Multicistronic chimeric protein expression system

Abstract

Described herein are multicistronic expression systems encoding chimeric proteins, in particular chimeric membrane-cleavable systems and chimeric antigen receptors. Nucleic acids, cells, and methods relating thereto are also described herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Nos. 63 / 333,483, filed April 21, 2022, 63 / 370,219, filed August 2, 2022, and 63 / 440,668, filed January 23, 2023, each of which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on XXXX, 20XX, is named XXX-XXX_sequencelisting.txt, and is ###,### bytes in size. [Background technology]

[0003] background Cell-based therapeutic platforms offer promising avenues for treating various diseases. One such promising platform is CAR-T-based therapy for the treatment of cancer. Given these promises, improvements in cell-based therapies are needed. An active area of ​​exploration is engineering cell-based therapies to produce and / or secrete effector molecules, such as cytokines, that enhance cell-based therapies, a process referred to as armoring. For example, unarmored CAR-T therapies have poor efficacy in solid tumors, and armoring can affect the entire cancer-immune cycle and enhance CAR-T activity. However, uncontrolled or unregulated armoring strategies can have negative effects on treatment, such as off-target effects and toxicity in subjects. Thus, additional methods for controlling and regulating the armoring of cell-based therapies, such as regulating the production and / or secretion of payload effector molecules, are needed. Summary of the Invention

[0004] overview Provided herein, in some embodiments, are cell-based therapeutic platforms, including chimeric antigen receptor (CAR)-based therapies (e.g., NK CARs), with regulated armoring of cell-based therapies, regulated secretion of payload effector molecules, etc. Also provided herein, in some embodiments, are combination cell-based immunotherapies, including regulated armoring for targeted treatment of cancer, such as ovarian cancer, breast cancer, colon cancer, lung cancer, and pancreatic cancer.

[0005] The treatments provided herein, however, can limit the systemic toxicity of armoring. For example, the immunotherapies provided herein can be tumor-specific and effective while limiting systemic toxicity and / or other off-target effects caused by armoring. These treatments deliver proteins of interest, such as immunomodulatory effector molecules, particularly IL-15, in a regulated manner, including modulation of secretion kinetics, cell state specificity, and cell or tissue specificity. The design of delivery vehicles has been optimized to improve overall function in cell-based therapies, such as cancer therapy, including, but not limited to, optimization of membrane cleavage sites, promoters, linkers, signal peptides, delivery methods, and the combination, regulation, and order of immunomodulatory effector molecules.

[0006] The design of the delivery vehicle is also optimized for the expression of multiple components for CAR-based therapy in a multicistronic system, including a chimeric antigen receptor (which activates both aCAR and inhibitory iCAR) and a membrane-cleavable chimeric protein.

[0007] A multicistronic expression system comprising an engineered nucleic acid, the engineered nucleic acid comprising: (A) an exogenous polynucleotide, oriented N-terminal to C-terminal, encoding a membrane-cleavable chimeric protein having the formula: SC-MT or MT-CS, wherein S comprises a secreted effector molecule, the secreted effector molecule comprises IL-15, C comprises a protease cleavage site, and MT comprises a cell membrane-tethering domain, wherein SC-MT or MT-CS is configured to be expressed as a single polypeptide; and (B) an exogenous polynucleotide encoding an inhibitory chimeric antigen receptor (iCAR), wherein iCAR comprises: (i) an antigen-binding domain specific for endomucin (EMCN), (ii) one or more intracellular inhibitory domains that inhibit an immune response, and (iii) a signal peptide, a transmembrane domain, a

[0014] Provided herein is a multicistronic expression system comprising: (A) an exogenous polynucleotide encoding an iCAR, the iCAR comprising one or more polypeptides selected from the group consisting of a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof; and (B) an exogenous polynucleotide encoding a bivalent activating chimeric antigen receptor (aCAR), the aCAR comprising (i) an antigen-binding domain specific for FLT3, (ii) an antigen-binding domain specific for CD33, (iii) one or more intracellular signaling domains that stimulate an immune response, and (iv) one or more polypeptides selected from the group consisting of a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

[0008] In some embodiments, the exogenous polynucleotides encoding the membrane-cleavable chimeric protein, the iCAR, and the bivalent aCAR are linked together by a polynucleotide linker encoding a 2A ribosome skipping element. In some embodiments, the 2A ribosome skipping element is selected from the group consisting of a T2A ribosome skipping element, an E2A ribosome skipping element, a P2A ribosome skipping element, an F2A ribosome skipping element, ribosome skipping element fusions thereof, and combinations thereof. In some embodiments, the ribosome skipping element fusion comprises an E2A / T2A ribosome skipping element. In some embodiments, the E2A / T2A ribosome 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).

