (MER019708), impas 5 peptidase (MER019712), impas 3 peptidase (MER019711), possible family A22 pseudogene (Homo sapiens chromosome 18) (MER029974), possible family A22 pseudogene (Homo sapiens chromosome 11) (MER023159), cathepsin V (MER004437), cathepsin X (MER004508), cathepsin F (MER004980), cathepsin L (MER000622), cathepsin S (MER000633), cathepsin O (MER001690), cathepsin K (MER000644), cathepsin W (MER003756), cathepsin H (MER000629), cathepsin B (MER000686), dipeptidyl peptidase I (MER001937), bleomycin hydrolase (animal) (MER002481), tubulointerstitial nephritis antigen (MER016137), tubulointerstitial nephritis antigen-related protein (MER021799),Cathepsin L-like pseudogene 1 (Homo sapiens) (MER002789), cathepsin B-like pseudogene (chromosome 4, Homo sapiens) (MER029469), cathepsin B-like pseudogene (chromosome 1, Homo sapiens) (MER029457), CTSLL2 gp (Homo sapiens) (MER005210), CTSLL3 gp (Homo sapiens) (MER005209), calpain-1 (MER000770), calpain-2 (MER000964), calpain-3 (MER001446), calpain-9 (MER004042), calpain-8 (MER021474), calpain-15 (MER004745), calpain-5 (MER002939), calpain-11 (MER005844), calpain-12 (MER029889), calpain-10 (MER013510), calpain-13 (MER0013511), calpain-14 (MER0013512), calpain-15 (MER0013513), calpain-16 (MER0013514), calpain-17 (MER0013515), calpain-18 (MER0013516), calpain-19 (MER0013517), calpain-20 (MER0013518), calpain-21 (MER0013519), calpain-22 (MER0013517), calpain-23 (MER0013518), calpain-24 (MER0013519), calpain-25 (MER001351 20139), calpain-14 (MER029744), Mername-AA253 peptidase (MER005537), calpain (calpamodulin) (MER000718), hypothetical protein 940251 (MER003201), ubiquitin hydrolase-L1 (MER000832), ubiquitin hydrolase-L3 (MER000836), ubiquitin hydrolase-BAP1 (MER003989), ubiquitin hydrolase-UCH37 (MER005539), ubiquitin-specific peptidase 5 (MER002066), ubiquitin-specific Peptidase 6 (MER000863), ubiquitin-specific peptidase 4 (MER001795), ubiquitin-specific peptidase 8 (MER001884), ubiquitin-specific peptidase 13 (MER002627), ubiquitin-specific peptidase 2 (MER004834), ubiquitin-specific peptidase 11 (MER002693), ubiquitin-specific peptidase 14 (MER002667), ubiquitin-specific peptidase 7 (MER002896), ubiquitin-specific peptidase 9X (MER005877), ubiquitin-specific peptidase 10 (MER004439), ubiquitin-specific peptidase 1 (MER002693). 4978), ubiquitin-specific peptidase 12 (MER005454), ubiquitin-specific peptidase 16 (MER005493), ubiquitin-specific peptidase 15 (MER005427), ubiquitin-specific peptidase 17 (MER002900), ubiquitin-specific peptidase 19 (MER005428), ubiquitin-specific peptidase 20 (MER005494), ubiquitin-specific peptidase 3 (MER005513), ubiquitin-specific peptidase 9Y (MER004314), ubiquitin-specific peptidase 18 (MER005641), ubiquitin-specific peptidase 21 (MER006258),Ubiquitin-specific peptidase 22 (MER012130), ubiquitin-specific peptidase 33 (MER014335), ubiquitin-specific peptidase 29 (MER012093), ubiquitin-specific peptidase 25 (MER011115), ubiquitin-specific peptidase 36 (MER014033), ubiquitin-specific peptidase 32 (MER014290), ubiquitin-specific peptidase 26 (Homo sapiens) (MER014292), ubiquitin-specific peptidase 24 (MER005706), ubiquitin-specific peptidase 42 (MER011852), ubiquitin-specific peptidase 46 (MER014629), ubiquitin-specific peptidase 37 (MER014633), ubiquitin-specific peptidase 28 ... 014634), ubiquitin-specific peptidase 47 (MER014636), ubiquitin-specific peptidase 38 (MER014637), ubiquitin-specific peptidase 44 (MER014638), ubiquitin-specific peptidase 50 (MER030315), ubiquitin-specific peptidase 35 (MER014646), ubiquitin-specific peptidase 30 (MER014649), Mername-AA091 peptidase (MER014743), ubiquitin-specific peptidase 45 (MER030314), ubiquitin-specific peptidase 51 (MER014769), ubiquitin-specific peptidase 34 (MER014780), ubiquitin-specific peptidase 48 (MER064620), ubiquitin-specific Peptidase 40 (MER015483), Ubiquitin-specific peptidase 41 (MER045268), Ubiquitin-specific peptidase 31 (MER015493), Mername-AA129 peptidase (MER016485), Ubiquitin-specific peptidase 49 (MER016486), Mername-AA187 peptidase (MER052579), USP17-like peptidase (MER030192), Ubiquitin-specific peptidase 54 (MER028714), Ubiquitin-specific peptidase 53 (MER027329), Ubiquitin-specific endopeptidase 39 [misleading] (MER064621), Mername-AA090 non-peptidase homolog (MER014739). 9), Ubiquitin-specific peptidase 43 [misleading] (MER030140), Ubiquitin-specific peptidase 52 [misleading] (MER030317), NEK2 pseudogene (MER014736), C19 pseudogene (Homo sapiens: chromosome 5) (MER029972), Mername-AA088 peptidase (MER014750), Autophagy protease-2 (MER013564), Autophagy protease-1 (MER013561), Autophagy protease-3 (MER014316), Autophagy protease-4 (MER064622), Cezanne deubiquitinating peptidase (MER029042), Cezanne-2 peptidase (MER029044),Tumor necrosis factor alpha-induced protein 3 (MER029050), trabid peptidase (MER029052), VCIP135 deubiquitinating peptidase (MER152304), otubain-1 (MER029056), otubain-2 (MER029061), CylD protein (MER030104), UfSP1 peptidase (MER042724), UfSP2 peptidase (MER060306), DUBA deubiquitinating enzyme (MER086098), KIAA0459 (Homo sapiens)-like protein (MER122467), Otud1 protein (MER125457), glycosyltransferase 28 domain-containing 1, isoform CRA_c (Homo sapiens)-like (MER123606), hin1L gp (Homo sapiens) (MER139816), ataxia-3 (MER099998), ATXN3L putative peptidase (MER115261), Josephin domain-containing 1 (Homo sapiens) (MER125334), Josephin domain-containing 2 (Homo sapiens) (MER124068), YOD1 peptidase (MER116559), legumin (plant α form) (MER044591), legumin (MER001800), glycosyl Phosphatidylinositol: protein transamidase (MER002479), legumin pseudogene (Homo sapiens) (MER029741), family C13 unspecified peptidase (MER175813), caspase-1 (MER000850), caspase-3 (MER000853), caspase-7 (MER002705), caspase-6 (MER002708), caspase-2 (MER001644), caspase-4 (MER001645), caspase-6 (MER001646), caspase-7 (MER001647), caspase-8 (MER001648), caspase-9 (MER001649), caspase-10 (MER001647), caspase-11 (MER001648), caspase-12 (MER001649), caspase-13 (MER001641), caspase-14 (MER001643), caspase-15 (MER001644), caspase-16 (MER001645), caspase-17 (MER001646), caspase-18 (MER001647), caspase-19 (MER001647), caspase-20 (MER001647), caspase-21 (MER001647), caspase-22 (MER001647), caspase-23 (MER001647), caspase-24 (MER001647), caspase-25 (MER001647), caspase-26 (MER001647), caspase-27 (MER001647), caspase-27 (MER001647), caspase-27 (MER001647), caspase- 01938), Caspase-5 (MER002240), Caspase-8 (MER002849), Caspase-9 (MER002707), Caspase-10 (MER002579), Caspase-14 (MER012083), Caspase-like (MER019325), Mername-AA143 Peptidase (MER021304), Mername-AA186 Peptidase (MER020516), Caspase (Homo sapiens) (MER021463), FLIP protein (MER003026), Mername-AA142 protein (MER021316), Caspase-12 pseudogene (Homo sapiens) (MER019698), Mername-AA093 caspase pseudogene (MER014766), subfamily C14A non-peptidase homolog (MER185329), subfamily C14A non-peptidase homolog (MER179956),Separase (Homo sapiens) (MER011775), separase-like pseudogene (MER014797), SENP1 peptidase (MER011012), SENP3 peptidase (MER011019), SENP6 peptidase (MER011109), SENP2 peptidase (MER012183), SENP5 peptidase (MER014032), SENP7 peptidase (MER014095), SENP8 peptidase (MER016161), SENP4 peptidase (MER005557), pyroglutamyl peptidase I (chordates) (MER011032), Mername-AA073 peptidase (MER029978), sonic hedgehog protein (MER011032), R002539), Indian hedgehog protein (MER002538), desert hedgehog protein (MER012170), dipeptidyl peptidase III (MER004252), Mername-AA164 protein (MER020410), LOC138971g.p. (Homo sapiens) (MER020074), Atp23 peptidase (MER060642), prenyl peptidase 1 (MER004246), aminopeptidase N (MER000997), aminopeptidase A (MER001012), leukotriene A4 hydrolase (MER001013), pyroglutamyl peptidase II (MER012221), cytoplasmic alanyl aminopeptidase (MER00274 6), cysteine ​​aminopeptidase (MER002060), aminopeptidase B (MER001494), aminopeptidase PILS (MER005331), arginyl aminopeptidase-like 1 (MER012271), leukocyte-derived arginine aminopeptidase (MER002968), aminopeptidase Q (MER052595), aminopeptidase O (MER019730), Tata binding protein-associated factor (MER026493), angiotensin-converting enzyme peptidase unit 1 (MER004967), angiotensin-converting enzyme peptidase unit 2 (MER001019), angiotensin-converting enzyme-2 (MER011061), and Mername-AA153 protein (MER002000). 514), phorate oligopeptidase (MER001737), neurolysin (MER010991), mitochondrial intermediate peptidase (MER003665), Mername-AA154 protein (MER021317), Leishmanolysin-2 (MER014492), Leishmanolysin-3 (MER180031), matrix metallopeptidase-1 (MER001063), matrix metallopeptidase-8 (MER001084), matrix metallopeptidase-2 (MER001080), matrix metallopeptidase-9 (MER001085), matrix metallopeptidase-3 (MER001068), matrix metallopeptidase-10 (Homo sapiens) (MER001072),Matrix metallopeptidase-11 (MER001075), matrix metallopeptidase-7 (MER001092), matrix metallopeptidase-12 (MER001089), matrix metallopeptidase-13 (MER001411), membrane-type matrix metallopeptidase-1 (MER001077), membrane-type matrix metallopeptidase-2 (MER002383), membrane-type matrix metallopeptidase-3 (MER002384), membrane-type matrix metallopeptidase-4 (MER002595), matrix metallopeptidase-20 (MER003021), matrix metallopeptidase-19 (MER002076), matrix metallopeptidase-23B (MER004766), membrane-type matrix metallopeptidase-5 (MER001077), membrane-type matrix metallopeptidase-2 (MER002383), membrane-type matrix metallopeptidase-3 (MER002384), membrane-type matrix metallopeptidase-4 (MER002595), matrix metallopeptidase-20 (MER003021), matrix metallopeptidase-19 (MER002076), matrix metallopeptidase-23B (MER004766), membrane-type matrix metallopeptidase-5 (MER001077), membrane-type matrix metallopeptidase-2 ER005638), membrane-type matrix metallopeptidase-6 (MER012071), matrix metallopeptidase-21 (MER006101), matrix metallopeptidase-22 (MER014098), matrix metallopeptidase-26 (MER012072), matrix metallopeptidase-28 (MER013587), matrix metallopeptidase-23A (MER037217), macrophage elastase homolog (chromosome 8, Homo sapiens) (MER030035), Mername-AA156 protein (MER021309), matrix metallopeptidase-like 1 (MER045280), subfamily M10A nonpeptidase homolog (MER175912), subfamily M 10A non-peptidase homolog (MER187997), subfamily M10A non-peptidase homolog (MER187998), subfamily M10A non-peptidase homolog (MER180000), transmembrane peptidase alpha subunit (MER001111), transmembrane peptidase beta subunit (MER005213), procollagen C-peptidase (MER001113), mammalian tolloid-like 1 protein (MER005124), mammalian tolloid-like 2 protein (MER005866), ADAMTS9 peptidase (MER012092), ADAMTS14 peptidase (MER016700), ADAMTS15 peptidase (MER017029) , ADAMTS16 peptidase (MER015689), ADAMTS17 peptidase (MER016302), ADAMTS18 peptidase (MER016090), ADAMTS19 peptidase (MER015663), ADAM8 peptidase (MER003902), ADAM9 peptidase (MER001140), ADAM10 peptidase (MER002382), ADAM12 peptidase (MER005107), ADAM19 peptidase (MER012241), ADAM15 peptidase (MER002386), ADAM17 peptidase (MER003094), ADAM20 peptidase (MER004725),ADAMDEC1 peptidase (MER000743), ADAMTS3 peptidase (MER005100), ADAMTS4 peptidase (MER005101), ADAMTS1 peptidase (MER005546), ADAM28 peptidase (Homo sapiens) (MER005495), ADAMTS5 peptidase (MER005548), ADAMTS8 peptidase (MER005545), ADAMTS6 peptidase (MER005893), ADAMTS7 peptidase (MER005894) , ADAM30 peptidase (MER006268), ADAM21 peptidase (Homo sapiens) (MER004726), ADAMTS10 peptidase (MER014331), ADAMTS12 peptidase (MER014337), ADAMTS13 peptidase (MER015450), ADAM33 peptidase (MER015143), oocyte astaxanthin (ovastacin) (MER029996), ADAMTS20 peptidase (Homo sapiens) (MER026906), procollagen I N-peptidase (MER004985), ADAM2 protein (MER003090), ADAM6 protein (MER047044), ADAM7 protein (MER005109), ADAM18 protein (MER012230), ADAM32 protein (MER026938), non-peptidase homolog (Homo sapiens chromosome 4) (MER029973), family M12 non-peptidase homolog (Homo sapiens chromosome 16) (MER047654), family M12 non-peptidase homolog (Homo sapiens chromosome 15) (MER047250), ADAM3B protein (Homo sapiens type) (M ER005199), ADAM11 protein (MER001146), ADAM22 protein (MER005102), ADAM23 protein (MER005103), ADAM29 protein (MER006267), protein similar to ADAM21 peptidase preproprotein (Homo sapiens) (MER026944), Mername-AA225 peptidase homolog (Homo sapiens) (MER047474), putative ADAM pseudogene (chromosome 4, Homo sapiens) (MER029975), ADAM3Ag.p. (Homo sapiens) (MER005200), ADAM1 gp (Homo sapiens) (MER003912), subfamily M12B non-peptidase homolog (MER188210), subfamily M12B non-peptidase homolog (MER188211), subfamily M12B non-peptidase homolog (MER188212), subfamily M12B non-peptidase homolog (MER188220), enkephalinase (MER001050), endothelin-converting enzyme 1 (MER001057), endothelin-converting enzyme 2 (MER004776),DINE peptidase (MER005197), enkephalin-2 (MER013406), Kell blood group protein (MER001054), PHEX peptidase (MER002062), i-AAA peptidase (MER001246), i-AAA peptidase (MER005755), parapleglin (MER004454), Afg3-like protein 2 (MER005496), Afg3-like protein 1A (MER014306), pappalysin-1 (MER002217), pappalysin-2 (MER014521), farnesylation protein convertase 1 (MER002646), metalloproteinase-related protein-1 (MER030873 ), Aminopeptidase AMZ2 (MER011907), Aminopeptidase AMZ1 (MER058242), Carboxypeptidase A1 (MER001190), Carboxypeptidase A2 (MER001608), Carboxypeptidase B (MER001194), Carboxypeptidase N (MER001198), Carboxypeptidase E (MER001199), Carboxypeptidase M (MER001205), Carboxypeptidase U (MER001193), Carboxypeptidase A3 (MER001187), Metallocarboxypeptidase D Peptidase Unit 1 (MER003781), Metallocarboxypeptidase Z (MER003428), Metallocarboxypeptidase D Peptidase Unit 2 (MER004963), Carboxypeptidase A4 (MER001 3421), carboxypeptidase A6 (MER013456), carboxypeptidase A5 (MER017121), metallocarboxypeptidase O (MER016044), cytoplasmic carboxypeptidase-like protein 5 (MER033174), cytoplasmic carboxypeptidase 3 (MER033176), cytoplasmic carboxypeptidase 6 (MER033178), cytoplasmic carboxypeptidase 1 (MER033179), cytoplasmic carboxypeptidase 2 (MER037713), metallocarboxypeptidase D non-peptidase unit (MER004964), adipocyte enhancer binding protein 1 (MER003889), carboxypeptidase-like protein X1 (MER013404), carboxypeptidase-like protein X2 (MER078764), cytoplasmic carboxypeptidase ... R026952), family M14 non-peptidase homolog (MER199530), trypsin (MER001214), mitochondrial processing peptidase β-subunit (MER004497), phenelzine lyase (MER003883), eupitrilysin (MER004877), mitochondrial processing peptidase non-peptidase α subunit (MER001413), ubiquinol-cytochrome c reductase core protein I (MER003543), ubiquinol-cytochrome c reductase core protein II (MER003544), ubiquinol-cytochrome c reductase core protein domain 2 (MER043998), trypsin unit 2 (MER046821),Phenelzine lyase unit 2 (MER046874), trypsin lysin unit 3 (MER078753), mitochondrial processing peptidase subunit alpha unit 2 (MER124489), phenelzine lyase unit 3 (MER142856), LOC133083g.p. (Homo sapiens) (MER021876), subfamily M16B non-peptidase homolog (MER188757), leucyl aminopeptidase (animal) (MER003100), peptidase-AA040 (M. ER003919), Leucyl Aminopeptidase-1 (Caenorhabditis elegans) (MER013416), Methionyl Aminopeptidase 1 (MER001342), Methionyl Aminopeptidase 2 (MER001728), Aminopeptidase P2 (MER004498), Xaa-Pro Dipeptidase (Eukaryotic) (MER001248), Aminopeptidase P1 (MER004321), Mitochondrial Intermediate-cleaving Peptidase 55 kDa (MER013463), Mitochondrial Methionyl Aminopeptidase (MER001728), 014055), Mername-AA020 peptidase homolog (MER010972), proliferation-associated protein 1 (MER005497), chromosome-specific transcription elongation factor 140 kDa subunit (MER026495), proliferation-associated protein 1-like (Homo sapiens chromosome X) (MER029983), Mername-AA226 peptidase homolog (Homo sapiens) (MER056262), Mername-AA227 peptidase homolog (Homo sapiens) (MER047 299), subfamily M24A non-peptidase homolog (MER179893), aspartyl aminopeptidase (MER003373), Gly-Xaa carboxypeptidase (MER033182), carnosine dipeptidase II (MER014551), carnosine dipeptidase I (MER015142), Mername-AA161 protein (MER021873), aminoacylase (MER001271), glutamate carboxypeptidase II (MER002104), NAALADASE L-peptidase (MER005239), glutamate carboxypeptidase III (MER005238), plasma glutamate carboxypeptidase (MER005244), Mername-AA103 peptidase (MER015091), Fxna peptidase (MER029965), transferrin receptor protein (MER002105), transferrin receptor 2 protein (MER005152), glutaminyl cyclase (MER015095), glutamate carboxypeptidase II (Homo sapiens) type non-peptidase homolog (MER026971), nicalin (MER044627), membrane dipeptidase (MER001260), membrane-bound dipeptidase-2 (MER013499), membrane-bound dipeptidase-3 (MER013496),Dihydroorotase (MER005767), dihydropyrimidinase (MER033266), dihydropyrimidinase-related protein-1 (MER030143), dihydropyrimidinase-related protein-2 (MER030155), dihydropyrimidinase-related protein-3 (MER030151), dihydropyrimidinase-related protein-4 (MER030149), dihydropyrimidinase-related protein-5 (MER030136), hypothetical protein-like 5730457F11RIK (MER033184), 1300019j08rik protein (MER033186), guanine aminohydrolase (MER037714), Kae1 putative peptidase (MER001577), O SGEPL1-like protein (MER013498), S2P peptidase (MER004458), subfamily M23B non-peptidase homolog (MER199845), subfamily M23B non-peptidase homolog (MER199846), subfamily M23B non-peptidase homolog (MER199847), subfamily M23B non-peptidase homolog (MER137320), subfamily M23B non-peptidase homolog (MER201557), subfamily M23B non-peptidase homolog (MER199417), subfamily M23B non-peptidase homolog (MER199418), subfamily M23B non-peptidase homolog (MER199419), subfamily M23B non-peptidase homolog Origin (MER199420), subfamily M23B non-peptidase homolog (MER175932), subfamily M23B non-peptidase homolog (MER199665), Poh1 peptidase (MER020382), Jab1 / MPN domain metalloenzyme (MER022057), Mername-AA165 peptidase (MER021865), Brcc36 isopeptidase (MER021890), histone H2A deubiquitinating enzyme MYSM1 (MER021887), AMSH deubiquitinating peptidase (MER030146), putative peptidase (Homo sapiens chromosome 2) (MER029970), Mername-AA168 protein (MER021890), R021886), COP9 signalosome subunit 6 (MER030137), 26S proteasome non-ATPase regulatory subunit 7 (MER030134), eukaryotic translation initiation factor 3 subunit 5 (MER030133), IFP38 peptidase homolog (MER030132), subfamily M67A non-peptidase homolog (MER191181), subfamily M67A unspecified peptidase (MER191144), granzyme B (Homo sapiens) (MER000168), testosterone (MER005212), tryptase beta (MER000136), kallikrein-related peptidase 5 (MER005544), corin (MER005881),Kallikrein-related peptidase 12 (MER006038), DESC1 peptidase (MER006298), tryptase gamma 1 (MER011036), kallikrein-related peptidase 14 (MER011038), hyaluronan-binding peptidase (MER003612), transmembrane peptidase, serine 4 (MER011104), intestinal serine peptidase (rodent) (MER016130), adrenal secretory serine peptidase (MER003734), tryptase delta 1 (homo sapiens) (MER005948), proteinase-3 (MER029902), dermazolin (MER006119), tryptase-6 (MER006118), Ovulation enzyme-1 domain 1 (MER099182), transmembrane peptidase, serine 3 (MER005926), kallikrein-related peptidase 15 (MER000064), Mername-AA031 peptidase (MER014054), TMPRSS13 peptidase (MER014226), Mername-AA038 peptidase (MER062848), Mername-AA204 peptidase (MER029980), cationic trypsin (Homo sapiens) (MER000020), elastase-2 (MER000118), mannan-binding lectin-related serine peptidase-3 (MER031968), cathepsin G ...064), TMPRSS13 peptidase (MER014226), Mername-AA038 peptidase (MER062848), Mername-AA204 peptidase (MER029980), cationic trypsin (Homo sapiens) (MER000020), elastase-2 (MER000118), mannan-binding lectin-related serine peptidase-3 (MER031968), cathepsin G (MER0000064), TMPRSS13 peptidase (MER014226), TMPRSS13 peptidase (MER014226), TMPRSS13 peptidase (MER014226), TMPRSS13 peptidase (MER014226), TMPRSS13 peptidase (MER014226), TMPR R000082), myeloblast protease (MER000170), granzyme A (MER001379), granzyme M (MER001541), chymotrypsin (Homo sapiens) (MER000123), tryptase α (MER000135), granzyme K (MER001936), granzyme H (MER000166), chymotrypsin B (MER000001), elastase-1 (MER003733), pancreatic endopeptidase E (MER000149), pancreatic elastase II (MER000146), enteropeptidase (MER002068), chymotrypsin C (MER000761), prostate protease (MER000135 ... 