[0009] In some aspects, the membrane-cleavable chimeric protein, iCAR, and 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, iCAR, and bivalent aCAR are encoded in the following order from 5' to 3': (i) membrane-cleavable chimeric protein, (ii) iCAR, and (iii) bivalent aCAR.

[0010] In some aspects, the secreted effector molecule comprises a signal peptide or signal anchor sequence. In some aspects, the signal peptide comprises a natural signal peptide that is natural to the secreted effector molecule. In some aspects, the signal peptide comprises a non-native signal peptide or the signal anchor sequence comprises a non-native signal anchor sequence that is non-native to the secreted effector molecule. In some aspects, the non-native signal peptide or non-native signal anchor sequence is selected from the group consisting of IgE, IL-12, IL-2, optimized IL-2, trypsiongen2, 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, serpinEl, GROalpha, CXCL12, IL-21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa. In some aspects, the non-native 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 embodiments, the IgE signal peptide is encoded by a polynucleotide sequence comprising the sequence ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCT (SEQ ID NO: 229).

[0011] In some aspects, the IL-15 comprises the amino acid sequence NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 224). In some aspects, the IL-15 is encoded by a polynucleotide sequence comprising the sequence AATTGGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAACAGCCTGTCCAGCAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACACCTCA (SEQ ID NO: 225).

[0012] 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, GGSGGGSGGGS, GGSGGSGGSGGS, GGSGGSGGSGGSGGS, GGS, GGGSGGGS, GGGSGGGSGGGSGGGS, GGGSGGGSGGGSGGGS, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGGS, GGGGSGGGGSGGGGGS, GGSSGGGGS, GSTSGSGKPGSGEGSTKG, and EAAAKEAAAKEAAAKEAAAK. In some aspects, the protease cleavage site further comprises the N-terminal peptide linker SGGGGSGGGGSG (SEQ ID NO: 230).

[0013] 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, GGSGGGSGGGS, GGSGGSGGSGGS, GGSGGSGGSGGSGGS, GGS, GGGSGGGS, GGGSGGGSGGGSGGGS, GGGSGGGSGGGSGGGS, GGGS, GGGGSGGGGS, GGGGSGGGGSGGGS, GGGGSGGGGSGGGGGS, GGSSGSGKPGSGEGSTKG, and EAAAKEAAAKEAAAKEAAAK. In some aspects, the protease cleavage site further comprises the C-terminal linker GGGSGGGGSGGGSLQ (SEQ ID NO: 231).

[0014] 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).

[0015] In some aspects, the cell membrane anchoring domain comprises a transmembrane domain selected from the group consisting of PDGFR-beta, CD8, CD28, CD3 zeta 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 cell membrane anchoring 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).

[0016] In some embodiments, the membrane-cleavable chimeric protein oriented N-terminal to C-terminal has the formula SC-MT. In some embodiments, 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).

[0017] In some aspects, an antigen binding domain specific for EMCN comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the EMCN-VH comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 291), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 296), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 298), and the EMCN-VL comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 291), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 296), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 298). The reference antibody comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of DENTYLN (SEQ ID NO: 299), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 300), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 301), and the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and 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).

[0018] In some aspects, the EMCN-VH and EMCN-VL are separated by a peptide linker. In some aspects, an antigen-binding domain specific for EMCN comprises the structure VH-L-VL or VL-L-VH, where L is the peptide linker. In some aspects, the peptide linker comprises the amino acid sequence SGGGGSGGGGSG, GGGSGGGGSGGGSLQ, GGS, GGSGGS, GGSGGGSGGGS, GGSGGSGGSGGS, GGSGGSGGGS, GGGS, GGGSGGGSGGGS, GGGSGGGSGGGSGGGS, GGGSGGGSGGGSGGGS, GGGGGS, GGGGSGGGGS, GGGGSGGGGSGGGS, GGGGSGGGGSGGGS, GGGGSGGGGSGGGS, GGGGSGGGGSGGGS, GSTSGSGKPGSGEGSTKG, or EAAAKEAAAKEAAAKEAAAK. In some embodiments, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 244). In some embodiments, the peptide linker is encoded by a polynucleotide sequence comprising the sequence GGAGGCGGAGGATCTGGTGCGGAGGAAGTGGCGGAGGCGGTTCT (SEQ ID NO: 253).

[0019] In some aspects, an antigen binding domain specific for EMCN comprises the structure VH-L-VL and comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQGTMVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQGTKLEIK (SEQ ID NO: 311).In some aspects, an antigen binding domain specific for EMCN is encoded by a polynucleotide sequence comprising the sequence (SEQ ID NO:312).

[0020] In some aspects, the intracellular inhibitory domain comprises an LIR1 intracellular inhibitory domain, wherein the intracellular inhibitory domain comprises an LIR1 intracellular inhibitory domain, optionally wherein the LIR1 intracellular inhibitory domain comprises the amino acid sequence LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 285), optionally wherein the LIR1 intracellular inhibitory domain comprises the sequence CTGCGGCACAGAAGGCAGGGCAAGCACTGGACAAGCACCCAGAGAAAGGCCGACTTTCAGCATCCTGCTGGCGCCGTTGGACCTGAGCCTACAGATAGAGGACTGCAGTGGCGGTCTAGCCCTGCCGCTGATGCCCAAGAGGAAAAT 8. The multicistronic expression system of any one of claims 1 to 7, encoded by a polynucleotide sequence comprising: CTTTACGCCGCCGTGAAGCACACCCAGCCTGAGGATGGCGTGGAAATGGACACCAGATCTCCCCACGATGAGGACCCTCAGGCCGTGACATACGCAGAAGTGAAGCACTCCAGACCTCGGAGAGAGATGGCAAGCCCTCCATCTCCTCTGAGCGGCGAGTTCCTGGACACCAAAGACAGACAGGCCGAAGAGGACAGACAGATGGATACCGAAGCCGCCGCTTCTGAAGCCCCACAGGATGTGACATATGCCCAGCTGCATAGCCTGACACTGCGGAGAGAAGCCACAGAGCCTCCACCTTCTCAAGAAGGCCCATCTCCTGCCGTGCCTTCCATCTATGCCACTCTGGCCATTCAC (SEQ ID NO: 286).In some embodiments, the LIR1 intracellular inhibitory domain has the sequence CTGCGGCACAGAAGGCAGGGCAAGCACTGGACAAGCACCCAGAGAAAGGCCGACTTTCAGCATCCTGCTGGCGCCGTTGGACCTGAGCCTACAGATAGAGGACTGCAGTGGCGGTCTAGCCCTGCCGCTGATGCCCAAGAGGAAAATCTTTACGCCGCCGTGAAGCACACCCAGCCTGAGGATGGCGTGGAAATGGACACCAGATCTCCCCACGATGAGGACCCTCAGGCCGTGACATACGCAGAAGTGAAGCAC TCCAGACCTCGGAGAGAGATGGCAAGCCCTCCATCTCCTCTGAGCGGCGAGTTCCTGGACACCAAAGACAGACAGGCCGAAGAGGACAGACAGATGGATACCGAAGCCGCCGCTTCTGAAGCCCCACAGGATGTGACATATGCCCAGCTGCATAGCCTGACACTGCGGAGAGAAGCCACAGAGCCTCCACCTTCTCAAGAAGGCCCATCTCCTGCCGTGCCTTCCATCTATGCCACTCTGGCCATTCAC (SEQ ID NO: 286).

[0021] In some aspects, a signal peptide is present in the iCAR. In some aspects, the signal peptide of the iCAR comprises a native signal peptide, a native or non-native signal peptide, and optionally the non-native signal peptide or non-native signal anchor sequence is selected from the group consisting of IgE, IL-12, IL-2, optimized IL-2, trypsiongen2, 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, serpinEl, GROalpha, CXCL12, IL-21, CD8, NKG2D, TNFR2, and GMCSF. In some aspects, the signal peptide of the iCAR comprises a CD8 signal peptide. In some aspects, the CD8 signal peptide comprises the 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).

[0022] In some aspects, the hinge domain is present in the iCAR comprising. In some aspects, the hinge domain of the iCAR is selected from the group consisting of a human Ig (immunoglobulin) hinge, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge, a KIR2DS2 hinge, an LNGFR hinge, an LIR1 hinge, a PDGFR-beta extracellular linker, and combinations thereof. In some aspects, the hinge domain of the iCAR comprises an LIR1 hinge. In some aspects, the LIR1 hinge comprises the amino acid sequence HPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV (SEQ ID NO: 417). In some embodiments, the LIR1 hinge is encoded by a polynucleotide sequence comprising the sequence CACCCATCCGATCCTCTCGAGCTGGTGGTTTCTGGACCTTCTGGCGGCCCTAGCAGCCCTACAACAGGACCTACAAGCACAAGCGGCCCTGAGGACCAACCTCTGACACCAACAGGCAGCGATCCTCAGTCTGGACTGGGGAGACATCTGGGCGTT (SEQ ID NO: 418). In some embodiments, the hinge domain of the iCAR comprises a CD8 hinge. In some embodiments, 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).