2460), kallikrein 1 (MER000093), kallikrein-related peptidase 2 (MER000094), kallikrein-related peptidase 3 (MER000115), midtrypsin (MER000022), complement component C1r-like peptidase (MER016352), complement factor D (MER000130), complement component activated C1r (MER000238), complement component activated C1s (MER000239), complement component C2a (MER000231), complement factor B (MER000229), mannan-binding lectin-associated serine peptidase 1 (MER000244), complement factor I (MER000228),Pancreatic endopeptidase E form B (MER000150), pancreatic elastase IIB (MER000147), coagulation factor XIIa (MER000187), plasma kallikrein (MER000203), coagulation factor Xia (MER000210), coagulation factor IXa (MER000216), coagulation factor Vila (MER000215), coagulation factor Xa (MER000212), thrombin (MER000188), protein C (activated) (MER000222), acrosin (MER000078), hepsin (MER000156), hepatocyte growth factor activator (MER000186) , mannan-binding lectin-related serine peptidase 2 (MER002758), u-plasminogen activator (MER000195), t-plasminogen activator (MER000192), plasmin (MER000175), kallikrein-related peptidase 6 (MER002580), neurotrypsin (MER004171), kallikrein-related peptidase 8 (MER005400), kallikrein-related peptidase 10 (MER003645), epitheliasin (MER003736), kallikrein-related peptidase 4 (MER005266), prosemin (MER004214), chymopas in (MER001503), kallikrein-related peptidase 11 (MER004861), kallikrein-related peptidase 11 (MER216142), trypsin-2A type (MER000021), HtrA1 peptidase (Homo sapiens) (MER002577), HtrA2 peptidase (MER208413), HtrA2 peptidase (MER004093), HtrA3 peptidase (MER014795), HtrA4 peptidase (MER016351), Tysnd1 peptidase (MER050461), TMPRSS12 peptidase (MER017085), HAT-like putative peptidase 2 (MER021884), trypsin Kallikrein C (MER021898), Kallikrein-related peptidase 7 (MER002001), Proteinase (MER003735), Kallikrein-related peptidase 13 (MER005269), Kallikrein-related peptidase 9 (MER005270), Proteinase-2 (MER005278), Umbilical vein peptidase (MER005421), LCLP peptidase (MER001900), Chiropeptidin (MER014385), Dermabellin-2 (MER021929), Complement factor D-like putative peptidase (MER056164), Ovoidase-2 (MER022410), HAT-like 4 peptidase (MER044589),Ovulation enzyme 1 domain 1 (MER022412), epidermis-specific SP-like putative peptidase (MER029900), testicular serine peptidase 5 (MER029901), Mername-AA258 peptidase (MER000285), polymerase-IA unit 1 (MER030879), polymerase-IA unit 2 (MER030880), testicular serine peptidase 2 (human type) (MER033187), putative acrosomal enzyme-like peptidase (Homo sapiens) (MER033253), HAT-like 5 peptidase (MER028215), polymerase-3 unit 1 (MER061763), polymerase-3 unit 2 (MER061748), with tryptophan / Serine protease-like peptidase (MER056263), polymerase-2 unit 1 (MER061777), Mername-AA123 peptidase (MER021930), HAT-like 2 peptidase (MER099184), hCG2041452-like protein (MER099172), hCG22067 (Homo sapiens) (MER099169), brain rescue factor-1 (Homo sapiens) (MER098873), hCG2041108 (Homo sapiens) (MER099173), polymerase-2 unit 2 (MER061760), polymerase-2 unit 3 (MER065694), Mername-AA201 (peptidase homolog) (MER000175), secreted trypsin-like serine peptidase homolog (MER030000), polymerase-1A unit 3 (MER029880), azurocidin (MER000119), haptoglobin-1 (MER000233), haptoglobin-related protein (MER000235), macrophage-stimulating protein (MER001546), hepatocyte growth factor (MER000185), protein Z (MER000227), TESP1 protein (MER047214), LOC136242 protein (MER016132), plasma kallikrein-like protein 4 (MER016346), PRSS35 protein (MER0001546), and PRSS35 protein (MER0001546). R016350), DKFZp586H2123-like protein (MER066474), apolipoprotein (MER000183), psi-KLK1 pseudogene (Homo sapiens) (MER033287), tryptase pseudogene I (MER015077), tryptase pseudogene II (MER015078), tryptase pseudogene III (MER015079), subfamily S1A unspecified peptidase (MER216982), subfamily S1A unspecified peptidase (MER216148), amidophosphoribosyltransferase precursor (MER003314), glutamine-fructose-6-phosphate aminotransferase 1 (MER003322),Glutamine: Fructose-6-phosphate amidotransferase (MER012158), Mername-AA144 protein (MER021319), Asparagine synthetase (MER033254), Family C44 non-peptidase homolog (MER159286), Family C44 unspecified peptidase (MER185625), Family C44 unspecified peptidase (MER185626), Isolated protein 1 (MER045376), Isolated protein 2 (MER064573), Isolated protein 3 (MER064582), Acid ceramidase precursor (MER100794), N-acylethanolamine acid amidase precursor (MER141667), Proteasome catalyzer Proteasome catalytic subunit 1 (MER000556), proteasome catalytic subunit 2 (MER002625), proteasome catalytic subunit 3 (MER002149), proteasome catalytic subunit 1i (MER000552), proteasome catalytic subunit 2i (MER001515), proteasome catalytic subunit 3i (MER000555), proteasome catalytic subunit 5t (MER026203), protein serine kinase c17 (MER026497), proteasome subunit α6 (MER000557), proteasome subunit α2 (MER000550), proteasome subunit α4 (MER000554), proteasome subunit α7 (MER033250 ), proteasome subunit α5 (MER000558), proteasome subunit α1 (MER000549), proteasome subunit α3 (MER000553), proteasome subunit XAPC7 (MER004372), proteasome subunit β3 (MER001710), proteasome subunit β2 (MER002676), proteasome subunit β1 (MER000551), proteasome subunit β4 (MER001711), Mername-AA230 peptidase homolog (Homo sapiens) (MER047329), Mername-AA231 pseudogene (Homo sapiens) (MER047172), Mername-AA232 pseudogene Gene (Homo sapiens) (MER047316), glycosylasparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622), threonine aspartase-1 (MER016969), γ-glutamyltransferase 5 (mammalian type) (MER001977), γ-glutamyltransferase 1 (mammalian type) (MER001629), γ-glutamyltransferase 2 (Homo sapiens) (MER001976), γ-glutamyltransferase-like protein 4 (MER002721), γ-glutamyltransferase-like protein 3 (MER016970), similar to γ-glutamyltransferase 1 precursor (Homo sapiens) (MER026204),Similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026205), Mername-AA211 putative peptidase (MER026207), gamma-glutamyltransferase 6 (MER159283), gamma-glutamyltranspeptidase homolog (chromosome 2, Homo sapiens) (MER037241), polycystin-1 (MER126824), KIAA1879 protein (MER159329), polycystic kidney disease 1-like 3 (ME R172554), γ-glutamyl hydrolase (MER002963), guanine 5″ monophosphate synthase (MER043387), carbamyl phosphate synthetase (Homo sapiens) (MER078640), dihydroorotase (N-terminal unit) (Homo sapiens) (MER060647), DJ-1 putative peptidase (MER003390), Mername-AA100 putative peptidase (MER014802), Mername -AA101 nonpeptidase homolog (MER014803), KIAA0361 protein (Homo sapiens type) (MER042827), F1134283 protein (Homo sapiens) (MER044553), nonpeptidase homolog chromosome 21 open reading frame 33 (Homo sapiens) (MER160094), family C56 nonpeptidase homolog (MER177016), family C56 nonpeptidase homolog (MER176613), family C56 Non-peptidase homolog (MER176918), mucin-like hormone receptor-like 2 containing EGF-like module (MER037230), CD97 antigen (human) (MER037286), mucin-like hormone receptor-like 3 containing EGF-like module (MER037288), mucin-like hormone receptor-like 1 containing EGF-like module (MER037278), mucin-like hormone receptor-like 4 containing EGF-like module (MER037294), EGF-cadherin LAG seven-way G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (Mus musculus) type protein (MER123205), GPR56 (Homo sapiens) type protein (MER122057), spider toxin receptor 2 (MER122199), spider toxin receptor-1 (MER126380), spider toxin receptor 3 (MER124612), protocadherin Flamingo2 (MER124239), ETL protein (MER126267), G protein-coupled receptor 112 (MER126114), seven transmembrane helical receptor (MER125448), Gpr114 protein (MER159320), GPR126 vascular induced G protein-coupled receptor (MER140015), GPR125 (Homo sapiens) type protein (MER159279), GPR116 (Homo sapiens) type G protein-coupled receptor (MER159280),GPR128 (Homo sapiens) type G protein-coupled receptor (MER162015), GPR133 (Homo sapiens) type protein (MER159334), GPR110 G protein-coupled receptor (MER159277), GPR97 protein (MER159322), KPG_006 protein (MER161773), KPG_008 protein (MER161835), KPG_009 protein (MER159335), unspecified homolog (MER166269), GPR113 protein (MER159352), brain-specific angiogenesis inhibitor 2 (MER159746), PIDD autoprocessing protein unit 1 (MER020001), PIDD autoprocessing protein unit 2 (MER063690), MUC1 self-cleaving mucin (MER074260), dystroglycan (MER054741), proprotein convertase 9 (MER022416), site-1 peptidase (MER001948), furin (MER000375), proprotein convertase 1 (MER000376), proprotein convertase 2 (MER000377), proprotein convertase 4 (MER028255), PACE4 proprotein convertase (MER000383), proprotein convertase 5 (MER002578), proprotein convertase 7 (MER002984), tripeptidyl peptidase II (MER000355), subfamily S8A nonpeptidase homolog (MER201339), subfamily S8A nonpeptidase homolog (MER191613), subfamily S8A unspecified Peptidase (MER191611), subfamily S8A unspecified peptidase (MER191612), subfamily S8A unspecified peptidase (MER191614), tripeptidyl peptidase I (MER003575), prolyl oligopeptidase (MER000393), dipeptidyl peptidase IV (eukaryotic) (MER000401), acyl aminoacyl peptidase (MER000408), fibroblast activation protein alpha subunit (MER000399), PREPL A protein (MER004227), dipeptidyl peptidase 8 (MER013484), dipeptidyl peptidase 9 (MER004923), FLJ1 putative peptidase (MER017240), Mername-AA194 putative peptidase (MER017353), Mername-AA195 putative peptidase (MER017367), Mername-AA196 putative peptidase (MER017368), Mername-AA197 putative peptidase (MER017371), C14orf29 protein (MER033244), hypothetical protein (MER033245), putative esterase / lipase / thioesterase (MER047309),Protein bat5 (MER037840), hypothetical protein flj40219 (MER033212), hypothetical protein flj37464 (MER033240), hypothetical protein flj33678 (MER033241), dipeptidyl peptidase homolog DPP6 (MER000403), dipeptidyl peptidase homolog DPP10 (MER005988), protein similar to Mus musculus chromosome 20 open reading frame 135 (MER037845), kynurenine carboxamidase (MER046020), thyroglobulin precursor (MER011604), acetylcholinesterase (MER033188), cholinesterase (MER033198), Carboxylesterase D1 (MER033213), hepatic carboxylesterase (MER033220), carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033226), bile salt-dependent lipase (MER033227), carboxylesterase-related protein (MER033231), neuroligin 3 (MER033232), neuroligin 4, X-linked (MER033235), neuroligin 4, Y-linked (MER033236), esterase D (MER043126), arylacetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone-sensitive lipase (MER033243). 274), neuroligin 1 (MER033280), neuroligin 2 (MER033283), family S9 non-peptidase homolog (MER212939), family S9 non-peptidase homolog (MER211490), subfamily S9C unspecified peptidase (MER192341), family S9 unspecified peptidase (MER209181), family S9 unspecified peptidase (MER200434), family S9 unspecified peptidase (MER209507), family S9 unspecified peptidase (MER209142), serine carboxypeptidase A (MER000430), yolk carboxypeptidase-like protein (MER005492), RISC peptidase (MER0109 60), family S15 unspecified peptidase (MER199442), family S15 unspecified peptidase (MER200437), family S15 unspecified peptidase (MER212825), lysosomal Pro-Xaa carboxypeptidase (MER000446), dipeptidyl peptidase II (MER004952), thymus-specific serine peptidase (MER005538), epoxide hydrolase-like putative peptidase (MER031614), Loc328574-like protein (MER033246), autohydrolase domain-containing protein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm-specific transcription protein (MER199890),Mesoderm-specific transcription protein (MER017123), cytoplasmic epoxide hydrolase (MER029997), cytoplasmic epoxide hydrolase (MER213866), similar to hypothetical protein FLJ22408 (MER031608), CGI-58 putative peptidase (MER030163), Williams-Beuren syndrome critical region protein 21 epoxide hydrolase (MER031610), epoxide hydrolase (MER031612), hypothetical protein 922408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), hypothetical protein (MER033249), valacyclovir hydrolysis enzyme (MER033259), Ccg1 interacting factor b (MER210738), glycosylasparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622), threonine aspartase-1 (MER016969), γ-glutamyltransferase 5 (mammalian type) (MER001977), γ-glutamyltransferase 1 (mammalian type) (MER001629), γ-glutamyltransferase 2 (Homo sapiens) (MER001976), γ-glutamyltransferase-like protein 4 (MER002721), γ-glutamyltransferase-like protein 3 (MER016970), similar to γ-glutamyltransferase 1 precursor ( Homo sapiens) (MER026204), similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026205), Mername-AA211 putative peptidase (MER026207), gamma-glutamyltransferase 6 (MER159283), gamma-glutamyltransferase homolog (chromosome 2, Homo sapiens) (MER037241), polycystin-1 (MER126824), KIAA1879 protein (MER159329), polycystic kidney disease 1-like 3 (MER172554), gamma-glutamyl hydrolase (MER002963), guanine 5″ monophosphate synthase (MER043387), carbamyl phosphate synthase (Homo sapiens type) (MER078 640), dihydroorotase (N-terminal unit) (Homo sapiens) (MER060647), DJ-1 putative peptidase (MER003390), Mername-AA100 putative peptidase (MER014802), Mername-AA101 non-peptidase homolog (MER014803), KIAA0361 protein (Homo sapiens) (MER042827), F1134283 protein (Homo sapiens) (MER044553), non-peptidase homolog chromosome 21 open reading frame 33 (Homo sapiens) (MER160094), family C56 non-peptidase homolog (MER177016), family C56 non-peptidase homolog (MER176613),Family C56 non-peptidase homolog (MER176918), mucin-like hormone receptor-like 2 with EGF-like module (MER037230), CD97 antigen (human) (MER037286), mucin-like hormone receptor-like 3 with EGF-like module (MER037288), mucin-like hormone receptor-like 1 with EGF-like module (MER037278), mucin-like hormone receptor-like 4 with EGF-like module (MER037294), cadherin EGF LAG seven-way G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (Mus musculus) type protein (MER123205), GPR56 (Homo sapiens) type protein (MER122057), spider toxin receptor 2 (MER122199), spider toxin receptor-1 (MER126380), spider toxin receptor 3 (MER124612), protocadherin Flamingo 2 (MER124239), ETL protein (MER126267), G protein-coupled receptor 112 (MER126114), seven transmembrane helical receptor (MER125448), Gpr114 protein (MER159320), GPR126 vascular-inducible G protein-coupled receptor (MER140015), GPR125 (Homo sapiens) type protein (MER159279), GPR116 (Homo sapiens) type G protein-coupled receptor (MER159280), GPR128 (Homo sapiens) type G protein-coupled receptor (MER162015), GPR133 (Homo sapiens) type protein (MER159334), GPR110 G protein-coupled receptor (MER159277), GPR97 protein (MER159322), KPG_006 protein (MER161773), KPG_008 protein (MER161835), KPG_009 protein (MER159335), unspecified homolog (MER166269), GPR113 protein (MER159352), brain-specific angiogenesis inhibitor 2 (MER159746), PIDD autologous processing protein unit 1 (MER020001), PIDD autologous processing protein unit 2 (MER063690), MU C1 self-cleaving mucin (MER074260), dystroglycan (MER054741), proprotein convertase 9 (MER022416), site-1 peptidase (MER001948), furin (MER000375), proprotein convertase convertase 1 (MER000376), proprotein convertase 2 (MER000377), proprotein convertase 4 (MER028255), PACE4 proprotein convertase (MER000383), proprotein convertase 5 (MER002578), proprotein convertase 7 (MER002984),Tripeptidyl peptidase II (MER000355), subfamily S8A non-peptidase homolog (MER201339), subfamily S8A non-peptidase homolog (MER191613), subfamily S8A unspecified peptidase (MER191611), subfamily S8A unspecified peptidase (MER191612), subfamily S8A unspecified peptidase (MER191614), tripeptidyl peptidase I (MER003575), prolyl oligopeptidase (MER000393), dipeptidyl peptidase IV (eukaryotic) (MER000401), acylaminoacyl peptidase (MER000408), fibroblast activation protein alpha subunit (MER000399), PREPL Protein A (MER004227), dipeptidyl peptidase 8 (MER013484), dipeptidyl peptidase 9 (MER004923), FLJ1 putative peptidase (MER017240), Mername-AA194 putative peptidase (MER017353), Mername-AA195 putative peptidase (MER017367), Mername-AA196 putative peptidase (MER017368), Mername-AA197 putative peptidase (MER017371) , C14orf29 protein (MER033244), hypothetical protein (MER033245), hypothetical esterase / lipase / thioesterase (MER047309), protein bat5 (MER037840), hypothetical protein flj40219 (MER033212), hypothetical protein flj37464 (MER033240), hypothetical protein flj33678 (MER033241), dipeptidyl peptidase homolog DPP6 (MER000403), dipeptidyl peptidase homolog DP P10 (MER005988), protein similar to Mus musculus chromosome 20 open reading frame 135 (MER037845), kynurenine carboxamidase (MER046020), thyroglobulin precursor (MER011604), acetylcholinesterase (MER033188), cholinesterase (MER033198), carboxylesterase D1 (MER033213), liver carboxylesterase (MER033220), carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033234). 226), bile salt-dependent lipase (MER033227), carboxylesterase-related protein (MER033231), neuroligin 3 (MER033232), neuroligin 4X-linked (MER033235), neuroligin 4Y-linked (MER033236), esterase D (MER043126), arylacetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone-sensitive lipase (MER033274),Neuroligin 1 (MER033280), Neuroligin 2 (MER033283), Family S9 non-peptidase homolog (MER212939), Family S9 non-peptidase homolog (MER211490), Subfamily S9C unspecified peptidase (MER192341), Family S9 unspecified peptidase (MER209181), Family S9 unspecified peptidase (MER200434), Family S9 unspecified peptidase (MER209507), Family S9 unspecified peptidase (MER209142), Serine carboxypeptide Enzyme A (MER000430), yolk carboxypeptidase-like protein (MER005492), RISC peptidase (MER010960), family S15 unspecified peptidase (MER199442), family S15 unspecified peptidase (MER200437), family S15 unspecified peptidase (MER212825), lysosomal Pro-Xaa carboxypeptidase (MER000446), dipeptidyl peptidase II (MER004952), thymus-specific serine peptidase (MER005538), putative epoxide hydrolase-like Peptidase (MER031614), Loc328574-like protein (MER033246), autohydrolase domain-containing protein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm-specific transcription protein (MER199890), mesoderm-specific transcription protein (MER017123), cytoplasmic epoxide hydrolase (MER029997), cytoplasmic epoxide hydrolase (MER213866), similar to hypothetical protein FLJ22408 (MER031608), C GI-58 putative peptidase (MER030163), Williams-Beuren syndrome critical region protein 21 epoxide hydrolase (MER031610), epoxide hydrolase (MER031612), hypothetical protein flj22408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), hypothetical protein (MER033249), valacyclovir hydrolase (MER033259), Ccg1 interacting factor b (MER210738).

[0221] The enzymatic activity of a protease can be regulated. For example, certain proteases can be inactivated by the presence or absence of a specific reagent (e.g., a reagent that binds to the protease, such as a specific small molecule inhibitor). Such proteases can be referred to as "repressible proteases." Exemplary inhibitors of certain proteases are listed in Table 4B. For example, the NS3 protease can be repressed by protease inhibitors including, but not limited to, simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovarprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiprevir. In another example, the protease catalytic activity can be regulated by regulating the expression of the protease itself, such as by engineering cells to express the protease using an inducible promoter system (e.g., Tet On / Off system) or a cell-specific promoter (promoters that can be used to express heterologous proteases are described in more detail in the section entitled "Promoters" herein). The protease can also comprise a degron, such as any degron described herein, and can be regulated using any degron system described herein.

[0222] The enzymatic activity of protease can also be regulated by selecting specific protease cleavage site.For example, protease cleavage site can be selected and / or engineered so that the sequence is shown by the desired rate of protease cleavage, such as the cutting kinetics that the endogenous sequence of the substrate of the desired protease natural cleavage is reduced. As another example, protease cleavage site can be selected and / or engineered so that the sequence is shown by the desired cleavage rate in a cell state specific manner. For example, various cell states (for example, after cell signal conduction such as immune cell activation) can affect the expression and / or location of some protease. As an illustrative example, known ADAM17 protein level and location are affected by signal conduction, for example, by protein kinase C (PKC) signal transduction pathway (for example, the activation carried out by PKC activator phorbol-12-myristate-13-acetate [PMA]). Therefore, protease cleavage site can be selected and / or engineered so that the cutting of protease cleavage site and the release of effector molecule subsequently are increased or reduced as required, depending on the protease characteristic (for example, expression and / or location) under the specific cell state. As another example, protease cleavage sites, particularly in combination with specific membrane tethering domains, can be selected and / or engineered to achieve optimal protein expression of the chimeric protein.

[0223] membrane tethering domain

[0224] The membrane-cleavable chimeric protein provided herein comprises a cell membrane tethering domain (referred to as "MT" in the formula S-C-MT or MT-C-S). Generally speaking, the cell membrane tethering domain can be any amino acid sequence motif that can direct the chimeric protein to be localized to the cell membrane of the cell expressing the chimeric protein (e.g., inserted into the cell membrane) or otherwise connected thereto. 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 comprise a post-translational modification tag or a motif that can modify the chimeric protein by post-translational modification to comprise a post-translational modification tag, wherein the post-translational modification tag allows connection to the cell membrane. Examples of post-translational modification tags include, but are not limited to, lipid anchor domains (e.g., GPI lipid anchors, myristoylation tags, or palmitoylation tags). Examples of cell membrane tethered domains include, but are not limited to, transmembrane-intracellular domains and / or transmembrane domains derived from PDGFR-β, CD8, CD28, CD3ζ chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, or BTLA. The cell membrane tethered domain can be a cell surface receptor or a cell membrane binding portion thereof.

[0225] 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

[0226] LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 204). In some embodiments, the B7-1 transmembrane domain is composed of a sequence having

[0227] TTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 252). In some embodiments, the B7-1 transmembrane domain is composed of

[0228] TTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 252) 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.

[0229] In some embodiments, the cell membrane tethering domain of the membrane cleavable chimeric protein (e.g., IL-15) comprises 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) comprises the sequence

[0230] LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 204).

[0231] In some embodiments, the B7-1 transmembrane domain of the membrane cleavable chimeric protein (e.g., IL-15) is composed of a protein having the sequence

[0232] The polynucleotide sequence of TTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 252) encodes. 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 TTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 252).

[0233] In some embodiments, the cell membrane tethering domain comprises a transmembrane domain derived from a CD8 polypeptide. Any suitable CD8 polypeptide can be used. Exemplary CD8 polypeptides include, but are not limited to, NCBI reference numbers 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

[0234] TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 271). In some embodiments, the CD8 hinge comprises the sequence

[0235] AAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 209).

[0236] In general, for all membrane-cleavable chimeric proteins described herein, the cell membrane tethering domain is either: (1) C-terminal to the protease cleavage site and N-terminal to any intracellular domain, if present (in other words, the cell membrane tethering domain is between the protease cleavage site and the intracellular domain, if present); or (2) N-terminal to the protease cleavage site and C-terminal to any intracellular domain, if present (also between the protease cleavage site and the intracellular domain, if present, but with the domain orientation reversed). In embodiments featuring a degron associated with the chimeric protein, the degron domain is a terminal cytoplasmic-oriented domain, particularly with respect to the cell membrane tethering (in other words, the cell membrane tethering domain is between the protease cleavage site and the degron). The cell membrane tethering domain can be linked to the protease cleavage site via a polypeptide linker (i.e., a polypeptide sequence not typically considered to be part of a cell membrane tethering domain or a protease cleavage site). The cell membrane tethered domain can be connected to the intracellular domain (if present) by a polypeptide linker (i.e., a polypeptide sequence that is not generally considered to be a part of the cell membrane tethered domain or the intracellular domain). The cell membrane tethered domain can be connected to the degron (if present) by a polypeptide linker (i.e., a polypeptide sequence that is not generally considered to be a part of the cell membrane tethered domain or the degron). The polypeptide linker can be any amino acid sequence that connects the first polypeptide sequence and the second polypeptide sequence. The polypeptide linker can be a flexible linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, a GSG linker (e.g., [GS]4GG [SEQ ID NO: 182]), A(EAAAK)3A (SEQ ID NO: 183), and a Whitlow linker (e.g., a "KEGS" 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, which is incorporated herein by reference). Other polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, and SEQ ID NO: 197. Other polypeptide linkers can be selected based on desired properties (e.g., length, flexibility, amino acid composition, etc.) and are known to those skilled in the art.

[0237] Generally speaking, the cell membrane tethering domain is oriented so that the secreted effector molecule and the protease cleavage site are exposed extracellularly after insertion into or association with the cell membrane, such that the protease cleavage site can be cleaved by its corresponding protease and the effector molecule is released ("secreted") into the extracellular space.

[0238] Degron systems and domains

[0239] In some embodiments, any protein described herein can comprise a degron domain, including but not limited to a component of a protease, a transcription factor, a promoter or a promoter system (e.g., ACP) and / or any membrane-cleavable chimeric protein described herein. Generally speaking, a degron domain can be any amino acid sequence motif that can direct regulated degradation (such as regulated degradation by an ubiquitin-mediated pathway). In the presence of an immunomodulatory drug (IMiD), the degron domain directs the degradation of a ubiquitin-mediated degron-fusion protein.

[0240] The degron domain can be a CRBN polypeptide substrate domain capable of binding to hydroxycerebroside (CRBN) in response to an immunomodulatory drug (IMiD), including but not limited to IKZF1, IKZF3, CK1a, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827, or fragments thereof, capable of drug-inducible binding to CRBN. The CRBN polypeptide substrate domain can be a chimeric fusion product of a native CRBN polypeptide sequence, such as an IKZF3 / ZFP91 / IKZF3 chimeric fusion product having the amino acid sequence of FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 175). Degron domains, and in particular the CRBN degron system, are described in more detail in International Application Publication No. WO 2019 / 089592 Al, which is incorporated herein by reference for all purposes.Other examples of degron domains include, but are not limited to, the HCV NS4 degron, PEST (two copies of residues 277-307 of human IκBα; SEQ ID NO: 161), GRR (residues 352-408 of human p105; SEQ ID NO: 162), DRR (residues 210-295 of yeast Cdc34; SEQ ID NO: 163), SNS (tandem repeats 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 repeats of SP1 and SP2 (SP2-SP1-SP2-SP1-SP2 of influenza A M2 protein; SEQ ID NO: 166), NS2 (three copies of residues 79-93 of influenza A NS protein; SEQ ID NO: 167). 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 containing the D433A and D434A point mutations), APC / C degron, COP1 E3 ligase-binding degron motif, CRL4-Cdt2-binding PIP degron, myosin-binding degron, KEAP1-binding degron, KLHL2- and KLHL3-binding degron, MDM2-binding motif, N-degron, hydroxyproline modification in hypoxia signaling, plant hormone-dependent SCF-LRR-binding degron, SCF ubiquitin ligase-binding phosphorylation degron, plant hormone-dependent SCF-LRR-binding degron, DSGxxS phosphorylation-dependent degron, Siah-binding motif, SPOP SBC docking motif or PCNA binding to the PIP box.

[0241] Regulated degradation can be drug-induced. The drug capable of mediating / regulating degradation can be a small molecule compound. The drug capable of mediating / regulating degradation can include "immunomodulatory drugs" (IMiDs). Generally speaking, as used herein, IMiDs refer to a class of small molecule immunomodulatory drugs containing an imide group. Hydroxycerebroside (CRBN) is a known IMiD target, and the binding of IMiDs to CRBN or a CRBN polypeptide substrate domain changes the substrate specificity of the CRBN E3 ubiquitin ligase complex, resulting in degradation of proteins having a CRBN polypeptide substrate domain (e.g., any secretable effector molecule or other protein of interest described herein). For degradation determinant domains having a CRBN polypeptide substrate domain, examples of imide-containing IMiDs include, but are not limited to, thalidomide, lenalidomide, or pomalidomide. IMiDs can be FDA-approved drugs.

[0242] The chimeric proteins described herein may comprise a degron domain (e.g., referred to as "D" in the formula S-C-MT-D or D-MT-C-S of the membrane-cleavable chimeric proteins described herein). In the absence of an IMiD, degron / ubiquitin-mediated degradation of the chimeric protein does not occur. After the chimeric protein is expressed and localized to the cell membrane, the protease cleavage site directs the cleavage of the chimeric protein, resulting in the release ("secretion") of the effector molecule into the extracellular space. In the presence of an immunomodulatory drug (IMiD), the degron domain directs ubiquitin-mediated degradation of the chimeric protein, thereby reducing or eliminating secretion of the effector molecule. Generally speaking, for membrane-cleavable chimeric proteins fused to a degron domain, the degron domain is the terminal cytoplasmic targeting domain, particularly relative to the cell membrane tethering domain, such as the C-terminal domain in the formula S-C-MT-D or the 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., not generally considered to be a part of a cell membrane tethering domain or a degron domain). The polypeptide linker can be any amino acid sequence connecting the first polypeptide sequence and the second polypeptide sequence. The polypeptide linker can be a flexible linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, a GSG linker (e.g., [GS]4GG [SEQ ID NO: 182]), A (EAAAK) 3A (SEQ ID NO: 183), and a Whitlow linker (e.g., a "KEGS" 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 a linker described in more detail in published U.S. Patent No. 5,990,275, which is incorporated herein by reference). Other polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, and SEQ ID NO: 197. Other polypeptide linkers can be selected based on desired properties (e.g., length, flexibility, amino acid composition, etc.) and are known to those skilled in the art. Generally, the degron is oriented relative to the cell membrane tethering domain such that the degron is exposed to the cytoplasm after localization to the cell membrane, such that the degron domain is capable of mediating degradation (e.g., exposed to the cytoplasm and cytoplasm) and is capable of mediating ubiquitin-mediated degradation.

[0243] For degron-fusion protein, the degron domain can be the N-terminal or C-terminal of a protein of interest (e.g., an effector molecule). The degron domain can be connected to a protein of interest by a polypeptide linker (i.e., a polypeptide sequence that is not generally considered to be a part of a protein of interest or a degron domain). A polypeptide linker can be any amino acid sequence connecting a first polypeptide sequence and a second polypeptide sequence. A polypeptide linker can be a flexible linker (e.g., a Gly-Ser-Gly sequence). The example of a polypeptide linker includes, but is not limited to, a GSG linker (e.g., [GS]4GG[SEQ ID NO:182]), A(EAAAK)3A(SEQ ID NO:183) and a Whitlow linker (e.g., "KEGS" linker such as amino acid sequence KESGSVSSEQLAQFRSLD(SEQ ID NO:184), an eGK linker such as amino acid sequence EGKSSGSGSESKST(SEQ ID NO:185) and a linker described in more detail in published U.S. Patent No. 5,990,275, which is incorporated herein by reference). Other polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196 and SEQ ID NO: 197. Other polypeptide linkers can be selected based on desired properties (e.g., length, flexibility, amino acid composition, etc.) and are known to those skilled in the art. The polypeptide linker can be cleavable, for example, by any of the protease cleavage sites described herein.

[0244] Homing molecules

[0245] The "tumor microenvironment" is the cellular environment in which a tumor resides, including surrounding blood vessels, immune cells, fibroblasts, myeloid-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM) (see, e.g., Pattabiraman, DR and Weinberg, RA, Nature Reviews Drug Discovery 13, 497–512 (2014); Balkwill, FR et al., J Cell Sci 125, 5591–5596, 2012; and Li, H. et al., J Cell Biochem 101(4), 805–15, 2007).

[0246] In some embodiments, engineered nucleic acids are configured to produce at least one homing molecule. For example, in the membrane-cleavable chimeric protein comprising secreted effector molecules as described herein, the secreted effector molecules can be homing molecules. "Home" refers to that cells actively navigate (migrate) to a target site (e.g., cells, tissues (e.g., tumors) or organs). "Homing molecules" refer to molecules that guide cells to a target site. In some embodiments, the function of a homing molecule is to identify and / or trigger the interaction of engineered cells with a target site. Non-limiting examples of homing molecules include CXCR1, CCR9, CXCR2, CXCR3, CXCR4, CCR2, CCR4, FPR2, VEGFR, IL6R, CXCR1, CSCR7, and PDGFR.

[0247] In some embodiments, the homing molecule is a chemokine receptor (a cell surface molecule that binds to a chemokine). Chemokines are small molecule cytokines or signaling proteins secreted by cells that can induce directional chemotaxis in cells. Chemokines can be divided into four main subfamilies: CXC, CC, CX3C, and XC, all of which play a biological role by selectively binding to chemokine receptors located on the surface of target cells. In some embodiments, the engineered nucleic acid is configured to produce CXCR4, which is a chemokine receptor that allows engineered cells to home along a chemokine gradient toward cells, tissues, or tumors expressing stromal cell-derived factor 1 (also known as SDF1, CXC motif chemokine 12, and CXCL12). Non-limiting examples of chemokine receptors that can 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 bound to CX3CL1), and XC chemokine receptors (e.g., XCR1). In some embodiments, the chemokine receptor is a member of the G protein-coupled transmembrane receptor or tumor necrosis factor (TNF) receptor superfamily (including but not limited to TNFRSF1A, TNFRSF1B). In some embodiments, the engineered nucleic acids are configured to produce CXCL8, CXCL9, and / or CXCL10 (promoting T cell recruitment), CCL3, and / or CXCL5, CCL21 (Th1 recruitment and polarization).

[0248] In some embodiments, the engineered nucleic acid is configured to produce a G protein coupled receptor (GPCR) (including but not limited to FPR2 and FPRL1) that detects an N-formylation containing oligopeptide.

[0249] In some embodiments, the engineered nucleic acid is configured to produce a receptor that detects an interleukin (including but not limited to IL6R).

[0250] In some embodiments, the 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).

[0251] In some embodiments, the homing molecule is an integrin. Integrins are transmembrane receptors that promote cell-extracellular matrix (ECM) adhesion. Integrins are obligate heterodimers with two subunits: α (alpha) and β (beta). The α subunit of integrin can be, but is not limited to: ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, IGTA7, ITGA8, ITGA9, IGTA10, IGTA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGA2B, ITGAX. The β subunit of integrin can be, but is not limited to: ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, and ITGB8. Engineered nucleic acids can be configured to produce any combination of integrin α and β subunits.

[0252] In some embodiments, the homing molecule is a matrix metalloproteinase (MMP). MMP is an enzyme that cuts the basement membrane components below the endothelial cell wall. Non-limiting examples of MMP include MMP-2, MMP-9, and MMP. In some embodiments, the engineered nucleic acid is configured to produce an inhibitor of a molecule (e.g., protein) that inhibits MMP. For example, the engineered nucleic acid can be configured to express an inhibitor (e.g., RNAi molecule) of membrane type 1 MMP (MT1-MMP) or TIMP metallopeptidase inhibitor 1 (TIMP-1).

[0253] In some embodiments, the homing molecule is a ligand that binds to a selectin on, for example, the endothelium of the target tissue (e.g., hematopoietic cell E- / L-selectin ligand (HCELL), Dykstran et al., Stem Cells. 2016 Oct;34(10):2501-2511).

[0254] The term "homing molecule" also encompasses transcription factors that modulate the production of molecules that improve / enhance cell homing.

[0255] In some embodiments, the homing molecule comprises an antibody, such as anti-integrin α4, β7 or anti-MAdCAM.

[0256] Chimeric Antigen Receptor (CAR)

[0257] Certain aspects of the present disclosure relate to chimeric receptors having any of the antigen-specific binding domains described herein (e.g., EMCN-specific, FLT3-specific, and / or CD33-specific binding domains) and capable of specifically binding to a protein, antigenic antigen, or antigenic epitope.

[0258] In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). In general, CAR is a chimeric protein, including an antigen binding domain and a polypeptide molecule heterologous to the antigen binding domain (such as a peptide heterologous to an antibody from which the antigen binding domain can be derived). The polypeptide molecule heterologous to the antigen binding domain can include but is not limited to a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer, one or more peptide linkers, or a combination thereof.

[0259] In some embodiments, CAR is an engineered receptor that is grafted with or imparts the specificity of a concern (e.g., EMCN, FLT3, or CD33) to an immune effector cell. In certain embodiments, CAR can be used to transplant the specificity of an antibody onto an immune response cell such as a T cell. In some embodiments, CAR of the present disclosure includes an extracellular antigen binding domain (e.g., scFv) fused to a transmembrane domain and fused to one or more intracellular signaling domains.

[0260] In some embodiments, the chimeric antigen receptor is an activating chimeric antigen receptor (aCAR, and unless otherwise specified, is also generally referred to as CAR). In some embodiments, the combination of the chimeric antigen receptor and its cognate ligand is sufficient to induce the activation of immunoreactive cells. In some embodiments, the combination of the chimeric antigen receptor and its cognate ligand is sufficient to induce immunoreactive cell stimulation. In some embodiments, the activation of immunoreactive cells leads to the killing of target cells. In some embodiments, the activation of immunoreactive cells leads to the expression and / or secretion of cytokines or chemokines by immunoreactive cells. In some embodiments, the stimulation of immunoreactive cells leads to the expression and / or secretion of cytokines or chemokines by immunoreactive cells. In some embodiments, the stimulation of immunoreactive cells leads to the differentiation of immunoreactive cells. In some embodiments, the stimulation of immunoreactive cells induces the proliferation of immunoreactive cells. In some embodiments, the activation and / or stimulation of immunoreactive cells can be a combination of the above reactions.

[0261] The number of ABDs in a binding molecule (such as a chimeric protein as described herein) defines the "valence" of the binding molecule. A binding molecule with a single ABD is "monovalent." A binding molecule with multiple ABDs is referred to as "multivalent." A multivalent binding molecule with two ABDs is "bivalent." A multivalent binding molecule with three ABDs is "trivalent." A multivalent binding molecule with four ABDs is "tetravalent." In various multivalent embodiments, all multiple ABDs have the same recognition specificity and can be referred to as "monospecific multivalent" binding molecules. In other multivalent embodiments, at least two of the multiple ABDs have different recognition specificities. Such binding molecules are multivalent and "multispecific." In a multivalent embodiment in which ABDs have two recognition specificities in common, the binding molecule is "bispecific." In a multivalent embodiment in which ABDs have three recognition specificities in common, the binding molecule is "trispecific." In a multivalent embodiment in which ABDs have multiple recognition specificities for different epitopes present on the same antigen in common, the binding molecule is "multiparatopic." Multivalent embodiments in which the ABD jointly recognize two epitopes on the same antigen are "biparatopic."

[0262] In various multivalent embodiments, the multivalency of the binding molecule improves the affinity of the binding molecule for a specific target. As used herein, "avidity" refers to the total strength of the interaction between two or more molecules (e.g., a multivalent binding molecule for a specific target), wherein the avidity is the cumulative strength of the 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 affinity of a binding molecule for a specific target is such that the interaction is a specific binding interaction, wherein the avidity between the two molecules has a value of less than 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 In some 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 affinities of the individual ABDs do not have KD values ​​consistent with specific binding of their respective antigens or epitopes to themselves. In some embodiments, 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 present on a single cell). In some embodiments, avidity is the cumulative strength of interaction provided by the affinities of multiple ABDs for separate epitopes on a shared single antigen.