[0023] In some aspects, a transmembrane domain of iCAR is present. In some aspects, the transmembrane domain of iCAR comprises a transmembrane domain selected from the group consisting of PDGFR-beta, CD8, CD28, CD3 zeta 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 iCAR comprises an LIR1 transmembrane domain. In some aspects, the LIR1 transmembrane domain comprises 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).

[0024] In some embodiments, the iCAR has the amino acid sequence MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQGTM VTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQGTK LEIKHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 430), or MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQGTMV TVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQGTKLEI KngaaHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 419).

[0025]

[0026] In some embodiments, the iCAR has the amino acid sequence MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQG TMVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQ GTKLEIKTTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACDVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 431), or MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQGT MVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYCLQGIHLPWTFGQGT KLEIKngaaTTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACDVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 421).

[0027]

[0028] 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 FLT3-VH comprises a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of GGTFSSYAIS (SEQ ID NO: 360), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of GIIPIFGTANYAQKFQG (SEQ ID NO: 361), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of FALFGFREQAFDI (SEQ ID NO: 362), and FLT3-VL comprises , a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASQSISSYLN (SEQ ID NO: 363), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of AASSLQS (SEQ ID NO: 364), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQSYSTPFT (SEQ ID NO: 365), wherein the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and 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 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCATFALFGFREQAFDIWGQGTTVTVSS (SEQ ID NO: 315), and the FLT3-VL comprises the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQSYSTPFTFGPGTKVDIK (SEQ ID NO: 316).

[0029] In some aspects, 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 complementarity determining region 1 (CDR-H1) having the amino acid sequence of DYNMH (SEQ ID NO: 402), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of YIYPYNGGTGYNQKFKSKA (SEQ ID NO: 403), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of GRPAMDYWGQ (SEQ ID NO: 404); It comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SVDNYGISFMN (SEQ ID NO: 405), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of AASNQGS (SEQ ID NO: 406), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQSKEVPWT (SEQ ID NO: 407), wherein the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and 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 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS (SEQ ID NO: 329) and the CD33-VL comprises the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 330).

[0030] In some aspects, the FLT3-VH, FLT3-VL, CD33-VH, and CD33-VL are separated by peptide linkers. In some aspects, the aCAR antigen binding domain comprises the structure (FLT3-VH)-L1-(CD33-VH)-L2-(CD33-VL)-L3-(FLT3-VL), where L1, L2, and L3 are the first peptide linker, the second peptide linker, and the third peptide linker, respectively. In some aspects, L1, L2, and / or L3 are each independently selected from the group consisting of SGGGGSGGGGSG, GGGSGGGGSGGGSLQ, GGS, GGSGGS, GGSGGGSGGGS, GGSGGSGGSGGS, GGSGGSGGSGGS, GGGS, GGGSGGGSGGGSGGGS, GGGSGGGSGGGSGGGS, GGGS, GGGGSGGGGS, GGGGGS, GGGGSGGGGSGGGSGGGS, GGGGSGGGGSGGGS, GGGGSGGGGSGGGGGS, GSTSGSGKPGSGEGSTKG, 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 GCGGCGGTGGCTCT (SEQ ID NO:254), or the L1 peptide linker GGGGSGGGGS (SEQ ID NO:243) is encoded by a polynucleotide sequence comprising the sequence GGAGGCGGAGGATCTTGGTGGTGGTGGATCT (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).

[0031] In some embodiments, the aCAR intracellular signaling domain that stimulates an immune response is selected from the group consisting of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278, 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, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD CD11d, ITGAE CD103, ITGAL CD11a, ITGAM CD11b, ITGAX The antigens are selected from the group consisting of 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, and combinations thereof. In some aspects, the aCAR intracellular signaling domain that stimulates an immune response comprises a CD28 costimulatory domain and a CD3ζ signaling domain.In some aspects, the CD28 costimulatory 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 CD3ζ signaling domain is encoded by a polynucleotide sequence comprising AGAGTGAAGTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 290).

[0032] In some aspects, the hinge domain of the aCAR is present. In some aspects, the hinge domain of the aCAR is selected from the group consisting of a human Ig (immunoglobulin) hinge, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge, a KIR2DS2 hinge, a LNGFR hinge, a LIR1 hinge, a PDGFR-beta extracellular linker, and combinations thereof. In some aspects, the hinge domain of the aCAR comprises a CD8 hinge. In some aspects, the CD8 hinge comprises 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).

[0033] In some aspects, the transmembrane domain of the aCAR is present. In some aspects, the transmembrane domain of the aCAR is selected from the group consisting of a human Ig (immunoglobulin) hinge, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge, a KIR2DS2 hinge, a LNGFR hinge, a LIR1 hinge, a PDGFR-beta extracellular linker, and combinations thereof. In some aspects, the transmembrane domain of the aCAR comprises a CD8 hinge. In some aspects, the CD8 transmembrane comprises 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).