[0263] In some embodiments, aCAR can be a bivalent bispecific CAR.In some embodiments, aCAR can be a bivalent bispecific CAR expressed on a cell (for example, an immune response cell) disclosed herein, as an OR logic gate (for example, an OR logic gate targeting multiple tumor targets) for increasing the potential target of an activation chimeric receptor. Bivalent bispecific aCAR can include antigen binding domains specific to FLT3 and antigen binding domains specific to CD33. Bivalent bispecific aCAR can include i) antigen binding domains specific to FLT3; (ii) antigen binding domains specific to CD33; (iii) one or more intracellular signaling domains stimulating an immune response; and (iv) one or more polypeptides including but not limited to the following: signal peptide, transmembrane domain, hinge domain, spacer, one or more peptide linkers and combinations thereof.

[0264] The CARs of the present disclosure may be first generation, second generation, or third generation CARs. A "first generation" CAR includes a single intracellular signaling domain typically derived from a T cell receptor chain. A "first generation" CAR typically has an intracellular signaling domain from the CD3-ζ (CD3ζ) chain, which is the primary transmitter of signals from endogenous TCRs. A "first generation" CAR may provide de novo antigen recognition and induce CD4 + T cells and CD8 + T cells are activated by their CD3 ζ chain signaling domains in a single fusion molecule, independent of HLA-mediated antigen presentation. " Second generation " CAR adds the second intracellular signaling domain from one of a variety of costimulatory molecules (such as CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of CAR, so that other signals are provided to T cells. " Second generation " CAR provides costimulation (for example, CD28 or 4-1BB) and activation (CD3 ζ). Preclinical studies have shown that " second generation " CAR can improve the anti-tumor activity of immunoreactive cells (such as T cells). " Third generation " CAR has multiple intracellular costimulatory signaling domains (for example, CD28 and 4-1BB) and intracellular activation signaling domains (CD3 ζ).

[0265] In some embodiments, the chimeric antigen receptor is a chimeric inhibitory receptor (iCAR). In some embodiments, the one or more chimeric inhibitory receptors bind to an antigen expressed on a non-tumor cell, and the non-tumor cell is derived from a tissue selected from the group consisting of brain, neuronal tissue, endocrine tissue, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gall bladder, pancreas, gastrointestinal tract, kidney, bladder, male reproductive organs, female reproductive organs, fat, soft tissue and skin.

[0266] In some embodiments, a chimeric inhibitory receptor (e.g., an EMCN-specific chimeric inhibitory receptor) can be used, for example, together with one or more activating chimeric receptors (e.g., activating chimeric TCR or CAR, such as FLT3 and / or CD33aCAR) expressed on a cell of the present disclosure (e.g., an immunoreactive cell), as a non-logic gate that controls, regulates, or inhibits one or more activities of one or more activating chimeric receptors. For example, if healthy cells express both an antigen recognized by a tumor-targeted chimeric receptor and an antigen recognized by a chimeric inhibitory receptor, the immunoreactive cells expressing tumor antigens can bind to healthy cells. In this case, the inhibitory chimeric antigen will also bind to the cognate ligand on the healthy cell, and the inhibitory function of the chimeric inhibitory receptor will reduce, reduce, prevent, or inhibit the activation of immunoreactive cells ("non-logic gating") by the tumor-targeted chimeric receptor. In some embodiments, a chimeric inhibitory receptor of the present disclosure can inhibit one or more activities of a cell of the present disclosure (e.g., an immunoreactive cell). In some embodiments, the immunoreactive cells may comprise one or more tumor-targeting chimeric receptors and one or more chimeric inhibitory receptors targeting antigens that are not expressed or generally believed to be expressed on tumors (e.g., EMCN). The combination of tumor-targeting chimeric receptors and chimeric inhibitory receptors in the same immunoreactive cell can be used to reduce on-target non-tumor toxicity.

[0267] In some embodiments, the extracellular antigen binding domain of the CAR of the present disclosure is at about 2×10 -7 M or smaller, approximately 1×10 -7 M or smaller, about 9×10 -8 M or smaller, approximately 1×10 -8 M or smaller, about 9×10 -9 M or smaller, about 5×10 -9 M or smaller, approximately 4×10 -9 M or smaller, about 3×10 -9 M or smaller, approximately 2×10 -9 M or smaller or about 1×10 -9 The dissociation constant (K d ) binds to one or more antigens (e.g., EMCN, FLT3, or CD33). In some embodiments, K d The range is from about 2×10 -7 M is about 1×10 -9 M.

[0268] The binding of the extracellular antigen binding domain of the CAR of the present disclosure can be detected by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), biolayer interferometry (e.g., Octet / ), surface plasmon resonance (SPR) technology (e.g., ) or Western blotting assay to measure. Each of these assays is typically detected by using a labeling agent (e.g., antibody or scFv) specific for the relevant complex to detect the presence of a protein-antibody complex of particular interest. For example, scFv can be radiolabeled and used in RIA determinations. Radioisotopes can be detected by means such as using a γ counter or scintillation counter or by automatic radiography. In certain embodiments, the extracellular antigen-binding domains of CAR are 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 domains of CAR are labeled with a second antibody specific for the extracellular antigen-binding domain, and wherein the second antibody is labeled (e.g., radiolabeled or labeled with a fluorescent marker).

[0269] In some embodiments, the CAR of the present disclosure includes an extracellular antigen binding domain that is bound to EMCN, FLT3 or CD33. In some embodiments, the CAR of the present disclosure includes an extracellular antigen binding domain that is bound to EMCN protein, EMCN derived antigen or EMCN derived epitope. In some embodiments, the CAR of the present disclosure includes an extracellular antigen binding domain that is bound to FLT3 protein, FLT3 derived antigen or FLT3 derived epitope. In some embodiments. In some embodiments, the CAR of the present disclosure includes an extracellular antigen binding domain that is bound to CD33 protein, CD33 derived antigen or CD33 derived epitope. The CAR of the present disclosure includes an extracellular antigen binding domain, a transmembrane domain and one or more intracellular signaling domains. In some embodiments, the extracellular antigen binding domain includes scFv. In some embodiments, the extracellular antigen binding domain includes a Fab fragment that may be cross-linked. In certain embodiments, the extracellular binding domain is a F(ab)2 fragment.

[0270] Extracellular antigen-binding domain

[0271] The antigen binding domain of the present disclosure may comprise any domain that binds to an antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, bispecific antibodies, conjugated antibodies, human antibodies, humanized antibodies, and functional fragments thereof, including but not limited to single domain antibodies (sdAb), such as heavy chain variable domains (VH), light chain variable domains (VL) and variable domains (VHH) of camel-derived nanobodies, and alternative scaffolds known in the art that function as antigen binding domains, such as recombinant fibronectin domains, T cell receptors (TCR), recombinant TCRs or fragments thereof with increased affinity, e.g., single chain TCRs, etc. In some cases, it is advantageous for the antigen binding domain to be derived from the same species in which the CAR is ultimately used.

[0272] 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.

[0273] 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.

[0274] In some embodiments, the extracellular antigen binding domain includes an scFv. In some embodiments, the extracellular antigen binding domain includes two single-chain variable fragments (scFv). In some embodiments, each of the two scFv binds to a different epitope on the same antigen. In some embodiments, the extracellular antigen binding domain includes a first scFv and a second scFv. In some embodiments, the first scFv and the second scFv bind to different 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).

[0275] In certain embodiments, the VH and VL are separated by a peptide linker.In certain embodiments, the peptide linker comprises any one of the amino acid sequences shown in Table 6.

[0276] In certain embodiments, scFv comprises the structure VH-L-VL or VL-L-VH, wherein VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain. In some embodiments, one or more scFvs each comprise the structure VH-L-VL or VL-L-VH, wherein VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain. When two or more scFvs are linked together, each scFv can be connected to the next scFv by a peptide linker. In some embodiments, one or more scFvs are each separated by a peptide linker.

[0277] In some embodiments, the peptide linker includes the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 244). In some embodiments, the peptide linker between the antigen binding domains of iCAR includes the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 244). In some embodiments, the peptide linker is encoded by a polynucleotide sequence comprising the sequence GGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCT (SEQ ID NO: 253). In some embodiments, the nucleic acid encoding the peptide linker includes a sequence 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).

[0278] In some embodiments, the peptide linker includes the amino acid sequence GGGGS (SEQ ID NO: 242). In some embodiments, the peptide linker between the antigen binding domains of aCAR includes the amino acid sequence GGGGS (SEQ ID NO: 242). In some embodiments, the 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, the nucleic acid encoding the 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).

[0279] In some embodiments, the peptide linker includes the amino acid sequence GGGGSGGGGS (SEQ ID NO: 243). In some embodiments, the peptide linker between the antigen binding domains of aCAR comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 243). In some embodiments, the 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, the nucleic acid encoding the peptide linker comprises 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).

[0280] In some embodiments, the peptide linker includes the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 247). In some embodiments, the peptide linker between the antigen binding domains of aCAR includes the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 247). In some embodiments, the peptide linker is encoded by a polynucleotide sequence comprising the sequence GGCTCTACATCTGGCTCTGGCAAACCTGGAAGCGGCGAGGGATCTACCAAGGGC (SEQ ID NO: 249). In some embodiments, the nucleic acid encoding the 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).

[0281] Table 6. Peptide linkers

[0282] In some embodiments, immune effector cells include 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 suitable antigen binding domains as described herein or known in the art. For example, the first antigen binding domain or the second antigen binding domain can be one or more antibodies, antigen binding fragments of antibodies, F (ab) fragments, F (ab') fragments, single chain variable fragments (scFv) or single domain antibodies (sdAb). In some embodiments, the antigen binding domain of the first chimeric receptor and / or the second chimeric receptor includes two single chain variable fragments (scFv). In some embodiments, each of the two scFvs is bound to different epitopes on the same antigen. In some embodiments, the antigen binding domain of the first chimeric receptor can be specific to EMCN, and the chimeric receptor can be specific to a second different antigen (such as a cancer antigen (for example, an antigen expressed on bone marrow cells (such as AML cells))).

[0283] 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.

[0284] In some embodiments, the CAR of the present disclosure may include 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, the two or more antigen binding domains each bind to the same antigen. In some embodiments, the two or more antigen binding domains each bind to different epitopes of the same antigen. In some embodiments, the two or more antigen binding domains each bind to different antigens.

[0285] In some embodiments, CAR includes two antigen binding domains. In some embodiments, the two antigen binding domains are connected to each other via a flexible joint. In some embodiments, the two antigen binding domains can each be independently selected from antibodies, antigen binding fragments of antibodies, scFv, sdAb, recombinant fibronectin domains, T cell receptors (TCR), recombinant TCR and single-chain TCR with increased affinity. In some embodiments, the CAR including two antigen binding domains is a bispecific CAR or a tandem CAR (tanCAR).

[0286] In certain embodiments, the bispecific CAR or tanCAR include an antigen binding domain including a bispecific antibody or antibody fragment (e.g., scFv). In some embodiments, in each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, VH can be upstream or downstream of VL. In some embodiments, an upstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH1), and its VH is upstream of its VL (VL1), and a downstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL2), and its VL is upstream of its VH (VH2), so that the entire bispecific antibody molecule has an arrangement mode VH1-VL1-VL2-VH2. In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) and its VL (VL1) are arranged together, and its VL is upstream of its VH (VH1), and the downstream antibody or antibody fragment (e.g., scFv) and its VH (VH2) are arranged together, and its VH is upstream of its VL (VL2), so that the entire bispecific antibody molecule has an arrangement VL1VH1-VL2-VH2. In some embodiments, if the construct is arranged as VH1-VL1-VL2-VH2, the linker is arranged between the two antibodies or antibody fragments (e.g., scFv), for example, between VL1 and VL2, or if the construct is arranged as VL1-VH1-VH2-VL2, the linker is arranged between VH1 and VH2. The linker can be a linker as described herein, for example, a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6. In general, the length of the joint between the two scFvs should be sufficient to avoid mismatching between the domains of the two scFvs. In some embodiments, the joint is arranged between the VL and VH of the first scFv. In some embodiments, the joint is arranged between the VL and VH of the second scFv. In the construct with multiple joints, any two or more of the joints can be the same or different. Therefore, in some embodiments, bispecific CAR or tanCAR include VL, VH, and may additionally include one or more joints arranged as described herein.

[0287] In an illustrative, non-limiting example, a CD33 / FLT3 bispecific, bivalent aCAR may have antigen binding domains encoded in the following order: (FLT3-VH)–L1–(CD33-VH)–L2–(CD33-VL)–L3–(FLT3-VL), wherein L1, L2, and L3 are the first, second, and third peptide linkers, respectively. L1, L2, and L3 may be the same peptide linker. L1, L2, and L3 may be the same peptide linker encoded by the same polynucleotide sequence. L1, L2, and L3 may be the same peptide linker encoded by different polynucleotide sequences. L1 and L3 may be the same peptide linker, and L2 may be a different peptide linker. L1 and L3 may be the same peptide linker encoded by the same polynucleotide sequence. L1 and L3 may be the same peptide linker encoded by different polynucleotide sequences. L1, L2, and L3 may each be a different peptide linker.

[0288] CAR transmembrane domain

[0289] In some embodiments, the transmembrane domain of CAR of the present disclosure (e.g., EMCN-specific, FLT3-specific and / or CD33-specific CARs described herein) comprises a hydrophobic alpha helix that spans at least a portion of the cell membrane. It has been demonstrated that different transmembrane domains can produce different receptor stabilities. After antigen recognition, the receptors aggregate and transmit the signal to the cell. In some embodiments, the transmembrane domain of CAR of the present disclosure may include a CD8 polypeptide, a CD28 polypeptide, a CD3-ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a transmembrane domain of a LIR-1 (LILRB1) polypeptide, or may be a synthetic peptide or any combination thereof.

[0290] In some embodiments, the transmembrane domain is derived from a CD8 polypeptide. Any suitable CD8 polypeptide can be used. Exemplary CD8 polypeptides include, but are not limited to, NCBI reference numbers NP_001139345 and AAA92533.1. In some embodiments, the transmembrane domain is derived from a CD28 polypeptide. Any suitable CD28 polypeptide can be used. Exemplary CD28 polypeptides include, but are not limited to, NCBI reference numbers NP_006130.1 and NP_031668.3. In some embodiments, the transmembrane domain is derived from a CD3-ζ polypeptide. Any suitable CD3-ζ polypeptide can be used. Exemplary CD3-ζ polypeptides include, but are not limited to, NCBI reference numbers NP_932170.1 and NP_001106862.1. In some embodiments, the transmembrane domain is derived from a CD4 polypeptide. Any suitable CD4 polypeptide can be used. Exemplary CD4 polypeptides include, but are not limited to, NCBI reference numbers NP_000607.1 and NP_038516.1. In some embodiments, the transmembrane domain is derived from a 4-1BB polypeptide. Any suitable 4-1BB polypeptide can be used. Exemplary 4-1BB polypeptides include, but are not limited to, NCBI reference numbers NP_001552.2 and NP_001070977.1. In some embodiments, the transmembrane domain is derived from an OX40 polypeptide. Any suitable OX40 polypeptide can be used. Exemplary OX40 polypeptides include, but are not limited to, NCBI reference numbers NP_003318.1 and NP_035789.1. In some embodiments, the transmembrane domain is derived from an ICOS polypeptide. Any suitable ICOS polypeptide can be used. Exemplary ICOS polypeptides include, but are not limited to, NCBI reference numbers NP_036224 and NP_059508. In some embodiments, the transmembrane domain is derived from a CTLA-4 polypeptide. Any suitable CTLA-4 polypeptide can be used. Exemplary CTLA-4 polypeptides include, but are not limited to, NCBI reference numbers 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, but are not limited to, NCBI reference numbers 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, but are not limited to, NCBI reference numbers 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, but are not limited to, NCBI reference numbers NP_057466.1 and NP_061199.2. In some embodiments, the transmembrane domain is derived from a BTLA polypeptide.Any suitable BTLA polypeptide can be used. Exemplary BTLA polypeptides include, but are not limited to, NCBI reference numbers NP_861445.4 and NP_001032808.2. Any suitable LIR-1 (LILRB1) polypeptide can be used. Exemplary LIR-1 (LLRB1) polypeptides include, but are not limited to, NCBI reference numbers NP_001075106.2 and NP_001075107.2.

[0291] In some embodiments, the transmembrane domain comprises a member comprising a member corresponding to NCBI reference numbers 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_00 5205.2, 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 a fragment thereof, 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 NP_05205.2, 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 a fragment thereof. In some embodiments, homology can be determined using standard software such as BLAST or FASTA. In some embodiments, the polypeptide can include 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 may have a NCBI reference number 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_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2, 180, at least 190, at least 200, at least 210, at least 220, at least 230, or at least 240 amino acids in length.

[0292] Additional examples of suitable polypeptides from which transmembrane domains can be derived include, but are not limited to, the transmembrane regions of the α, β, or ζ chains of the T cell receptor, CD27, CD3ε, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, CD2, CD27, LFA-1 (CD11a, CD18), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, I TGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(tactile), CE ACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​PAG / Cbp, NKG2D and NG2C.

[0293] 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).

[0294] In some embodiments, the iCAR transmembrane domain includes a LIR1 transmembrane domain. In some embodiments, the iCAR transmembrane domain includes a LIR1 transmembrane domain having the sequence VIGILVAVILLLLLLLFLI (SEQ ID NO: 259). In some embodiments, the iCAR transmembrane domain includes a LIR1 transmembrane domain encoded by a polynucleotide sequence having the sequence GTGATCGGCATTCTGGTCGCCGTGATCCTGCTCCTGTTGCTCCTGCTGCTTCTGTTCCTGATC (SEQ ID NO: 260). In some embodiments, the iCAR transmembrane domain includes a LIR1 transmembrane domain encoded by a polynucleotide sequence having the sequence GTGATCGGCATTCTGGTCGCCGTGATCCTGCTCCTGTTGCTCCTGCTGCTTCTGTTCCTGATC (SEQ ID NO: 260) 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 GTGATCGGCATTCTGGTCGCCGTGATCCTGCTCCTGTTGCTCCTGCTGCTTCTGTTCCTGATC (SEQ ID NO: 260).

[0295] In some embodiments, the aCAR transmembrane domain includes a CD8 transmembrane domain. In some embodiments, the aCAR transmembrane domain includes a CD8 transmembrane domain having a sequence IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 206). In some embodiments, the aCAR transmembrane domain includes a CD8 transmembrane domain encoded by a polynucleotide sequence having a sequence ATCTATATCTGGGCCCCTCTGGCTGGCACATGCGGAGTTCTGCTGCTCAGCCTGGTCATCACCCTGTACTGCAACCACAGA (SEQ ID NO: 261). In some embodiments, the aCAR transmembrane domain includes a CD8 transmembrane domain encoded by a polynucleotide sequence 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 ATCTATATCTGGGCCCCTCTGGCTGGCACATGCGGAGTTCTGCTGCTCAGCCTGGTCATCACCCTGTACTGCAACCACAGA (SEQ ID NO: 261).

[0296] spacer

[0297] In some embodiments, CAR of the present disclosure (for example, EMCN specificity, FLT3 specificity and / or CD33 specificity CAR as described herein) can also include a spacer, which connects the extracellular antigen-binding domain to the transmembrane domain. The flexibility of the spacer may be sufficient to allow the antigen-binding domains to be oriented in different directions to promote antigen recognition. In some embodiments, the spacer can be a hinge from a human protein. For example, the hinge can be a human Ig (immunoglobulin) hinge, including but not limited to IgG4 hinge, IgG2 hinge, CD8a hinge or IgD hinge. In some embodiments, the spacer can include an IgG4 hinge, IgG2 hinge, IgD hinge, CD28 hinge, KIR2DS2 hinge, LNGFR hinge or PDGFR-β extracellular joint. In some embodiments, the spacer is positioned between the antigen-binding domain and the transmembrane domain. In some embodiments, the spacer can comprise any one 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 one of the amino acid sequences listed in Table 7. In some embodiments, the nucleic acid encoding any one of the spacers of the present disclosure can comprise any one 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 one of the nucleic acid sequences listed in Table 8.

[0298] In some embodiments, the aCAR hinge domain includes a CD8 hinge. In some embodiments, the aCAR hinge domain includes a CD8 hinge having the amino acid sequence ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 272). In some embodiments, the aCAR hinge domain includes a CD8 hinge encoded by a polynucleotide sequence having the sequence GCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTGCCCGCCAAGCCTACAACAACCCCTGCTCCTAGACCACCTACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCCGAAGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCATACAAGAGGACTGGATTTTGCCTGCGAC (SEQ ID NO: 284). In some embodiments, the aCAR hinge domain includes a CD8 hinge encoded by a polynucleotide sequence 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 GCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTGCCCGCCAAGCCTACAACAACCCCTGCTCCTAGACCACCTACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCCGAAGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCATACAAGAGGACTGGATTTTGCCTGCGAC (SEQ ID NO: 284).

[0299] In some embodiments, the iCAR hinge domain includes a CD8 hinge. In some embodiments, the iCAR hinge domain includes a CD8 hinge having an amino acid sequence of TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 271). In some embodiments, the iCAR hinge domain includes a CD8 hinge encoded by a polynucleotide sequence having a sequence of ACAACAACACCCGCACCTCGGCCTCCAACTCCAGCTCCAACAATTGCACTGCAACCCCTGAGTCTGAGGCCCGAGGCCTGTAGGCCAGCAGCTGGCGGAGCTGTTCACACTAGAGGCCTGGACTTTGCCTGTGAC (SEQ ID NO: 283). In some embodiments, the iCAR hinge domain comprises a CD8 hinge encoded by a polynucleotide sequence 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 ACAACAACACCCGCACCTCGGCCTCCAACTCCAGCTCCAACAATTGCACTGCAACCCCTGAGTCTGAGGCCCGAGGCCTGTAGGCCAGCAGCTGGCGGAGCTGTTCACACTAGAGGCCTGGACTTTGCCTGTGAC (SEQ ID NO: 283).