[0034] In some embodiments, an aCAR signal peptide of the aCAR is present. In some embodiments, the signal peptide of the aCAR is selected from the group consisting of IgE, IL-12, IL-2, optimized IL-2, trypsiongen2, 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, serpinEl, GROalpha, CXCL12, IL-21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa. In some embodiments, the signal peptide of the aCAR comprises a GM-CSFRa signal peptide. In some embodiments, the GM-CSFRa signal peptide comprises the 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).

[0035] In some embodiments, the aCAR comprises the amino acid sequence (SEQ ID NO: 414).

[0036] In some embodiments, the aCAR comprises the amino acid sequence (SEQ ID NO: 415).

[0037] Also provided herein are expression vectors comprising any of the multicistronic 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.

[0038] Also provided herein are isolated cells comprising any of the multicistronic expression systems provided herein or any of the expression vectors provided herein. In some aspects, the cells comprise immune cells. In some aspects, the cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, bone marrow cells, macrophages, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs), and iPSC-derived cells. In some aspects, the cells are NK cells.

[0039] 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.

[0040] Also provided herein is a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:425.

[0041] Also provided herein is a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:426.

[0042] Also provided herein is a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:427.

[0043] Also provided herein is a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:428.

[0044] Also provided herein is a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:429.

[0045] Also provided herein is a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:432.

[0046] Also provided herein is a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:433.

[0047] Also provided herein are methods of treating a subject in need thereof, comprising administering a therapeutically effective dose of any of the isolated cells provided herein or any of the cells provided herein.

[0048] Also provided herein are methods of treating a subject having cancer, the methods comprising administering to the subject a therapeutically effective dose of any of the isolated cells provided herein, or any of the polynucleotides provided herein.

[0049] Also provided herein are methods for stimulating a cell-mediated immune response against tumor cells in a subject, comprising administering to a tumor-bearing subject 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.

[0050] Also provided herein are methods of making an engineered cell, the methods comprising transducing an isolated cell with any of the multicistronic expression systems provided herein, any of the expression vectors provided herein, or any of the expression vectors provided herein. [Brief explanation of the drawings]