[0300] In some embodiments, the iCAR hinge domain comprises a LIR1 hinge. In some embodiments, the iCAR hinge domain comprises a LIR1 hinge having the amino acid sequence HPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV (SEQ ID NO: 417). In some embodiments, the iCAR hinge domain comprises a LIR1 hinge encoded by a polynucleotide sequence having the sequence CACCCATCCGATCCTCTCGAGCTGGTGGTTTCTGGACCTTCTGGCGGCCCTAGCAGCCCTACAACAGGACCTACAAGCACAAGCGGCCCTGAGGACCAACCTCTGACACCAACAGGCAGCGATCCTCAGTCTGGACTGGGGAGACATCTGGGCGTT (SEQ ID NO: 418). In some embodiments, the iCAR hinge domain comprises a LIR1 hinge encoded by a polynucleotide sequence 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 CACCCATCCGATCCTCTCGAGCTGGTGGTTTCTGGACCTTCTGGCGGCCCTAGCAGCCCTACAACAGGACCTACAAGCACAAGCGGCCCTGAGGACCAACCTCTGACACCAACAGGCAGCGATCCTCAGTCTGGACTGGGGAGACATCTGGGCGTT (SEQ ID NO:418).

[0301] Table 7. Interval amino acid sequences

[0302]

[0303] Table 8. Interval nucleic acid sequences

[0304]

[0305] In some embodiments, the CAR of the present disclosure may further include a short oligopeptide or polypeptide linker having a length between 2 and 10 amino acid residues, and a linkage may be formed 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 includes the amino acid sequence of GGCKJSGGCKJS (SEQ ID NO: 208).

[0306] In some aspects, the transmembrane domain also comprises at least a portion of the extracellular domain of the same protein.

[0307] Intracellular signaling domain

[0308] In some embodiments, CAR of the present disclosure (e.g., EMCN-specific, FLT3-specific and / or CD33-specific CARs described herein) includes one or more cytoplasmic domains or cytoplasmic regions. The cytoplasmic domain or cytoplasmic region of CAR may include an intracellular signaling domain.

[0309] Examples of suitable intracellular signaling domains that can be used in the CARs of the present disclosure include, but are not limited to, cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act synergistically to regulate signal transduction upon antigen receptor engagement, as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capability.

[0310] Without wishing to be bound by theory, it is believed that the signal generated by TCR alone is not enough to fully activate T cells, and therefore secondary and / or costimulatory signals are generally required to fully activate. Therefore, T cell activation can be mediated by two different types of cytoplasmic signaling sequences, i.e., those sequences that initiate antigen-dependent primary activation by TCR (primary intracellular signaling domain), and those sequences that play a role in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains). In addition, T cell signaling and function (e.g., activation signaling cascades) can be negatively regulated by inhibitory receptors present in T cells through intracellular inhibitory co-signaling domains.

[0311] In some embodiments, the intracellular signaling domain of the CAR of the present disclosure may include an inhibitory intracellular signaling domain. 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, SIRPα, HAVR, GITR, PD-L1, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, CD94, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2 and SIGLEC-10. Tables 10 and 11 provide amino acid sequences and nucleotide sequences of exemplary inhibitory intracellular signaling domains, respectively. In some embodiments, the inhibitory intracellular signaling domain includes one or more intracellular inhibitory co-signaling domains (e.g., see Table 10 and Table 11. In some embodiments, the one or more intracellular inhibitory co-signaling domains are connected to other domains (e.g., transmembrane domains) 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 connected by 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 Inhibitory domain. In some embodiments, the one or more intracellular inhibitory co-signaling domains of the chimeric protein comprise one or more ITIM-containing proteins or fragments thereof. ITIM is a conserved amino acid sequence found in the cytoplasmic tails of many inhibitory immunoreceptors. Examples of ITIM-containing proteins include, but are not limited to, PD-1, TIGIT, BTLA and LIR-1 (LILRB1), TIM3, KIR3DL1, NKG2A, LAG3, LAIR1, SIRPα, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, CD94, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, and SIGLEC-10.

[0312] Table 10 - Amino acid sequences of exemplary inhibitory intracellular signaling domains

[0313]

[0314]

[0315] Table 11 - Nucleic acid sequences of exemplary inhibitory intracellular signaling domains

[0316]

[0317]

[0318]

[0319]

[0320]

[0321] In some embodiments, the iCAR comprises a LIR1 intracellular inhibitory domain. In some embodiments, the iCAR comprises a LIR1 intracellular inhibitory domain having the amino acid sequence LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 285). In some embodiments, the iCAR comprises a sequence consisting of CTGCGGCACAGAAGGCAGGGCAAGCACTGGACAAGCACCCAGAGAAAGGCCGACTTTCAGCATCCTGCTGGCCCGTTGGACCTGAGCCTACAGATAGAGGACTGCAGTGGC GGTCTAGCCCTGCCGCTGATGCCCAAGAGGAAAATCTTTACGCCGCCGTGAAGCACACCCAGCCTGAGGATGGCGTGGAAATGGACACCAGATCTCCCCACGATGAGGACCCTCAGGCCGTGACATACGCAG The polynucleotide sequence of AAGTGAAGCACTCCAGACCTCGGAGAGATGGCAAGCCCTCCATCTCCTCTGAGCGGCGAGTTCCTGGACACCAAAGACAGACAGGCCGAAGAGGACAGACAGATGGATACCGAAGCCGCCGCTTCTGAAGCCCCACAGGATGTGACATATGCCCAGCTGCATAGCCTGACACTGCGGAGAGAAGCCACAGAGCCTCCACCTTCTCAAGAAGGCCCATCTCCTGCCGTGCCTTCCATCTATGCCACTCTGGCCATTCAC (SEQ ID NO: 286) encodes the LIR1 intracellular inhibitory domain.In some embodiments, the iCAR comprises a combination of CTGCGGCACAGAAGGCAGGGCAAGCACTGGACAAGCACCCAGAGAAAGCCCGACTTTCAGCATCCTGCTGGCGCCGTTGGACCTGAGCCTACAGATAGAGGACTGCAGTGGCGG TCTAGCCCTGCCGCTGATGCCCAAGAGGAAAATCTTTACGCCGCCGTGAAGCACACCCAGCCTGAGGATGGCGTGGAAATGGACACCAGATCTCCCCACGATGAGGACCCTCAGGCCGTGACATACGCAGA 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 AGTGAAGCACTCCAGACCTCGGAGAGAGATGGCAAGCCCTCCATCTCCTCTGAGCGGCGAGTTCCTGGACACCAAAGACAGACAGGCCGAAGAGGACAGACAGATGGATACCGAAGCCGCCGCTTCTGAAGCCCCACAGGATGTGACATATGCCCAGCTGCATAGCCTGACACTGCGGAGAGAAGCCACAGAGCCTCCACCTTCTCAAGAAGGCCCATCTCCTGCCGTGCCTTCCATCTATGCCACTCTGGCCATTCAC (SEQ ID NO: 286) encodes a LIR1 intracellular inhibitory domain.

[0322] In some embodiments, the one or more intracellular inhibitory co-signaling domains comprise one or more non-ITIM scaffold proteins or fragments 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.

[0323] The inhibitory intracellular signaling domain may further comprise an enzymatic inhibitory domain. In some embodiments, the enzymatic inhibitory domain comprises an enzymatic catalytic domain. In some embodiments, the enzymatic 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 are given in Pavel Otáhal 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). A more detailed description is provided in the literature (Immunity, 2012 Sep;137(1):1-19), each of which is incorporated herein by reference for all purposes.

[0324] In some embodiments, the intracellular signaling domain of CAR of the present disclosure may include a primary signaling domain, which regulates the primary activation of TCR complexes in a stimulating manner or an inhibitory manner. The primary intracellular signaling domain that plays a role in a stimulating manner can contain a signaling motif, which is referred to as an activation motif (ITAM) based on immunoreceptor tyrosine. The example of a suitable ITAM-containing primary intracellular signaling domain that can be used in CAR of the present disclosure includes but is not limited to CD3-ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), FcεRI, DAP10, DAP12 and CD66d intracellular signaling domain.

[0325] In some embodiments, the CAR of the present disclosure comprises an intracellular signaling domain, such as a primary signaling domain of a CD3-zeta polypeptide. The 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 number NP_932170 or NP_001106864.2 or a fragment thereof. In some embodiments, the CD3-zeta polypeptide may include 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 contiguous 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.

[0326] In other embodiments, the primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain having altered (e.g., increased or decreased) activity compared to a native ITAM domain. In one embodiment, the primary signaling domain comprises a primary intracellular signaling domain comprising a modified ITAM, e.g., a primary intracellular signaling domain comprising an optimized and / or truncated ITAM. In one embodiment, the primary signaling domain comprises one, two, three, four, or more ITAM motifs.

[0327] In some embodiments, the intracellular signaling domain of the CAR of the present disclosure may itself include a CD3-ζ signaling domain or it may be combined with any other desired intracellular signaling domain that can be used in the case of the CAR of the present disclosure. For example, the intracellular signaling domain of the CAR may include a CD3-ζ chain portion and a costimulatory signaling domain. The costimulatory signaling domain may refer to a portion of a CAR that includes an intracellular domain of a costimulatory molecule. The costimulatory molecules of the present disclosure are cell surface molecules other than antigen receptors or their ligands that may be required for the effective response of lymphocytes to antigens. Examples of suitable costimulatory molecules include but are not limited to CD97, CD2, ICOS, CD27, CD154, CD8, OX40, 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 ligands that specifically bind to CD83, MHC Class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, CDS, ICAM-1, (CD11a / CD18), BAFFR, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGA X, CD11c, 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, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, etc.

[0328] In some embodiments, the aCAR intracellular signaling domain includes a CD28 costimulatory domain. In some embodiments, the aCAR intracellular signaling domain includes a CD28 costimulatory domain having an amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 287). In some embodiments, the aCAR intracellular signaling domain includes a CD28 costimulatory domain encoded by a polynucleotide sequence comprising the sequence AGAAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCCGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGATTTCGCCGCCTACCGGTCC (SEQ ID NO: 288). In some embodiments, the aCAR intracellular signaling domain includes a CD28 costimulatory domain encoded by a polynucleotide sequence 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 AGAAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCCGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGATTTCGCCGCCTACCGGTCC (SEQ ID NO: 288).

[0329] In some embodiments, the aCAR intracellular signaling domain comprises a CD3 zeta signaling domain. In some embodiments, the aCAR intracellular signaling domain comprises a CD3 zeta signaling domain having the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 289). In some embodiments, the aCAR intracellular signaling domain includes a CD3 zeta signaling domain encoded by a polynucleotide sequence having the sequence AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 290).In some embodiments, the aCAR intracellular signaling domain comprises a motif consisting of: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO:290) 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 a polynucleotide sequence encoded by a CD3 zeta signaling domain.

[0330] In some embodiments, the aCAR intracellular signaling domain includes a CD28 costimulatory domain and a CD3 zeta signaling domain. In some embodiments, the aCAR intracellular signaling domain includes a CD28 costimulatory domain having the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 287) and a CD3 zeta signaling domain having the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 289).

[0331] In some embodiments, the intracellular signaling sequences in the cytoplasmic portion of the CAR of the present disclosure can be connected to each other in a random or specified order. In some embodiments, for example, a short oligopeptide or polypeptide linker of length between 2 amino acids and 10 amino acids (for example, 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) can form a linkage between intracellular signaling sequences. In one embodiment, glycine-serine doublets can be used as suitable joints. In one embodiment, a single amino acid, such as alanine or glycine, can be used as a suitable joint.

[0332] In some embodiments, the intracellular signaling domain includes two or more costimulatory signaling domains, for example, 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 includes two costimulatory signaling domains. In some embodiments, two or more costimulatory signaling domains are separated by a linker of the present disclosure (for example, any one 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.

[0333] In some embodiments, the cells of the present disclosure express a CAR that includes an antigen binding domain, a transmembrane domain, a primary signaling domain, and one or more costimulatory signaling domains.

[0334] 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 comprises a transmembrane domain and at least one intracellular inhibitory co-signaling domain, each intracellular inhibitory co-signaling domain is derived from a protein selected from the group consisting of PD-1, CTLA4, TIGIT, BTLA and LIR1 (LILRB1), TIM3, KIR3DL1, NKG2A, LAG3, SLAP1, SLAP2, Dok-1, Dok-2, LAIR1, GRB-2, CD200R, SIRPα, HAVR, GITR, PD-L1, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, CD94, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, and SIGLEC-10.

[0335] 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 is different from the first protein.

[0336] Natural Killer Cell Receptor (NKR) CAR

[0337] In some embodiments, the CAR of the present disclosure comprises one or more components of a natural killer cell receptor (NKR), thereby forming a NKR-CAR. The NKR component can be a transmembrane domain, hinge domain, or cytoplasmic domain from any suitable natural killer cell receptor, including but not limited to killer cell immunoglobulin-like receptors (KIRs), such as KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3 ...4, KIR2DL IR3DL3, KIR2DP1 and KIRSDPI; natural cytotoxicity receptors (NCRs), such as NKp30, NKp44, NKp46; the signaling lymphocyte activation molecule (SLAM) family of immune cell receptors, such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME and CD2F-10; Fc receptors (FcRs), such as CD16 and CD64; and Ly49 receptors, such as LY49A and LY49C. In some embodiments, NKR-CAR can interact with an adapter molecule or an intracellular signaling domain (such as DAP12). Exemplary configurations and sequences of CARs including NKR components are described in International Patent Publication WO2014 / 145252, published on September 18, 2014.

[0338] Immunoreactive cells

[0339] Certain aspects of the present disclosure relate to a cell, such as an immunoreactive cell, that has been genetically engineered to contain 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 to treat myeloid malignancies (e.g., AML).

[0340] 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 is a bone marrow cell, a blood cell, a skin cell, a 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 but are not limited to embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), 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 derived from or differentiated by stem cells of the present disclosure. In some embodiments, the cell is an immune cell. The immune cell of the present disclosure can be separated or differentiated by stem cells of the present disclosure (e.g., by ESC or iPSC). Exemplary immune cells include but are not limited to T cells (e.g., helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, autologous killer T cells, αβT cells and γδT cells), B cells, natural killer (NK) cells, dendritic cells, myeloid cells, macrophages and monocytes. In some embodiments, the cells are neuronal cells. The neuronal cells of the present disclosure can be separated or differentiated by stem cells of the present disclosure (e.g., by ESC or iPSC). Exemplary neuronal cells include but are not limited to 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.

[0341] In some embodiments, the cell is an immune response cell. The immune response cells of the present disclosure can be separated or differentiated by stem cells of the present disclosure (e.g., by ESC or iPSC). Exemplary immune response cells of the present disclosure include, but are not limited to, lymphoid lineage cells. Lymphoid lineages (including B cells, T cells, and natural killer (NK) cells) provide the production of antibodies, the regulation of the cellular immune system, the detection of foreign substances in the blood, the detection of host foreign cells, and the like. Examples of immune response cells of lymphoid lineages include, but are not limited to, T cells, natural killer (NK) cells, embryonic stem cells, pluripotent stem cells, and induced pluripotent stem cells (e.g., those stem cells from which lymphoid cells can be derived or differentiated). T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. In some embodiments, the T cells of the present disclosure can be any type of T cell, including, but not limited to, 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: for example, TEM cells and T EMRA Cells, regulatory T cells (also called suppressor T cells), natural killer T cells, mucosal-associated invariant T cells and gamma delta T cells.Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic cells or tumor cells. The patient's own T cells can be genetically modified to target specific antigens by introducing one or more chimeric receptors (such as chimeric TCR or CAR).

[0342] Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and function during the innate immune response. NK cells do not require prior activation in order to carry out their cytotoxic effects on target cells.

[0343] In some embodiments, the immune response cells of the present disclosure are T cells. The T cells of the present disclosure can be autologous, allogeneic, or derived in vitro from engineered progenitor cells or stem cells.

[0344] In some embodiments, the immune response cells of the present disclosure are universal T cells lacking TCR-α β. In the art, methods for developing universal T cells are described in, for example, Valton et al., Molecular Therapy (2015); 23 9, 1507-1518, and Torikai et al., Blood 2012 119: 5697-5705.

[0345] In some embodiments, the immune response cells of the present disclosure are isolated immune response cells comprising one or more chimeric receptors of the present disclosure. In some embodiments, the immune response cells comprise 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.

[0346] In some embodiments, the immune response cell is a T cell. In some embodiments, the immune response cell is a natural killer (NK) cell.

[0347] In some embodiments, immune response cells express or can express immune receptors.Immunoreceptors are generally capable of inducing changes in signal transduction or protein expression in cells expressing immune receptors, thereby regulating immune responses (e.g., regulation, activation, startup, stimulation, increase, prevention, weakening, suppression, reduction, reduction, obstruction or suppression of immune responses) when bound to cognate ligands. For example, when CD3 chains are present in TCR / CAR clusters in response to ligand binding, a signal transduction cascade mediated by an activation motif (ITAM) based on immunoreceptor tyrosine is generated. Specifically, in certain embodiments, when endogenous TCR, exogenous TCR, chimeric TCR or CAR (particularly activating CAR) are bound to their respective antigens, an immune synapse is formed, and the formation is included in the aggregation of many molecules near the bound receptors (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.). The aggregation of this membrane-bound signaling molecule allows the ITAM motif phosphorylation included in the CD3 chain, which then can start T cell activation pathways and ultimately activate transcription factors (such as NF-κB and AP-1). These transcription factors are able to induce global gene expression in T cells to increase IL-2 production, thereby promoting the proliferation and expression of master regulatory T cell proteins, thereby initiating T cell-mediated immune responses such as cytokine production and / or T cell-mediated killing.

[0348] Cells expressing multiple chimeric receptors

[0349] In some embodiments, the cells of the present disclosure (e.g., immunoreactive cells) include two or more chimeric receptors of the present disclosure. In some embodiments, the cells include two or more chimeric receptors, wherein one of the two or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cells include 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 cells include 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 cells include five or more chimeric receptors, wherein at least one of the five or more chimeric receptors is a chimeric inhibitory receptor.

[0350] In some embodiments, each of the two or more chimeric receptors comprises a different antigen binding domain, e.g., an antigen binding domain that binds to the same antigen or a different antigen. In some embodiments, each antigen bound by two or more chimeric receptors is expressed on the same cell, such as a bone marrow cell type (e.g., the same AML cell type). In some embodiments, each antigen bound by two or more chimeric receptors is an AML-associated antigen (e.g., FLT3, CD33, CD123, CLEC12A, CXCR4, EphA3, etc.).

[0351] In embodiments where cells of the present disclosure (e.g., immunoreactive cells) express two or more different chimeric receptors, the antigen binding domains of each of the different chimeric receptors may be designed such that the antigen binding domains do not interact with each other. For example, cells of the present disclosure (e.g., immunoreactive cells) express a first chimeric receptor (e.g., an EMCN-specific chimeric receptor) and a second chimeric receptor, and the cell may comprise a first chimeric receptor comprising an antigen binding domain that does not associate 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, and the antigen binding domain of the second chimeric receptor may comprise a VHH.

[0352] Without wishing to be bound by theory, it is believed that in cells having multiple chimeric membrane-embedded receptors each comprising an antigen binding domain, interactions between the antigen binding domains of each receptor may be undesirable because such interactions may inhibit the ability of one or more of the antigen binding domains to bind to their cognate antigens. Thus, in embodiments where cells of the present disclosure (e.g., immune response cells) express two or more chimeric receptors, the chimeric receptors include 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, such as a camel, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence.

[0353] In some embodiments, when present on the surface of a cell, the 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, the 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 the 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 each other to a lesser extent than when both are scFv antigen binding domains. In some embodiments, the antigen binding domains of the first chimeric receptor and the second chimeric receptor associate with each other to a lesser extent than when both are scFv antigen binding domains.

[0354] Chimeric inhibitory receptors

[0355] In some embodiments, cells of the present disclosure (e.g., immunoreactive cells) comprise 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 to an antigen that is typically expressed on normal cells (e.g., cells that are typically considered healthy) but not on tumor cells (such as AML cells). In some embodiments, the chimeric inhibitory receptor comprises an antigen binding domain that binds to EMCN (e.g., an EMCN-specific antigen binding domain having one or more of the amino acid sequences listed in Table 1).

[0356] In some embodiments, the one or more chimeric inhibitory receptors bind to an antigen expressed on a non-tumor cell derived from a tissue selected from the group consisting of brain, neuronal tissue, endocrine tissue, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gall bladder, pancreas, gastrointestinal tract, kidney, bladder, male reproductive organ, female reproductive organ, adipose, soft tissue, and skin.

[0357] In some embodiments, a chimeric inhibitory receptor (e.g., an EMCN-specific chimeric inhibitory receptor) can be used, for example, together 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 immunoreactive cell) as a NOT logic gate that controls, regulates, or inhibits one or more activities of one or more activating chimeric receptors. In some embodiments, a chimeric inhibitory receptor of the present disclosure can inhibit one or more activities of a cell of the present disclosure (e.g., an immunoreactive cell).

[0358] In some embodiments, the cells of the present disclosure comprise one or more chimeric inhibitory receptors of the present disclosure and further comprise a tumor-targeting chimeric receptor that binds to one or more tumor-associated antigens. In some embodiments, the one or more tumor-associated antigens comprise AML-associated antigens. In some embodiments, the one or more tumor-associated antigens comprise CD33. In some embodiments, the one or more tumor-associated antigens comprise FLT3. In some embodiments, the one or more tumor-associated antigens comprise CD33 and FLT3.

[0359] co-stimulatory ligands

[0360] In some embodiments, the cell of the present disclosure (for example, immune response cell) can further include one or more recombinant or exogenous costimulatory ligands.For example, the cell can be further transduced with one or more costimulatory ligands so that the cell co-expresses or is induced to co-express one or more chimeric receptors of the present disclosure (for example, EMCN specificity as described herein, FLT3 specificity and / or CD33 specific CAR) and one or more costimulatory ligands. Without wishing to be bound by theory, it is believed that the interaction between one or more chimeric receptors and one or more costimulatory ligands can provide non-antigen specific signals important for the complete activation of cells. The example of suitable costimulatory ligands includes but is not limited to members of the tumor necrosis factor (TNF) superfamily and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates acute phase reactions. Its main function is to regulate immune cells. Members of the TNF superfamily have many common features. Most TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminal) containing short cytoplasmic segments and relatively long extracellular regions. Examples of suitable TNF superfamily members include, but are not limited to, nerve growth factor (NGF), CD40L (CD40L) / CD154, CD137L / 4-1BBL, TNF-α, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor beta (TNFβ) / lymphotoxin alpha (LTa), lymphotoxin beta (LTP), CD257 / B cell activating factor (BAFF) / Blys / THANK / Tall-1, glucocorticoid-inducible TNF receptor ligand (GITRL) and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF 14). The immunoglobulin (Ig) superfamily is a large group of cell surface soluble proteins that participate in the recognition, binding or adhesion processes of cells. These proteins share structural features with immunoglobulins and have immunoglobulin domains (folds). Examples of suitable immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86 (both ligands of CD28), PD-L1 / (B7-H1), which is a ligand of PD-1. In certain embodiments, the one or more co-stimulatory ligands are selected from 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof.

[0361] Chemokine receptors

[0362] In some embodiments, the cell of the present disclosure (for example, immunoreactive cell) comprises one or more chimeric receptors (for example, EMCN specificity as described herein, FLT3 specificity and / or CD33 specific CAR) and may also include one or more chemokine receptors. For example, the transgenic expression of chemokine receptor CCR2b or CXCR2 in cells such as T cells enhances transportation to solid tumors secreting CCL2 or secreting CXCL1 (Craddock et al., "Journal of Immunotherapy (J Immunother.)" October 2010; 33 (8): 780-8, and Kershaw et al., "Human Gene Ther. (Hum Gene Ther.)" November 1, 2002; 13 (16): 1971-80). Without wishing to be bound by theory, it is believed that the chemokine receptors expressed on cells expressing chimeric receptors disclosed herein can recognize chemokines secreted by tumors and improve cell targeting to tumors, which can promote cell infiltration into tumors and enhance the anti-tumor efficacy of cells. The chemokine receptors of the present disclosure may include naturally occurring chemokine receptors, recombinant chemokine receptors, or chemokine binding fragments thereof. Examples of suitable chemokine receptors that can be expressed on cells of the present disclosure include, but are not limited to, CXC chemokine receptors, such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7; CC chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11; CX3C chemokine receptors, such as CX3CR1; XC chemokine receptors, such as XCR1; and chemokine binding fragments thereof. In some embodiments, the chemokine receptor to be expressed on the cell is selected based on the chemokines secreted by the tumor.

[0363] Chimeric receptor regulation

[0364] Some embodiments of the present disclosure relate to regulating one or more chimeric receptor activities (e.g., EMCN-specific CARs described herein) of cells expressing chimeric receptors of the present disclosure. There are several methods for regulating chimeric receptor activity. In some embodiments, an adjustable chimeric receptor may be required, in which one or more chimeric receptor activities can be controlled to optimize the safety and / or efficacy of chimeric receptor therapy. For example, apoptosis is induced using caspase fused to a dimerization domain (see, e.g., Di et al., New England Journal of Medicine (N Engl. J. Med.) November 3, 2011; 365 (18): 1673-1683) can be used as a safety switch in chimeric receptor therapy. In some embodiments, the cells expressing the chimeric receptor of the present disclosure may also express inducible caspase-9 (iCaspase-9) which induces caspase-9 activation and leads to apoptosis upon administration of a dimeric drug such as rimiducid (IUPAC name: [(1R)-3-(3,4-dimethoxyphenyl)-1-[3-[2-[2-[2-[3-[(1R)-3-(3,4-dimethoxyphenyl)-1-[(2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butyryl]piperidine-2-carbonyl]oxypropyl]phenoxy]acetyl]amino]ethylamino]-2-oxoethoxy]phenyl]propyl](2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butyryl]piperidine-2-carboxylate). In some embodiments, iCaspase-9 contains a binding domain comprising a chemical dimerization inducer (CID), which mediates dimerization in the presence of the CID, resulting in inducible and selective depletion of cells expressing the chimeric receptor.

[0365] Alternatively, in some embodiments, the chimeric receptors of the present invention can be regulated by utilizing small molecules or antibodies that inactivate or otherwise inhibit the activity of the chimeric receptor. For example, antibodies can deplete cells expressing the chimeric receptor by inducing antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, cells expressing chimeric receptors of the present invention can further express antigens recognized by molecules that can induce cell death through ADCC or complement-induced cell death. For example, cells expressing chimeric receptors of the present invention can further express receptors that can be targeted by antibodies or antibody fragments. Examples of suitable receptors that can be targeted by antibodies or antibody fragments include, but are not limited to, EpCAM, VEGFR, integrins (e.g., ανβ3, α4, α13 / 4β3, α4β7, α5β1, ανβ3, αν), TNF receptor superfamily members (e.g., TRAIL-R1 and TRAIL-R2), PDGF receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD 5. CD11, CD11a / 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, as well as truncated forms thereof.

[0366] In some embodiments, the chimeric receptor-expressing cells of the present disclosure may also express a truncated epidermal growth factor receptor (EGFR) that lacks signaling capability but retains an epitope recognized by a molecule capable of inducing ADCC (eg, WO 2011 / 056894).

[0367] In some embodiments, the cells expressing chimeric receptors of the present invention further comprise a highly expressed tight marker / suicide gene that combines target epitopes from both CD32 and CD20 antigens in cells expressing chimeric receptors, thereby binding anti-CD20 antibodies (e.g., rituximab), resulting in selective depletion of cells expressing the chimeric receptors by ADCC. Other methods for depleting cells expressing chimeric receptors of the present invention may include, but are not limited to, administering monoclonal anti-CD52 antibodies that selectively bind to and target cells expressing chimeric receptors to destroy by inducing ADCC. In some embodiments, chimeric receptor ligands such as anti-atopic antibodies may be used to selectively target cells expressing chimeric receptors. In some embodiments, the anti-atopic antibodies can cause effector cell activity, such as ADCC or ADC activity. In some embodiments, the chimeric receptor ligands may be further coupled to agents such as toxins that induce cell killing. In some embodiments, cells expressing chimeric receptors of the present invention may further express target proteins recognized by cell depletion agents of the present invention. 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 as soon as it is desired to reduce or eliminate cells expressing the chimeric receptor. In some embodiments, the cell depleting agent is an anti-CD52 antibody.

[0368] In some embodiments, the regulated chimeric receptor comprises a set of polypeptides in which the components of the chimeric receptor of the present disclosure are distributed among separate polypeptides or members. For example, the set of polypeptides can include a dimerization switch that, in the presence of a dimeric molecule, allows the polypeptides to couple to each other to form a functional chimeric receptor.

[0369] Endothelial mucin-specific antigen-binding domain

[0370] The present disclosure provides an antigen binding domain (e.g., single-chain variable fragment) that is bound to endothelial mucin (EMCN), a chimeric protein (e.g., any one of the iCARs described herein) comprising an antigen binding domain that is bound to EMCN, and nucleic acids encoding such antigen binding domains and chimeric proteins. Without wishing to be bound by theory, EMCN is a sialic acid glycoprotein that interferes with the assembly of focal adhesion complexes and inhibits the interaction between cells and the extracellular matrix. The EMCN-specific antigen binding domain is bound to human EMCN (e.g., Uniprot Q9ULC0, which is incorporated herein by reference for all purposes) or an epitope fragment thereof. EMCN can be expressed on cells that are generally considered healthy, such as healthy hematopoietic stem cells (HSC), HSPC, healthy multipotent progenitor cells (MPP), healthy lympho-myeloid sensitized progenitor cells (LMPP), and healthy hematopoietic progenitor cells (HPC). EMCN can be expressed on hematopoietic stem cells and progenitor cells (HSPC). EMCN can be expressed on HSC. EMCN can be represented on MPP. EMCN can be expressed on LMPP. 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., May 2002, 160(5):1669-1681, which is incorporated herein by reference for all purposes.

[0371] The present disclosure provides an EMCN-specific antigen-binding domain comprising one or more of the amino acid sequences listed in Table 1.

[0372] Table 1.

[0373]

[0374]

[0375] In some embodiments, an antigen binding domain that is 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 complementary determining region 1 (CDR-H1) having an amino acid sequence of RYDMH (SEQ ID NO: 291); a heavy chain complementary determining region 2 (CDR-H2) having an amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 296); and a heavy chain complementary determining region 3 (CDR-H3) having an amino acid sequence of RIKD (SEQ ID NO: 298), and the EMCN-VL comprises a light chain complementary determining region 1 (CDR-L1) having an amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 299); a light chain complementary determining region 2 (CDR-L2) having an amino acid sequence of QVSKLDS (SEQ ID NO: 291). and a light chain complementary 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.

[0376] In some embodiments, an antigen binding domain specific for EMCN comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: EMCN-VH comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQGTMVTVSS (SEQ ID NO: 302); and EMCN-VL comprises the amino acid sequence DVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQGTKLEIK (SEQ ID NO: 310).

[0377] In some embodiments, an antigen binding domain specific for EMCN comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQGTMVTVSSGGGGSGGGGSGGGSDVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQGTKLEIK (SEQ ID NO: 311).In some embodiments, the antigen-binding domain specific for EMCN is encoded by a polynucleotide sequence comprising SEQ ID NO: 312: GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGATACGATATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGTGATCTGGGGCAACGGCAACACACACTACCACAGCGCCCTGAAGTCCCGGTTCACCATCTCCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCACCCTGAGAATCAAGGATTGGGGCCAGGGCACCATGGTCACCGTTTCTTCTGGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCTGACGTGGTCATGACACAGAGCCCTCTGAGCCTGCCTGTGACACTGGGACAGCCTGCCAGCATCAGCTGCAAGTCTAGCCAGTCTCTGGTGGCCAGCGACGAGAACACCTACCTGAACTGGTTCCAGCAGAGGCCCGGACAGTCTCCTAGACGGCTGATCTACCAGGTGTCCAAGCTGGATAGCGGCGTGCCCGATAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGAAGATCAGCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTCTGCAAGGCATCCATCTGCCTTGGACCTTTGGCCAGGGCACAAAGCTGGAAATCAAGIn 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 (SEQ ID NO:312).

[0378] FLT3 and CD33-specific antigen-binding domains

[0379] The present disclosure provides antigen binding domains (e.g., single-chain variable fragments) that bind to FLT3 and CD33, chimeric proteins comprising 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.

[0380] In some embodiments, the chimeric receptor comprises one or more amino acid sequences listed in Table A1 or Table A2. In some embodiments, the activating chimeric receptor (aCAR) comprises one or more amino acid sequences listed in Table A1 or Table A2. In some embodiments, the bispecific bivalent aCAR comprises one or more amino acid sequences listed in Table A1 or Table A2. Table A1 provides the variable domains of antibody heavy or light chains. CDRs are determined using the Kabat method and each variable heavy chain or variable light chain is bold italicized in Table A1 and is shown in Table A2. In some embodiments, the nucleic acid encoding any one of the chimeric receptors of the present disclosure comprises one or more nucleic acid sequences listed in Table B.

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387] In some embodiments, an 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 complementary determining region 1 (CDR-H1) having an amino acid sequence of DYNMH (SEQ ID NO: 402); a heavy chain complementary determining region 2 (CDR-H2) having an amino acid sequence of YIYPYNGGTGYNQKFKSKA (SEQ ID NO: 403); and a heavy chain complementary determining region 3 (CDR-H3) having an amino acid sequence of GRPAMDYWGQ (SEQ ID NO: 404), and the CD33-VL comprises: a light chain complementary determining region 1 (CDR-L1) having an amino acid sequence of RASESVDNYGISFMN (SEQ ID NO: 405). NO:405); a light chain complementary determining region 2 (CDR-L2), the CDR-L2 having the amino acid sequence of AASNQGS (SEQ ID NO:406); and a light chain complementary determining region 3 (CDR-L3), the 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.

[0388] In some embodiments, an antigen binding domain specific for CD33 includes a CD33-VH having the amino acid sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS (SEQ ID NO: 329), and a CD33-VL having the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 330).

[0389] In some embodiments, an 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 complementary determining region 1 (CDR-H1) having an amino acid sequence of GGTFSSYAIS (SEQ ID NO: 360); a heavy chain complementary determining region 2 (CDR-H2) having an amino acid sequence of GIIPIFGTANYAQKFQG (SEQ ID NO: 361); and a heavy chain complementary determining region 3 (CDR-H3) having an amino acid sequence of FALFGFREQAFDI (SEQ ID NO: 362), and the FLT3-VL comprises: a light chain complementary determining region 1 (CDR-L1) having an amino acid sequence of RASQSISSYLN (SEQ ID NO: 363). NO: 363); a light chain complementary determining region 2 (CDR-L2) having the amino acid sequence of AASSLQS (SEQ ID NO: 364); and a light chain complementary 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.

[0390] In some embodiments, an antigen binding domain specific for FLT3 includes a FLT3-VH having the amino acid sequence of EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCATFALFGFREQAFDIWGQGTTVTVSS (SEQ ID NO: 315), and a FLT3-VL having the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQSYSTPFTFGPGTKVDIK (SEQ ID NO: 316).

[0391] In some embodiments, the bivalent bispecific aCAR includes an antigen binding domain specific for FLT3 and an antigen binding domain specific for CD33.

[0392] In some embodiments, a bivalent bispecific aCAR specific for FLT3 and CD33 comprises the amino acid sequence MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCATFALFGFREQAFDIWGQGTTVTVSSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSGSTSGSGKPGSGEGSTKGDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQSYSTPFTFGPGTKVDIKALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 414).

[0393] In some embodiments, a bivalent, bispecific aCAR specific for FLT3 and CD33 comprises the amino acid sequence (SEQ ID NO: 415).

[0394] Engineered nucleic acids

[0395] Provided herein are polycistronic expression systems comprising engineered nucleic acids encoding: (i) a membrane-cleavable chimeric protein; (ii) a bivalent aCAR; and (iii) an iCAR.

[0396] In certain embodiments described herein, the polycistronic expression systems include an engineered nucleic acid encoding an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding each of: (i) a membrane-cleavable chimeric protein; (ii) an aCAR; and (iii) an iCAR.

[0397] The promoter is operably linked to the expression cassette of the polycistronic system such that the exogenous polynucleotide sequence encoding each of the membrane-cleavable chimeric protein, the bivalent aCAR, and the iCAR is constructed to be expressed as a single polypeptide.

[0398] "Engineered nucleic acid" is a nucleic acid that does not exist in nature. However, it should be understood that although engineered nucleic acid as a whole is not naturally occurring, it can include nucleotide sequences that exist in nature. In some embodiments, engineered nucleic acid includes nucleotide sequences from different organisms (e.g., from different species). For example, in some embodiments, engineered nucleic acid includes murine nucleotide sequences, bacterial nucleotide sequences, human nucleotide sequences, and / or viral nucleotide sequences. The term "engineered nucleic acid" includes recombinant nucleic acids and synthetic nucleic acids. "Recombinant nucleic acid" refers to a molecule that is constructed by connecting nucleic acid molecules and can be replicated in living cells in some embodiments. "Synthetic nucleic acid" refers to a molecule that is amplified or chemically synthesized or synthesized by other means. 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 internucleotide bonds and non-natural nucleic acids. Modifications are described in more detail in U.S. Patent No. 6,673,611 and U.S. Application Publication No. 2004 / 0019001, each of which is incorporated by reference in its entirety. The modified internucleotide bond can be a phosphorodithioate or a phosphorothioate bond. Non-natural nucleic acids can be locked nucleic acids (LNA), peptide nucleic acids (PNA), glycol nucleic acids (GNA), phosphorodiamidate morpholino oligomers (PMO or "morpholino") and threose nucleic acids (TNA). Non-natural nucleic acids are further described in detail in International Application WO 1998 / 039352, U.S. Application Publication No. 2013 / 0156849 and U.S. Patent No. 6,670,461; No. 5,539,082; No. 5,185,444, each of which is incorporated herein by reference in its entirety. Recombinant nucleic acids and synthetic nucleic acids also include those molecules produced by replication of any of the foregoing. The engineered nucleic acids of the present disclosure can be encoded by a single molecule (for example, contained in the same plasmid or other vector) or by a plurality of different molecules (for example, a plurality of different independently replicated molecules). Engineered nucleic acids can be isolated nucleic acids. Isolated nucleic acids include, but are not limited to, cDNA polynucleotides, RNA polynucleotides, RNAi oligonucleotides (e.g., siRNA, miRNA, antisense oligonucleotides, shRNA, etc.), mRNA polynucleotides, circular plasmids, linear DNA fragments, vectors, minicircles, ssDNA, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs), and oligonucleotides.

[0399] The engineered nucleic acids of the present disclosure can be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). Cloning generates engineered nucleic acid constructs (see, eg, Gibson, DG et al., Nature Methods, 343-345, 2009; and Gibson, DG et al., Nature Methods, 901-903, 2010, each of which is incorporated herein by reference). GIBSON Typically, three enzymatic activities are used in a single-tube reaction: a 5' exonuclease, a gamma extension activity of a DNA polymerase, and a DNA ligase activity. The 5' exonuclease activity cuts back the 5' end sequence and exposes the complementary sequence for annealing. The polymerase activity then fills the gap in the annealed region. DNA ligase then seals the nick and covalently joins the DNA fragments together. The overlapping sequences of adjacent fragments are much longer than those used in Golden Gate Assembly, thus achieving a higher percentage of correct assembly. In some embodiments, IN- Engineered nucleic acid constructs were generated by cloning (Clontech).

[0400] promoter

[0401] In general, in all embodiments described herein, the engineered nucleic acid encoding membrane-cleavable chimeric protein, bivalent aCAR and iCAR contains an expression cassette containing a promoter. In some embodiments, engineered nucleic acid (for example, an engineered nucleic acid comprising an expression cassette) includes a promoter operably connected to a nucleotide sequence (for example, an exogenous polynucleotide sequence) encoding at least two different proteins. For example, engineered nucleic acid can include a promoter operably connected to a nucleotide sequence encoding at least three, at least four, at least five, at least six, at least seven, at least eight, at least eight, at least nine or at least ten different proteins. In some embodiments, engineered nucleic acid includes a promoter operably connected to a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different proteins. In some embodiments, engineered nucleic acid (for example, an engineered nucleic acid comprising an expression cassette) includes a promoter operably connected to a nucleotide sequence (for example, an exogenous polynucleotide sequence) encoding at least two membrane-cleavable chimeric proteins. For example, the engineered nucleic acid can 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, the 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.

[0402] "Promoter" refers to the control region of a nucleic acid sequence in which the transcription initiation and rate of the remainder of the nucleic acid sequence are controlled. The promoter may also contain subregions to which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. The promoter may be constitutive, inducible, repressible, tissue-specific, or any combination thereof. The promoter drives the expression or transcription of the nucleic acid sequence it regulates. In this article, a promoter is considered to be "operably connected" when it is in the correct functional position and orientation relative to the nucleic acid sequence that it regulates to control ("drive") the transcription initiation and / or expression of the sequence.

[0403] A promoter can be a promoter naturally associated with a gene or sequence, such as one that can be obtained by isolating a 5' non-coding sequence upstream of the coding segment of a given gene or sequence. Such a promoter can be referred to as "endogenous". In some embodiments, the coding nucleic acid sequence can be under the control of a recombinant or heterologous promoter, which refers to a promoter that is not typically associated with a coding sequence in its natural environment. Such promoters can include promoters of other genes; promoters isolated from any other cell; and non-"naturally occurring" synthetic promoters or enhancers, such as promoters or enhancers that contain different elements and / or mutations of different transcriptional regulatory regions that alter expression by genetic engineering methods known in the art. In addition to synthesizing nucleic acid sequences for promoters and enhancers, sequences can also be generated using recombinant cloning and / or nucleic acid amplification techniques, including polymerase chain reaction (PCR) (see, for example, U.S. Patent No. 4,683,202 and U.S. Patent No. 5,928,906).

[0404] The promoter of the engineered nucleic acid can be an "inducible promoter," which refers to a promoter characterized by regulating (e.g., initiating or activating) transcriptional activity in the presence of a signal, the influence of a signal, or contact with a signal. The signal can be an endogenous or normal exogenous condition (e.g., light), a compound (e.g., a chemical or non-chemical compound), or a protein (e.g., a cytokine) that contacts the inducible promoter in a certain manner, thereby actively regulating the transcriptional activity of the inducible promoter. Activation of transcription can involve acting directly on the promoter to drive transcription or indirectly acting on the promoter by inactivating a repressor that prevents the promoter from driving transcription. Conversely, inactivation of transcription can involve acting directly on the promoter to prevent transcription or indirectly acting on the promoter by activating a repressor that then acts on the promoter.