[0051] [Figure 1] A schematic diagram illustrating the components and organization of the entire FLT3 OR CD33 NOT EMCN logic-gated CAR-NK cell line is provided. [Figure 2] 1 provides a schematic illustrating the components evaluated in the optimization of the FLT3 OR CD33 NOT EMCN logic-gated CAR-NK cell line. [Figure 3] 1 shows the gating strategy for assessing iCAR and aCAR expression by flow cytometry. [Figure 4]aCAR and iCAR expression at day 15 for the indicated set of RetroVec constructs is shown. [Figure 5] Shown is aCAR and iCAR expression at day 15 for the indicated series of self-inactivating (SIN) gamma-retroviral SINvec constructs. [Figure 6] Representative flow cytometry plots of membrane-bound IL-15 expression at day 15 for the indicated series of RetroVec constructs are shown. [Figure 7] Representative flow cytometry plots of membrane-bound IL-15 expression at day 15 for the indicated series of SINvec constructs are shown. [Figure 8] 1 shows a first series comparison of aCAR, iCAR expression, and membrane-bound IL-15 for the indicated series of RetroVec and SINvec constructs. [Figure 9] A second series of comparisons of aCAR, iCAR expression, and membrane-bound IL-15 for the indicated series of RetroVec and SINvec constructs is shown. [Figure 10] A third series comparison of aCAR, iCAR expression, and membrane-bound IL-15 for the indicated series of RetroVec and SINvec constructs is shown. [Figure 11] A summary of aCAR and iCAR expression at day 15 as assessed by MFI for the indicated set of RetroVec and SINvec constructs is shown. [Figure 12] A general overview of the manifestation considerations for the various components and configurations evaluated is provided. [Figure 13] Total cytotoxicity against a target cell line expressing FLT3 / CD33 (AML cell line MV4-11) and a target cell line engineered to also express the NOT-gated target antigen EMCN (MV4-11 + EMCN) for constructs using the LIR1 ICD is shown for both aCAR to iCAR constructs (top panel) and iCAR to aCAR constructs (bottom panel). [Figure 14]Normalized cytotoxicity against a target cell line expressing FLT3 / CD33 (AML cell line MV4-11) and a target cell line engineered to also express the NOT-gated target antigen EMCN (MV4-11+EMCN) for constructs using the LIR1 ICD is shown. [Figure 15] Shown are total cytotoxicity at day 17 for constructs ordered from aCAR to iCAR configuration against a target cell line expressing FLT3 / CD33 (AML cell line MV4-11) and a target cell line engineered to also express the NOT-gated target antigen EMCN (MV4-11 + EMCN) at the effector:target ratios shown for constructs using the LIR1 ICD. [Figure 16] Normalized cytotoxicity at day 10 against a target cell line expressing FLT3 / CD33 (AML cell line MV4-11) and a target cell line engineered to also express the NOT-gated target antigen EMCN (MV4-11 + EMCN) at the effector:target ratios shown for constructs using the LIR1 ICD, both for aCAR to iCAR constructs (top panel) and for constructs ordered from iCAR to aCAR constructs (bottom panel). [Figure 17] 1 illustrates a schematic diagram of the membrane-cleavable system described herein, in which a desired payload is expressed as a chimeric protein in which a protease cleavage site is inserted between the payload and the membrane-tethering domain. The left panel illustrates a schematic diagram of a membrane-cleavable system using a transmembrane structure (e.g., the chimeric protein includes a transmembrane domain). The right panel illustrates a schematic diagram of a membrane-associated system using a membrane-associated structure (e.g., the chimeric protein includes a post-translational modification tag that enables association with the cell membrane). [Figure 18]Figure 1 illustrates a representative membrane-cleavable system for regulated secretion of IL-15, in which IL-15 is expressed as a chimeric protein with a cleavage site capable of cleavage by TACE inserted between the IL-15 payload and the membrane-tethering domain. The left panel illustrates a type I transmembrane structure (e.g., SC-MT) through the use of type I transmembrane domains, such as PDGFR-beta and CD8. The right panel illustrates a type II transmembrane structure (e.g., MT-CS) through the use of type II transmembrane domains, such as NKG2D and TNFR2. [Figure 19] The components and their order of the indicated constructs encoding either bivalent FLT3 OR CD33 aCARs or monospecific aCARs are shown. [Figure 20] 1 shows survival curves in the MV4-11 mouse tumor model for bivalent FLT3 OR CD33 CAR-NK cells and monospecific CAR-NK cells. [Figure 21] Representative tumor imaging in the MV4-11 mouse tumor model for bivalent FLT3 OR CD33 CAR-NK cells and monospecific CAR-NK cells is shown. [Figure 22] Various tagged and untagged combinations of aCAR and iCAR are illustrated. [Figure 23] Representative flow cytometry plots of the "no virus" control used to evaluate tagged and untagged CARs are shown. [Figure 24] Representative flow cytometry plots of untagged constructs SB07401 and SB07405 at day 8 are shown. [Figure 25] Representative flow cytometry plots of the iCAR tag-only construct SB06467 at day 8 are shown. [Figure 26] 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 is shown. [Figure 27]A summary of the expression of each of the CD33, FLT3, and EMCN CARs for the indicated constructs with the Loop4 linker is shown. [Figure 28] A summary of the expression of each of the CD33, FLT3, and EMCN CARs for the indicated constructs with the Loop 6 linker is shown. [Figure 29] Shown is total cytotoxicity against a target cell line expressing FLT3 / CD33 (AML cell line MV4-11) and a target cell line engineered to also express the NOT-gated target antigen EMCN (MV4-11 + EMCN) at an effector:target ratio of 1:2 for constructs using the LIR1 ICD. [Figure 30] Shown are normalized cytotoxicity (normalized to no virus control) against a target cell line expressing FLT3 / CD33 (AML cell line MV4-11) and a target cell line engineered to also express the NOT-gated target antigen EMCN (MV4-11 + EMCN) at an effector:target ratio of 1:2 for constructs using the LIR1 ICD. [Figure 31] Shown are normalized cytotoxicity (normalized to no virus control) against a target cell line expressing FLT3 / CD33 (AML cell line MV4-11) and a target cell line engineered to also express the NOT-gated target antigen EMCN (MV4-11 + EMCN) at an effector:target ratio of 1:4 for constructs using the LIR1 