[0405] A promoter is "responsive" or "regulated" by a local tumor condition (e.g., inflammation or hypoxia) or signal if, in the presence of that condition or signal, transcription from the promoter is activated, inactivated, increased, or decreased. In some embodiments, the promoter comprises a response element. A "response element" is a short DNA sequence within the promoter region that binds to a specific molecule (e.g., a transcription factor) that regulates (modulates) gene expression from the promoter. Response elements that can be used in accordance with the present disclosure include, but are not limited to, phloretin regulatable control element (PEACE), zinc finger DNA binding domain (DBD), interferon-gamma activation sequence (GAS) (Decker, T. et al. J Interferon Cytokine Res. 1997 Mar;17(3):121-34, incorporated by reference), interferon stimulated response element (ISRE) (Han, KJ et al. J Biol Chem. 2004 Apr 9;279(15):15652-61, incorporated by reference), NF-κB response element (Wang, V. et al. Cell Reports. 2012;2(4):824-839, incorporated by reference), and STAT3 response element (Zhang, D. et al. J Biol Chem. 1996;271:9503-9509, incorporated 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 the sensitivity of the response element to its cognate binding molecule. Tandem repeats can be labeled 2X, 3X, 4X, 5X, etc. to indicate the number of repeats present.

[0406] Non-limiting examples of responsive promoters (also known as "inducible promoters") (e.g., TGF-β responsive promoters) are listed in Table 5A, which shows the design of the promoter and transcription factor, and shows the effect of the inducer molecule on the transcription factor (TF) and transgene transcription (T) (B, binding; D, dissociation; nd, not determined) (A, activation; DA, inactivation; DR, derepression) (see Horner, M. and Weber, W. FEBS Letters 586 (2012) 20784-2096m, and references cited therein). Non-limiting examples of components of an inducible promoter include those presented in Table 5B.

[0407] Table 5A. Examples of responsive promoters

[0408]

[0409]

[0410] Table 5B. Exemplary components of inducible promoters

[0411]

[0412]

[0413] Non-limiting examples of promoters include the cytomegalovirus (CMV) promoter, the elongation factor 1-alpha (EF1a) promoter, the elongation factor (EFS) promoter, the MND promoter (a synthetic promoter containing the U3 region of a modified MoMuLV LTR with a 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 5C).

[0414] Table 5C. Exemplary constitutive promoters

[0415]

[0416]

[0417]

[0418]

[0419]

[0420] The promoter can be a tissue-specific promoter. Generally, a tissue-specific promoter directs transcription of a nucleic acid (e.g., an engineered nucleic acid encoding a chimeric protein, such as a membrane-cleavable chimeric protein having the formula S-C-MT or MT-C-S)) such that expression is restricted 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), specific promoters for T-cell receptors (Winoto and Baltimore, (1989)) and immunoglobulins; Banerji et al., (1983); Queen and Baltimore, 1983), neuron-specific promoters (e.g., neurofilament promoter; Byrne and Ruddle, 1989), pancreas-specific promoters (Edlund et al., (1985)), or mammary gland-specific promoters (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 2003). 249:374-379 (1990)) or the alpha-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3:537-546 (1989)), the contents of each of which are incorporated herein by reference in their entirety. Promoters can be constitutive in a particular cell type, organelle, or tissue. Tissue-specific promoters and / or regulatory elements may also include a promoter from a liver-type fatty acid binding (FAB) protein gene that is specific for colon epithelial cells; an insulin gene that is specific for pancreatic cells; transthyretin, α1-antitrypsin, type 1 plasminogen activator inhibitor (PAI-I), apolipoprotein AI, and LDL receptor genes that are specific for hepatocytes; myelin basic protein (MBP) genes that are specific for oligodendrocytes; glial fibrillary acidic protein (GFAP) genes that are specific for glial cells; OPSIN that specifically targets the eye; and a neuron-specific enolase (NSE) promoter that is specific for neural cells. Examples of tissue-specific promoters include, but are not limited to, creatine kinase promoters for directing expression in muscle and cardiac tissues and immunoglobulin heavy or light chain promoters for expression in B cells. Other tissue-specific promoters include human smooth muscle α-actin promoters.Exemplary tissue-specific expression elements for the liver include, but are not limited to, the HMG-COA reductase promoter, sterol regulatory element 1, phosphoenolpyruvate carboxykinase (PEPCK) promoter, human C-reactive protein (CRP) promoter, human glucokinase promoter, cholesterol L 7-α hydroxylase (CYP-7) promoter, β-galactosidase α-2,6 sialyltransferase promoter, insulin-like growth factor binding protein (IGFBP-I) promoter, aldolase B promoter, human transferrin promoter, and type I collagen promoter. Exemplary tissue-specific expression elements for the prostate include, but are not limited to, the prostatic acid phosphatase (PAP) promoter, the prostate secretory protein 94 (PSP 94) promoter, the prostate-specific antigen complex promoter, and the 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 α subunit promoter. Exemplary tissue-specific expression elements for the pancreas include, but are not limited to, the pancreatitis-associated protein promoter (PAP), the elastase 1 transcriptional enhancer, the pancreatic-specific amylase and elastase enhancer promoter, and the 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, the cholesterol side chain cleavage (SCC) promoter. Exemplary tissue-specific expression elements for the general nervous system include, but are not limited to, the γ-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-1 / granzyme B promoter, terminal deoxytransferase (TdT), λ5, VpreB, and lck (lymphocyte-specific tyrosine protein kinase p561ck) promoters, the human 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, the pp60c-src tyrosine kinase promoter, the organ-specific neoantigen (OSN) promoter, and the colon-specific antigen-P promoter. Tissue-specific expression elements for mammary cells include, but are not limited to, the human α-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.

[0421] In some embodiments, the promoters disclosed herein are regulated by signals within the tumor microenvironment. A tumor microenvironment is considered to regulate a promoter if, in the presence of a tumor microenvironment, the activity of the promoter increases or decreases by at least 10% relative to the activity of the promoter in the absence of the tumor microenvironment. In some embodiments, the activity of the promoter increases or decreases 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%, or at least 100% relative to the 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 the activity of the promoter in the absence of a tumor microenvironment.

[0422] In some embodiments, the activity of the promoter is increased or decreased by at least 2 times (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100 times) relative to the activity of the promoter in the absence of a tumor microenvironment. For example, the activity of the promoter is increased or decreased by at least 3 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times relative to the activity of the promoter in the absence of a tumor microenvironment. In some embodiments, the activity of the promoter is increased or decreased by 2 to 10 times, 2 to 20 times, 2 to 30 times, 2 to 40 times, 2 to 50 times, 2 to 60 times, 2 to 70 times, 2 to 80 times, 2 to 90 times, or 2 to 100 times relative to the activity of the promoter in the absence of a tumor microenvironment.

[0423] In some embodiments, the promoter of the present disclosure is activated under hypoxic conditions. "Hypoxic conditions" are conditions in which the body or an area of ​​the body lacks sufficient oxygen supply at the tissue level. Hypoxic conditions can lead to inflammation (e.g., inflammatory cytokine levels increase under hypoxic conditions). In some embodiments, the promoter activated under hypoxic conditions is operably linked to a nucleotide encoding a chimeric protein that reduces the expression of inflammatory cytokine activity, thereby reducing the inflammation caused by hypoxic conditions. In some embodiments, the promoter activated under hypoxic conditions includes a hypoxia response element (HRE). "Hypoxia response element (HRE)" is a response element that responds to hypoxia inducible factor (HIF). In some embodiments, HRE comprises a consensus motif NCGTG (wherein N is A or G).

[0424] Conditionally activated control polypeptide (ACP) promoter system

[0425] In some embodiments, the synthetic promoter is a promoter system comprising a conditional activation control polypeptide (ACP) binding domain sequence and a promoter sequence. This system is also referred to as an "ACP response promoter" in this article. Generally speaking, the ACP promoter system comprises a first expression cassette encoding a conditional activation control polypeptide (ACP) and a second expression cassette encoding an ACP response promoter operably connected to an exogenous polynucleotide sequence, such as an exogenous polynucleotide sequence encoding a membrane-cleavable chimeric protein described herein or any other protein of interest (e.g., a protease). In some embodiments, the first expression cassette and the 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 response promoter can be operably connected to a nucleotide sequence encoding a single protein of interest or a plurality of proteins of interest.

[0426] The promoter of the ACP promoter system (e.g., a promoter that drives ACP expression or a promoter sequence of an ACP-responsive promoter) can comprise any promoter sequence described herein (see "Promoter" above). The ACP-responsive promoter can be derived from minP, NFkB response element, CREB response element, NFAT response element, SRF response element 1, SRF response element 2, AP1 response element, TCF-LEF response element promoter fusion, hypoxia response element, SMAD binding element, STAT3 binding site, minCMV, YB-TATA, minTK, inducer molecule response promoter, and tandem repeats thereof. In some embodiments, the ACP-responsive promoter comprises a minimal promoter.

[0427] In some embodiments, the ACP binding domain comprises one or more zinc finger binding sites. In some embodiments, the ACP response promoter comprises a minimal promoter and the ACP binding domain comprises one or more zinc finger binding sites. The ACP binding domain may comprise 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 comprises 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 comprises 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 consists of a zinc finger array (ZFA). The zinc finger array comprises a plurality of zinc finger protein motifs connected together. Each zinc finger motif binds to a different nucleic acid motif. This creates a ZFA with specificity for any desired nucleic acid sequence, for example, a ZFA with desired specificity for an ACP binding domain having a particular 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, the ZFA is an array, string, or chain of ZF motifs arranged in series. The ZFA can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 zinc finger motifs. A ZFA can have 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. A ZF protein domain can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more ZFAs. A ZF domain can have 1-10, 1-15, 1-2, 1-3, 1-

[0428] 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.

[0429] In some embodiments, the ACP is a transcriptional regulator. 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 recognition receptor of the present disclosure.

[0430] ACP can further include effector domains, such as transcription effector domains. For example, the transcription effector domain can be an effector domain or activation domain of a transcription factor. The transcription factor activation domain is also referred to as a transactivation domain and acts as a scaffold domain of a protein, such as a transcription co-regulator for activating or repressing the transcription of a gene. Any suitable transcription effector domain can be used in an ACP, including but not limited to herpes simplex virus protein 16 (VP16) activation domain; an activation domain consisting of four tandem copies of VP16, i.e., VP64 activation domain; p65 activation domain of NFκB; Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising VP64, p65, and Rta activation domains, referred to as a VPR activation domain; the histone acetyltransferase (HAT) core domain of human E1A-associated protein p300, referred to as p300 HAT core activation domain; Krüppel-associated box (KRAB) repressor domain; repressor element silencing transcription factor (REST) ​​repressor domain; WRPW motif of hairy-related basic helix-loop-helix repressor protein, which motif is called WRPW repressor domain; DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repressor domain; and HP1α chromosome shadow repressor domain or any combination thereof.

[0431] In some embodiments, the effector domain is a transcriptional effector domain selected from the group consisting of: the herpes simplex virus protein 16 (VP16) activation domain; an activation domain composed of four tandem copies of VP16, namely the VP64 activation domain; the p65 activation domain of NFκB; the Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, p65, and Rta activation domains, referred to as the VPR activation domain; and the histone acetyltransferase (HAT) core domain of the human E1A-associated protein p300, referred to as the p300 activation domain. The HAT core activation domain; the Krüppel-associated box (KRAB) repressor domain; the repressor element silencing transcription factor (REST) ​​repressor domain; the WRPW motif of the hairy-related basic helix-loop-helix repressor protein, which is called the WRPW repressor domain; the DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repressor domain; and the HP1α chromosome shadow repressor domain.

[0432] In some embodiments, ACP is a small molecule (e.g., drug) inducible polypeptide. For example, in some embodiments, ACP can be induced by tetracycline (or its derivatives) and comprises a TetR domain and a VP16 effector domain. In some embodiments, ACP includes estrogen receptor variants, such as ERT2, and can be regulated by tamoxifen or its metabolites (such as 4-hydroxy-tamoxifen [4-OHT], N-desmethyltamoxifen, tamoxifen-N-oxide or endoxifen) by nuclear localization controlled by tamoxifen.

[0433] In some embodiments, ACP is a small molecule (e.g., drug) inducible polypeptide comprising one or more homologous cleavage sites of a repressible protease and a repressible protease. In some embodiments, the repressible protease is active (cutting the homologous cleavage site) in the absence of a specific reagent, and inactive (not cutting the homologous cleavage site) in the presence of a specific reagent. In some embodiments, the specific reagent is a protease inhibitor. In some embodiments, the protease inhibitor specifically suppresses a given repressible protease of the present disclosure. The repressible protease can be any protease described herein that can be inactivated by the presence or absence of a specific reagent (about exemplary repressible protease, homologous cleavage site and protease inhibitor, referring to "protease cleavage site" above).

[0434] In some embodiments, the ACP has a degron domain (for exemplary degron sequences, see "Degron Systems and Domains"). The degron domains can be in any order or position relative to the individual domains of the ACP. For example, the degron domain can be the N-terminus of a repressor protease, the C-terminus of a repressor protease, the N-terminus of a ZF protein domain, the C-terminus of a ZF protein domain, the N-terminus of an effector domain, or the C-terminus of an effector domain.

[0435] Polycistronic and multi-promoter systems

[0436] In some embodiments, the engineered nucleic acid (e.g., an engineered nucleic acid comprising an expression cassette) is configured to produce a plurality of chimeric proteins. For example, the nucleic acid can be configured to produce 2 to 20 different chimeric proteins. In some embodiments, the nucleic acids are configured to produce 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 20, 4 to 19, 4 to 18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 20, 5 to 19, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 20, 6 to 19, 6 to 18, 6 to 17, 6 to 16, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, 6 to 8 , 6 to 7, 7 to 20, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 7 to 8, 8 to 20, 8 to 19, 8 to 18, 8 to 17, 8 to 16, 8 to 15, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9 to 20, 9 to 19, 9 to 18, 9 to 17, 9 to 16, 9 to 15, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 9 to 10, 10 to 20, 10 to 19 10 to 18, 10 to 17, 10 to 16, 10 to 15, 10 to 14, 10 to 13, 10 to 12, 10 to 11, 11 to 20, 11 to 19, 11 to 18, 11 to 17, 11 to 16, 11 to 15, 11 to 14, 11 to 13, 11 to 12, 12 to 20, 12 to 19, 12 to 18, 12 to 17, 12 to 16, 12 to 15, 12 to 14, 12 to 13, 13 to 20, 13 to 19, 13 to 18, 13 to 17, 13 to 16, 13 to 15, 13 to 14, 14 to 20,In some embodiments, the nucleic acid is 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.

[0437] Generally, the engineered nucleic acids described herein are polycistronic, ie, as described above.

[0438] Engineered nucleic acids can also use multiple promoters to express genes from multiple ORFs, i.e., more than one individual mRNA transcript 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. Generally speaking, any number of promoters can be used to express any number of chimeric proteins. In some embodiments, at least one ORF...

Claims

1. A polycistronic expression system comprising engineered nucleic acid, the engineered nucleic acid comprising: (A) An exogenous polynucleotide encoding a membrane-cleavable chimeric protein whose orientation from the N-terminus to the C-terminus has the following formula: S–C–MT or MT–C–S in S includes a secretable effector molecule, wherein the secretable effector molecule includes IL-15, C contains a protease cleavage site, and MT contains a cell membrane tethering domain, wherein S–C–MT or MT–C–S is constructed 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 endothelial mucin (EMCN); (ii) one or more intracellular inhibitory domains that inhibit the 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, one or more peptide linkers, and combinations thereof; and (C) An exogenous polynucleotide encoding a bivalent activating chimeric antigen receptor (aCAR), the bivalent activating chimeric antigen receptor comprising: (i) An antigen-binding domain specific for FLT3; (ii) 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, one or more peptide linkers, and combinations thereof.

2. The polycistronic expression system of claim 1, wherein the exogenous polynucleotide encoding each of the membrane-cleavable chimeric protein, the iCAR, and the bivalent aCAR passes through Polynucleotide linkers encoding a 2A ribosome jumping element are linked together, optionally wherein the 2A ribosome jumping element is selected from the group consisting of: T2A ribosome jumping element, E2A ribosome jumping element, P2A core Glycosome jumping elements, F2A ribosome jumping elements, ribosome jumping element fusions thereof, and combinations thereof, optionally wherein said ribosome jumping element fusions comprise E2A / T2A ribosome jumping elements, optionally wherein said The E2A / T2A ribosomal jumping element contains the amino acid sequence QCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO:221), optionally wherein said E2A / T2A ribosome jumping element consists of the sequence CAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATCTAATCCTGGACCTGGATCT GGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACGTGGAGGAAAACCCTGGACCT (SEQ ID NO:222) or the sequence CAGTGCACAAATTATGCACTGCTGAAGCTCGCCGGGGATGTCGAGAGTAACCCAGGACCTGGAAGC GGAGAAGGTCGTGGTAGTCTACTAACGTGTGGTGATGTAGAAGAAAATCCTGGACCT The polynucleotide sequence encoding (SEQ ID NO:223).

3. The polycistronic expression system of claim 1 or 2, wherein the membrane-cleavable chimeric protein, the iCAR and the bivalent aCAR are encoded in the following order from 5' to 3': (i ) membrane-cleavable chimeric protein; (ii) bivalent aCAR; and (iii) iCAR; or encoded in the following order from 5' to 3': (i) membrane-cleavable chimeric protein; (ii) iCAR; and ( iii) Bivalent aCAR.

4. The polycistronic expression system of any one of claims 1-3, wherein the secretable effector molecule comprises a signal peptide or signal anchor sequence, optionally wherein the signal peptide comprises a signal for the secretable effector molecule. A natural signal peptide native to the secreted effector molecule, optionally wherein the signal peptide comprises a non-natural signal peptide, or the signal anchor The sequence comprises a non-natural signal anchor sequence that is non-native to the secretable effector molecule, optionally wherein the non-natural signal peptide or the non-natural signal anchor sequence is selected from the group consisting of: IgE, IL-12 , IL-2, optimized IL-2, trypsinogen-2, Gaussia luciferase, C D5, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL-6, IL-8, CCL2, TIMP2, VEGFB, bone protection protein, serpin E1, GROα, C XCL12, IL-21, CD8, NKG2D, TNFR2, GMCSF and GM-CSFRa, optionally wherein said non-natural signal peptide comprises an IgE signal peptide, optionally wherein said 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 ATGGACTGGACTTGGATACTCTTTCTGGTCCGCTGCCGCCACACGGGTGCACTCT (SEQ ID NO:229).

5. The polycistronic expression system of any one of claims 1-4, wherein the protease cleavage site further comprises an N-terminal peptide linker, optionally selected from the group consisting of: SGGGGSGGGGSG;GGGSGGGGSGGGSLQ;GGS;GGSGGS;GGSGGSGGS; GGSGGSGGSGGS;GGGSGGSGGSGGSGGS;GGGS;GGGSGGGS;GGGSGGGSGGGS; GGGSGGGSGGGSGGGS;GGGSGGGSGGGSGGGSGGGS;GGGGS;GGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGSGGGGS; GGGGSGGGGSGGGGSGGGGSGGGGS; NO: 230), optionally wherein the protease cleavage site further comprises a C-terminal peptide linker optionally selected from the group consisting of: SGGGGSGGGGSG; GGGSGGGGSGGGSLQ; GGS; GGSGGS; GGSGGSGGS; GGSGGSGGSGGS;GGGSGGSGGSGGSGGS;GGGS;GGGSGGGS;GGGSGGGSGGGS; GGGSGGGSGGGSGGGS;GGGSGGGSGGGSGGGSGGGS;GGGGS;GGGGSGGGGS; GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGSGGGGS; GGGGSGGGGSGGGGSGGGGSGGGGS; NO: 231), optionally wherein the protease cleavage site comprises a tumor necrosis factor-alpha 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 said TACE-specific cleavage site comprises the amino acid sequence SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ (SEQ ID NO:250), optionally wherein said TACE-specific cleavage site consists of a sequence comprising TCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGTGACCCCTGAGCCTATCTTCAGCCTGATC GGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAA The polynucleotide sequence encoding (SEQ ID NO:251).

6. The polycistronic expression system of any one of claims 1-5, wherein the cell membrane tethered domain comprises a transmembrane domain selected from the group consisting of: PDGFR-β, CD8 , CD28, CD3ζ-chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, N KG2D, EpoR, TNFR2, LIR1, B7-1 and BTLA, optionally wherein said cell membrane tethering domain comprises a B7-1 transmembrane domain, optionally wherein said B7-1 transmembrane domain comprises amino acids Sequence LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV(SEQ ID NO:204), optionally wherein said B7-1 transmembrane domain consists of the sequence TTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGACC TACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAGAGAAAGCGTGCGG CCTGTG (SEQ ID NO:252) encodes a polynucleotide sequence, optionally wherein the membrane-cleavable chimeric protein is oriented from the N-terminus to the C-terminus of the formula: S-C-MT, optionally wherein the membrane-cleavable chimeric protein has the following formula: S-C-MT, optionally wherein the membrane-cleavable chimeric protein has the following formula: The cleavable chimeric protein contains the amino acid sequence MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV(SEQ IDNO:226), optionally wherein said membrane-cleavable chimeric protein consists of the sequence comprising ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCTAATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAACAGCCTGTCCAGCAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACACCTCATCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGTGACCCCTGAGCCTATCTTCAGCCTGATCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAATTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAGAGAAAGCGTGCGGCCTGTG The polynucleotide sequence encoding (SEQ ID NO:227).