ICD. [Figure 32] Figure 1 shows the specific cytotoxicity of target cell lines expressing CD33 (SEM) and target cell lines also expressing the NOT-gated target antigen (SEM + EMCN) at an effector:target ratio of 1:2. The figures provide SEM EMCN- in the left column and EMCN+ in the right column, respectively. [Figure 33A] Specific cytotoxicity of target cell lines expressing CD33 and also expressing the NOT gated target antigen (SEM+EMCN) at an effector:target ratio of 1:2 in a 24-hour continuous killing assay is shown. [Figure 33B]Specific cytotoxicity of target cell lines expressing CD33 and also expressing the NOT gated target antigen (SEM+EMCN) at an effector:target ratio of 1:2 in a 48-hour continuous killing assay is shown. [Figure 33C] Specific cytotoxicity of target cell lines expressing CD33 and also expressing the NOT gated target antigen (SEM+EMCN) at an effector:target ratio of 1:2 in a 96-hour continuous killing assay is shown. EMCN+ right column, respectively. [Figure 34A] A general schematic of the in vivo experiment to evaluate NOT logic-gated CAR-NK cells is shown. [Figure 34B] FIG. 34B shows the characterization of SEM CD33+ and a 50:50 mixture of SEM CD33+ / EMCN+ cells shown in FIG. 34A. [Figure 35] Results of an in vivo experiment in which mice were injected with a 1:1 mixture of SEM + / - EMCN and treated with NK cells transduced with the respective vectors are shown. The top row shows BLI images at day 11, while the bottom panel shows BLI images 19 days after tumor injection. [Figure 36] Regions of interest (ROIs) are quantified on days 11 and 19, and the results are shown in Figure 35. [Figure 37A] Figure 1 shows the percentage 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 each construct. The circled area compares the percentage of EMCN+SEM cells between OR / NOT GATED and OR-GATED CAR-NK cell treatments, showing a significantly higher percentage of EMCN+SEM cells in animals treated with OR / NOT GATED CAR-NK cells, demonstrating NOT GATE protection of EMCN+ cells. [Figure 37B] Shown is the percentage of EMCN+ SEM cells at day 21 of the study shown in Figure 37A. [Figure 37C] Shown is the percentage of EMCN+ SEM cells at day 27 of the study shown in Figure 37A. [Figure 38A]Shown are the mean BLI measurements of mice on each day of the in vivo study in which mice were injected with MV4-11 cells and treated with NK cells transduced with each construct. [Figure 38B] 38A shows the quantified BLI signal of the mouse shown in FIG. 38A over the course of the in vivo study shown in FIG. 38A. [Figure 39A] BLI measurements of mice are shown on each day of an in vivo study in which mice were injected with MV4-11 cells and treated with NK cells transduced with each construct. [Figure 39B] Representative images are shown 55 days after tumor implantation of mice treated with PBS, untransduced NK cells, or NK cells transduced with SB07412 or SB07418. [Figure 39C] Survival curves and media survival (days) for the treatment groups tested are shown. [Figure 39D] Data was collected through day 120, showing a continuation of the study shown in Figure 39C. [Figure 40] 1 shows testing of NK cells expressing aCAR and different iCAR intracellular domains in a cytotoxicity assay. [Figure 41A] 1 shows a comparison of cell-mediated cytotoxicity against MOLM-13 using unmanipulated or manipulated NK cells. [Figure 41B] 1 shows a comparison of cell-mediated cytotoxicity against MV4-11 using unmanipulated or manipulated NK cells. [Figure 41C] 1 shows a comparison of cell-mediated cytotoxicity against SEM-CD33 using unmanipulated or manipulated NK cells. [Figure 41D] NK cytotoxicity of MOLM-13 cells at multiple E:T ratios. [Figure 41E] NK cytotoxicity of MV4-11 at multiple E:T ratios. [Figure 41F] Shows NK cytotoxicity of engineered and unengineered NK cells from multiple experiments plotted against CD33 CAR expression. [Figure 42]Heatmaps for visualizing fold expression analysis of cytokine production by NK cells after co-culture with MV4-11 or MOLM-13 are shown. [Figure 43A] NK cell cytotoxicity of engineered and unengineered NK cells from donor A co-cultured with primary AML samples. [Figure 43B] NK cell cytotoxicity of engineered and unengineered NK cells from donor B co-cultured with primary AML samples. [Figure 44] 1 shows a heatmap analysis of NK activation markers analyzed by flow cytometry. [Figure 45A] Figure 1 shows the expression of surface-associated IL15 on transduced NK cells derived from different donors. [Figure 45B] Figure 1 shows the expression of secreted IL15 in transduced NK cells derived from different donors. [Figure 45C] 1 shows the expression of CD33 / FLT3 bivalent aCAR on transduced NK cells derived from different donors. [Figure 45D] 1 shows the expression of EMCN iCAR in engineered NK cells derived from different donors. [Figure 46-1] Figure 46A shows the results of a cytotoxicity assay of engineered NK cells incubated with EMCN-expressing human stem cells, and Figure 46B shows the results of a cytotoxicity assay of engineered NK cells incubated with AML (leukemia cells). [Figure 46-2] Figure 46C shows the results of a cytotoxicity assay of engineered NK cells incubated with human stem cell progenitor cells. [Figure 46-3] Figure 46D shows the results of a cytotoxicity assay of engineered NK cells incubated with primary healthy MPPs. Results are shown for the entire healthy MPP population. [Figure 46-4] Figure 46E shows the results of a cytotoxicity assay of engineered NK cells incubated with primary healthy MPPs. Results for the healthy EMCN+ MPP population are shown. [Figure 47]Figure 1 shows EMCN expression on various hematopoietic stem and progenitor cell (HSPC) subpopulations in CD34-enriched primary human bone marrow cells across two representative donors, including hematopoietic progenitor cells (HPCs), lymphomyelo-primed progenitor cells (LMPPs), and multipotent progenitor cells (MPPs) in addition to HSCs. [Figure 48] Flow cytometry gating strategy for enumerating the number of viable, remaining EMCN+ HSCs after the killing assay is shown. From left to right and top to bottom, (1) cell size parameters, (2) single cells, (3) CD56-negative cells (i.e., not NK cells), (4) viable CD34+ undifferentiated cells, (5) CD45RA-CD38- cells (i.e., HSCs and MPPs), (6) CD90+ cells (i.e., HSCs), and (7) a progressive gate on EMCN+ HSCs. Gates for CD90 and EMCN were set using FMO controls. DETAILED DESCRIPTION OF THE INVENTION