7. The polycistronic expression system of any one of claims 1-6, wherein the antigen-binding domain specific to EMCN comprises a heavy chain variable (VH) region and a light chain variable (VH) region. VL) area, in: The EMCN-VH contains: Heavy chain complementarity determining region 1 (CDR-H1), said CDR-H1 has the amino acid sequence of RYDMH (SEQ ID NO: 291), Heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 296), and Heavy chain complementarity determining region 3 (CDR-H3), said CDR-H3 having the amino acid sequence of RIKD (SEQ ID NO: 298), and The EMCN-VL contains: Light chain complementarity determining region 1 (CDR-L1), said CDR-L1 has the amino acid sequence of KSSQSLVASDENTYLN (SEQ ID NO: 299), Light chain complementarity determining region 2 (CDR-L2), said CDR-L2 having QVSKLDS (SEQ ID NO: 300) amino acid sequence, and Light chain complementarity determining region 3 (CDR-L3), said CDR-L3 having LQGIHLPWT (SEQ ID NO: 301) amino acid sequence, 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 said EMCN-VH and said EMCN-VL are separated by a peptide linker, optionally wherein said antigen binding domain specific for EMCN comprises the structure VH-L-VL or VL-L-VH, wherein L is said peptide linker, optionally wherein The peptide linker includes the amino acid sequence SGGGGSGGGGSG; GGGSGGGGSGGGSLQ;GGS;GGSGGS;GGSGGSGGS;GGSGGSGGSGGS; GGSGGSGGSGGSGGS;GGGS;GGGSGGGS;GGGSGGGSGGGS;GGGSGGGSGGGSGGGS;GGGSGGGSGGGSGGGSGGGS;GGGGS;GGGGSGGGGS;GGGGSGGGGSGGGGS; GGGGSGGGGSGGGGSGGGGS;GGGGSGGGGSGGGGSGGGGSGGGGS; GSTSGSGKPGSGEGSTKG; or EAAAKEAAAKEAAAKEAAAK, 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) .

8. The polycistronic expression system of any one of claims 1-7, wherein the intracellular inhibitory domain comprises a LIR1 intracellular inhibitory domain, optionally wherein the LIR1 intracellular inhibitory domain Contains amino acid sequence LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH(SEQ ID NO:285), optionally wherein said LIR1 intracellular inhibitory domain consists of the sequence CTGCGGCACAGAAGGCAGGGCAAGCACTGGACAAGCACCCAGAGAAAGGCCGACTTTCAGCATCCTGCTGGCGCCGTTGGACCTGAGCCTACAGATAGAGGACTGCAGTGGCGGTCTAGCCCTGCCGCTGATGCCCAAGAGGAAAATCTTTACGCCGCCGTGAAGCACACCCAGCCTGAGGATGGCGTGGAAATGGACACCAGATCTCCCCACGATGAGGACCCTCAGGCCGTGACATACGCAGAAGTGAAGCACTCCAGACCTCGGAGAGAGATGGCAAGCCCTCCATCTCCTCTGAGCGGCGAGTTCCTGGACACCAAAGACAGACAGGCCGAAGAGGACAGACAGATGGATACCGAAGCCGCCGCTTCTGAAGCCCCACAGGATGTGACATATGCCCAGCTGCATAGCCTGACACTGCGGAGAGAAGCCACAGAGCCTCCACCTTCTCAAGAAGGCCCATCTCCTGCCGTGCCTTCCATCTATGCCACTCTGGCCATTCAC The polynucleotide sequence encoding (SEQ ID NO:286).

9. The polycistronic expression system of any one of claims 1-8, wherein (A) the signal peptide is present in the iCAR, optionally wherein the signal peptide of the iCAR comprises a native signal Peptide, natural or non-natural signal peptide, optionally wherein said non-natural signal The peptide or said non-natural signal anchor sequence is selected from the group consisting of: IgE, IL-12, IL-2, optimized IL-2, trypsinogen-2, Gaussia luciferase, CD5, human IgKVII, mouse IgKVII, VSV-G, Prolactin, serum albumin preprotein, azurosin preprotein, osteonectin, CD33, IL-6, IL-8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, CXCL12, IL-2 1. 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 said CD8 signal peptide consists of the sequence ATGGCTCTGCCCGTGACAGCTTTGCTGCTGCCTTTGGCACTGCTGCTGCATGCTGCTAGACCA (SEQ IDNO:416); and / or wherein (B) a hinge domain is present in the iCAR, optionally wherein the hinge domain of the iCAR is selected from the group consisting of: human Ig (immunoglobulin) hinge, IgG4 hinge, IgG2 hinge, CD 8a hinge or IgD hinge, KIR2DS2 hinge, LNGFR hinge, LIR1 hinge, PDGFR-β extracellular linker and combinations thereof, optionally wherein the hinge domain of the iCAR comprises a LIR1 hinge, optionally wherein the LIR1 hinge comprises amino acid sequence HPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV (SEQ ID NO:417), optionally wherein the LIR1 hinge consists of the sequence CACCCATCCGATCCTCTCGAGCTGGTGGTTTCTGGACCTTCTGGCGGCCCTAGCAGCCCTACAACA GGACCTACAAGCACAAGCGGCCCTGAGGACCAACCTCTGACACCAACAGGCAGCGATCCTCAGTCTGGACTGGGGAGACATCTGGGCGTT (SEQ ID NO: 418) The polynucleotide sequence encoding, the hinge domain of the ICAR is the CD8 hinge, which contains the CD8 hinge containing the amino acid sequence TTTPAPAPRPAPAPACRPAAVHTRGLDFACD (SEQ I D no: 271), any Selected land, the CD8 hinge contains the sequence ACAACAACACCCGCACCTCGGCCTCCAACTCCAGCTCCAACAATTGCACTGCAACCCCTGAGTCTG AGGCCCGAGGCCTGTAGGCCAGCAGCTGGCGGAGCTGTTCACACTAGAGGCCTGGACTTTGCCTGT GAC (SEQ ID NO: 283), and / or wherein (C) there is a transmembrane domain of the iCAR, optionally wherein the transmembrane domain of the iCAR comprises a polynucleotide selected from the group consisting of Transmembrane domain: PDGFR-β, CD8, CD28, CD3ζ-chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, P D-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, LIR1 and BTLA, optionally wherein the transmembrane domain of said iCAR comprises the LIR1 transmembrane domain, optionally wherein said The LIR1 transmembrane domain contains the amino acid sequence VIGILVAVILLLLLLLLLFLI (SEQ ID NO:259), optionally wherein said LIR1 transmembrane domain consists of the sequence comprising GTGATCGGCATTCTGGTCGCCGTGATCCTGCTCCTGTTGCTCCTGCTGCTTCTGTTCCTGATC The polynucleotide sequence encoding (SEQ IDNO:260).

10. The polycistronic expression system of any one of claims 1-9, wherein the iCAR comprises the amino acid sequence: MALPPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQGT MVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQG TKLEIKngaaHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAAD AQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH(SEQ ID NO:419), optionally wherein said iCAR consists of a sequence comprising ATGGCTCTGCCCGTGACAGCTTTGCTGCTGCCTTTGGCACTGCTGCTGCATGCTGCTAGACCAGAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGATACGATATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCGTGATCTGGGGCAACGGCAACACACACTACCACAGCGCCCTGAAGTCCCGGTTCACCATCTCCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCACCCTGAGAATCAAGGATTGGGGCCAGGGCACCATGGTCACCGTTTCTTCTGGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCTGACGTGGTCATGACACAGAGCCCTCTGAGCCTGCCTGTGACACTGGGACAGCCTGCCAGCATCAGCTGCAAGTCTAGCCAGTCTCTGGTGGCCAGCGACGAGAACACCTACCTGAACTGGTTCCAGCAGAGGCCCGGACAGTCTCCTAGACGGCTGATCTACCAGGTGTCCAAGCTGGATAGCGGCGTGCCCGATAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGAAGATCAGCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTCTGCAAGGCATCCATCTGCCTTGGACCTTTGGCCAGGGCACAAAGCTGGAAATCAAGAATGGCGCTGCACACCCATCCGATCCTCTCGAGCTGGTGGTTTCTGGACCTTCTGGCGGCCCTAGCAGCCCTACAACAGGACCTACAAGCACAAGCGGCCCTGAGGACCAACCTCTGACACCAACAGGCAGCGATCCTCAGTCTGGACTGGGGAGACATCTGGGCGTTGTGATCGGCATTCTGGTCGCCGTGATCCTGCTCCTGTTGCTCCTGCTGCTTCTGTTCCTGATCCTGCGGCACAGAAGGCAGGGCAAGCACTGGACAAGCACCCAGAGAAAGGCCGACTTTCAGCATCCTGCTGGCGCCGTTGGACCTGAGCCTACAGATAGAGGACTGCAGTGGCGGTCTAGCCCTGCCGCTGATGCCCAAGAGGAAAATCTTTACGCCGCCGTGAAGCACACCCAGCCTGAGGATGGCGTGGAAATGGACACCAGATCTCCCCACGATGAGGACCCTCAGGCCGTGACATACGCAGAAGTGAAGCACTCCAGACCTCGGAGAGAGATGGCAAGCCCTCCATCTCCTCTGAGCGGCGAGTTCCTGGACACCAAAGACAGACAGGCCGAAGAGGACAGACAGATGGATACCGAAGCCGCCGCTTCTGAAGCCCCACAGGATGTGACATATGCCCAGCTGCATAGCCTGACACTGCGGAGAGAAGCCACAGAGCCTCCACCTTCTCAAGAAGGCCCATCTCCTGCCGTGCCTTCCATCTATGCCACTCTGGCCATTCAC (SEQ ID NO: 420) polynucleotide sequence encoding.

11. The polycistronic expression system of any one of claims 1-10, wherein the antigen-binding domain specific for FLT3 comprises a heavy chain variable (VH) region and a light chain variable (VH) region. VL) area, in: The FLT3-VH contains: Heavy chain complementarity determining region 1 (CDR-H1), said CDR-H1 has the amino acid sequence of GGTFSSYAIS (SEQ ID NO: 360), Heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of GIIPIFGTANYAQKFQG (SEQ ID NO: 361), and Heavy chain complementarity determining region 3 (CDR-H3), the CDR-H3 having the amino acid sequence of FALGFFREQAFDI (SEQ ID NO: 362), and The FLT3-VL contains: Light chain complementarity determining region 1 (CDR-L1), said CDR-L1 has the amino acid sequence of RASQSISSYLN (SEQ ID NO: 363), Light chain complementarity determining region 2 (CDR-L2), said CDR-L2 has AASSLQS (SEQ ID NO: 364) amino acid sequence, and Light chain complementarity determining region 3 (CDR-L3), said CDR-L3 has QQSYSTPFT (SEQ ID NO: 365) amino acid sequence, 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 where: The FLT3-VH contains the amino acid sequence and The FLT3-VL contains the amino acid sequence DIQMTQSPSSSLSASVGDRVTITCRASQSISLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDLATYYCQQSYSTPFTFGPGTKVDIK (SEQ ID NO: 316), optionally wherein said FLT3-VH and said FLT3-VL are separated by a peptide linker.

12. The polycistronic expression system of any one of claims 1-11, wherein the antigen-binding domain specific for CD33 comprises a heavy chain variable (VH) region and a light chain variable (VH) region. VL) area, in: The CD33-VH contains: Heavy chain complementarity determining region 1 (CDR-H1), said CDR-H1 has the amino acid sequence of DYNMH (SEQ ID NO: 402), Heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of YIYPYNGGTGYNQKFKSKA (SEQ ID NO: 403), and Heavy chain complementarity determining region 3 (CDR-H3), the CDR-H3 having the amino acid sequence of GRPAMDYWGQ (SEQ ID NO: 404), and The CD33-VL contains: Light chain complementarity determining region 1 (CDR-L1), said CDR-L1 has the amino acid sequence of RASESVDNYGISFMN (SEQ ID NO: 405), Light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of AASNQGS (SEQ ID NO: 406), and Light chain complementarity determining region 3 (CDR-L3), the 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 where: The CD33-VH contains the amino acid sequence and The CD33-VL contains the amino acid sequence DIQMTQSPSSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRF SGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 330), optionally wherein said CD33-VH and said CD33-VL are separated by a peptide linker.

13. The polycistronic expression system of any one of claims 1-12, wherein the aCAR antigen-binding domain comprises the structure (FLT3-VH)-L1-(CD33-VH)-L2-(CD33 -VL) -L3-(FLT3-VL), wherein L1, L2 and L3 are the first peptide linker, the second peptide linker and the third peptide linker respectively; optionally wherein said L1, L2 and / or L3 are each independently selected from The group consisting of: SEQ ID Nos 230-248, optionally wherein said L1 peptide linker is the amino acid sequence GGGGS (SEQ ID NO:242) or GGGGSGGGS (SEQ ID NO:243), optionally wherein said L1 peptide linker GGGGS (SEQ ID NO: 242) encoded by a polynucleotide sequence comprising the sequence GCGGCGGGTGGCTCT (SEQ ID NO: 254), or the L1 peptide linker GGGSGGGGS (SEQ ID NO:243) consists of sequence GGAGGCGGAGGATCTGGTGGTGGTGGATCT (SEQ ID NO:256) encodes a polynucleotide sequence, optionally wherein the L2 peptide linker is the amino acid sequence GSTGSSGKPGSGEGSTKG (SEQ ID NO:247), optionally wherein the L2 peptide linker consists of the sequence GGCTCTACATCTGGCTCTGGCAAACCTGGAAGCGGCGAGGGATCTACCAAGGGC (SEQ ID NO:249) encodes a polynucleotide sequence, optionally wherein the L2 peptide linker is the amino acid sequence GGGGSGGGGS (SEQ ID NO:243), optionally wherein the L2 peptide linker is comprised of the sequence GGTGGCGGAGGAAGTGGCGGCGGAGGCTCT (SEQ ID NO: 257), optionally wherein the L3 peptide linker is the amino acid sequence GGGGS (SEQ ID NO:242) or GGGSGGGGS (SEQ ID NO:243), optionally wherein said 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) consists of the sequence GGCGGTGGCGGATCTGGCGGAGGTGGCAGT (SEQ ID NO:258) polynucleotide sequence encoding.

14. The polycistronic expression system of any one of claims 1-13, wherein the aCAR intracellular signaling domain that stimulates an immune response is selected from the group consisting of: CD3-ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d, CD97, CD2, ICOS, CD27, CD15 4. CD8, OX40, 4-1BB, CD28, ZAP40, CD30, GITR, HVEM, DAP10, DAP12, MyD88, 2B4, CD40, PD-1, LFA-1, CD7, LIGHT, NKG2C, B7-H3, MHC Class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, SLAM proteins, activated NK cell receptors, BTLA, Toll ligand receptors, CDS, ICAM-1, (CD11a / CD 18), BAFFR, KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, IL2Rβ, IL2Rγ, IL7Rα, IT GA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a , ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(C D226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile type), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (C D162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a and combinations thereof, optionally wherein said aCAR intracellular signaling domain that stimulates an immune response comprises a CD28 costimulatory domain and CD3ζ signaling domain, optionally wherein said CD28 costimulatory domain comprises the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:287), optionally wherein said CD28 costimulatory domain consists of the sequence AGAAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCCGGACCT ACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGATTTCGCCGCCTACCGGTCC (SEQ ID NO: 288) The polynucleotide sequence encoding, the optional place where the CD3ζ signal conduction domain contains the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 289), optionally wherein said CD3ζ signaling domain is comprised of the sequence AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO:290) encodes a polynucleotide sequence, 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 IgD hinge, KIR2DS2 hinge, LNGFR hinge, LIR1 hinge, PDGFR-β extracellular linker and combinations thereof, optionally wherein the hinge domain of the aCAR comprises a CD8 hinge, optionally wherein the CD8 hinge comprises an amino acid sequence ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO: 272), optionally wherein said CD8 hinge consists of a sequence comprising the sequence GCCCTGAGCAACAGCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTGCCCGCCAAGCCTACAACAACCCCTGCCCTAGACCTACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCCGAAGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCATACAAGAGGACTGGATTTTGCCTGCGAC (SEQ ID NO: 272) NO:284) polynucleotide sequence coding, and / or wherein (B) There is a transmembrane domain of said aCAR, optionally wherein said transmembrane domain of aCAR is selected from the group consisting of: human Ig (immunoglobulin) hinge, IgG4 hinge, IgG2 hinge, CD8a hinge or IgD hinge, KIR2DS2 hinge, LNGFR hinge, LIR1 hinge, PDGFR-β extracellular linker and combinations thereof, optionally wherein the transmembrane domain of the aCAR comprises a CD8 hinge, optionally wherein the CD8 transmembrane comprises an amino acid sequence IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO:206), optionally wherein said CD8 transmembrane is comprised of the sequence ATCTATATCTGGGCCCCTCTGGCTGGCACATGCGGAGTTCTGCTGCTCAGCCTGGTCATCACCCTGTACTGCAACCACAGA (SEQ ID NO: 261) The polynucleotide sequence encoding; and / or (C) There is an ACAR signal peptide in the existence of ACAR, and the ACAR signal peptide of the ACAR signaling is selected. IGE, IL-12, IL-2, Optimized IL-2, Intoctinase Origin-2, GAUSSIA fluorescent enzyme, CD5, human Igkvii, mouse Igkvii, vsv-g Prime, bone adhesion, CD33, IL-6, IL-8, CCL2, TIMP2, VEGFB, osteopopin, lingine protease inhibitors E1, GROα, CXCL12, IL-21, NKG2D, TNFR2, GMCSF, and GMCSF and GM-CSFRA, the selected place, the ACAR's signal peptide contains the GM-CSFRA signal peptide, and the selected place where the GM-CSFRA signal peptide contains the amino acid sequence MLLLVTSLLLLLPAFLLIP (SEQ ID NO: 423). GM-CSFRA signal peptide contains sequences ATGCTGCTGCTGGTTACATCTCTGCTGCTGTGCGAGCTGCCCCATCCTGCCTTTCTGCTTATTCCT (SEQ IDNO: 424) The polynucleotide sequence encoding, the optional place where the ACAR contains amino acid sequence MllvtsllCelphPAFLLLLIPEVQLVQLVQLVQLVQLVQLVKKPGSSVSCKASGGTFSSWVRQAPGQG LEWMGGIPGTanyaqkFQGRVTEDTAVYCALFGFREQAFD IWGQGTTVTVSSSSQGSQLVQLVQLVQSGAEVKPGSSVSCKVSCKASGYTDYNMHWVRQAPGQGLEW TVSSGSGSGSGSGEGSGDIQSPSPSPSPSSASVGDRVTCRVDNYGISFMNWFQQKPKAAASNQSGSGSGSGSGSGSGSGTDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDIGSL GTKVEIKGGGSDISDQSPSPSPSSASVGDRVTCRASQSISSYLNWYQQKPKAPKLLIYAA YFSHFVPVFLPAPTTPAPRPTPAPTIASQPLRPEAGGAVHTRGLDFACDIYI YRSRVKFSRSADADADADAYKQGQNQLYNLGRGRREEYDVLDVLDPEMGRDPEMGRKNPQEGLYNELQKDKMAEAEIGMKGHDGLTKDALHMQALHMQALHMQALHMQALHMQALHMQALHMQALHMQALHMQALHMQALT , Optional, the ACAR contains amino acid sequence MllvtsllCelphPAFLLLLIPEVQLVQLVQLVQLVQLVQLVKKPGSSVSCKASGGTFSSWVRQAPGQG LEWMGGIPGTanyaqkFQGRVTEDTAVYCALFGFREQAFD IWGQGTTVTVSSSSSGGGSGGSQLVQLVQLVQSGAEVKPGSCKVSCKASGYTDYNMHWVRQAPG QGTLVTVSSSGGGGGSDIQMTQSPSPSSSASVGDRVTCRASESVDNYGISFMNWFQQKPKAAASNQGSGSGSGSGSGSLQPDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD TKVEIKGGGSGGSGGSDIQMTQSPSSSSSSVGDRVTCRasqsqsqsylnwyqqkpkapkll NSIMYFSHFVPVFVFAKPTTPAPRPPAPAPTIASQPLRPEAGAVHTRDFAC DFAYRSRVKFSRVKFSADAPAYKQGQNQlynelnlgrRGRDVLDPEMGRDPEMGKNPQEGLYNEGDKDKMAEIGMKERRRRGHDGLTKDALPPR (seq ID NO: 4 15).

15. An expression carrier, which contains the multi-smooth reflux expression system as described in any of the claims 1-14. : 426, SEQ ID NO: 427, SEQID NO: 428, SEQ ID NO: 429, SEQ ID NO: 433, or SEQ ID NO:

432.

16. An isolated cell, which contains the Duoshun reflux expression system or the expression carrier described in any claim 1-14. Cells include immune cells, which are selected in which the cells are selected below the following groups: T cells, natural killing (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, natural killing T (NKT ) Cells, osteomycosis, macrophages, human embryo stem cells (ESC), ESC derivatives, polyo -stem cells, and induced polyo -stem cells (IPSC), and IPSC derivatives. The choice of the cells are NK cells.

17. A polynucleotide, which contains the nucleic acid sequence of the group composed of the following paragraph: SEQ IDNO: 425, SEQ ID NO: 426, SEQ ID NO: 427, SEQ ID NO: 428, SEQ ID NO: 429, SEQ ID NO: 433, SEQ ID NO: 432, and SEQ ID NO:

433.

18. A method for a subject needed for the treatment includes any separate cells or claims as described as described in any claim 17 described by the right requirements of the active dose of the treatment. Among the places, the subjects have cancer, and the separated cells are derived from the same exotic as the subject or relative to the subject.

19. A method of immune response to tumor cells that stimulate the cells that stimulate the cells in the subject. The polynucleotide of cells or claims 17, the separated cells are derived from the same kind of exotic or relative to the subject from the subject.

20. A method for preparing engineering cells that include the multi-smooth reflux expression system as described as described in any of the claims 1-14 The cells of polynucleotide are separated.

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