[0052] Detailed Description The present disclosure provides a multicistronic expression system encoding (i) a membrane-cleavable chimeric protein, (ii) a bivalent aCAR, and (iii) an iCAR. The multicistronic expression system comprises a membrane-cleavable chimeric protein encoding an FLT3 OR CD33 NOT EMCN logic-gated CAR and IL-15.

[0053] Multicistronic expression systems are provided that include engineered nucleic acids encoding: (A) an exogenous polynucleotide, the exogenous polynucleotide encoding a membrane-cleavable chimeric protein oriented N-terminal to C-terminal and having the formula: SC-MT or MT-CS, wherein S comprises a secreted effector molecule, the secreted effector molecule comprises IL-15, C comprises a protease cleavage site, and MT comprises a cell membrane anchoring domain, wherein SC-MT or MT-CS is configured to be expressed as a single polypeptide; (B) an exogenous polynucleotide encoding an inhibitory chimeric antigen receptor (iCAR), wherein the iCAR comprises: (i) an antigen-binding domain specific for endomucin (EMCN), (ii) one or more intracellular inhibitory domains that inhibit an immune response, and (iii) one or more polypeptides selected from the group consisting of a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof; and (C) An exogenous polynucleotide encoding a bivalent activating chimeric antigen receptor (aCAR), wherein the aCAR comprises: (i) an antigen-binding domain specific for FLT3; (ii) an antigen-binding domain specific for CD33; (iii) one or more intracellular signaling domains that stimulate an immune response; and (iv) one or more polypeptides selected from the group consisting of a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

[0054] The expression systems described herein are multicistronic, meaning that more than one separate polypeptide (e.g., multiple chimeric proteins) can be produced from a single mRNA transcript. The engineered nucleic acid can be multicistronic through the use of various linkers, for example, a polynucleotide sequence encoding a first chimeric protein can be linked to a nucleotide sequence encoding a second chimeric protein (e.g., in a 5' to 3' direction: first gene: linker: second gene, etc.). 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 a 2A ribosomal skipping element, including, but not limited to, an E2A / T2A ribosomal skipping element. In certain embodiments, the E2A / T2A ribosome skipping element comprises the amino acid sequence of GSGQCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 219). One exemplary nucleic acid encoding an E2A / T2A ribosome skipping element is GGTAGCGGCCAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATCTAATCCTGGACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACGTGGAGGAAAACCCTGGACCT (SEQ ID NO: 220). In certain embodiments, the nucleic acid encoding the E2A / T2A ribosome 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 an 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). One exemplary nucleic acid encoding an E2A / T2A ribosomal skipping element is CAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATCTAATCCTGGACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACGTGGAGGAAAACCCTGGACCT (SEQ ID NO: 222). Another exemplary nucleic acid encoding an 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.

[0055] The linker can encode a cleavable linker polypeptide sequence, such as a furin cleavage site or a TEV cleavage site, and following expression, the cleavable linker polypeptide is cleaved so as to produce separate polypeptides encoded by the first and second genes. The cleavable linker can include a polypeptide sequence, such as a flexible linker (e.g., a Gly-Ser-Gly sequence), that further facilitates cleavage.

[0056] The linker can encode an internal ribosome entry site (IRES) so that separate polypeptides encoded by the first and second genes are produced during translation. The linker can encode a splice acceptor, such as a viral splice acceptor.

[0057] The linker can be a combination of linkers, such as a furin-2A linker, that can produce separate polypeptides through 2A ribosomal skipping followed by further cleavage of the furin site, allowing for complete removal of the 2A residue. In some embodiments, the linker combination can include a furin sequence, a flexible linker, and a 2A linker. Thus, in some embodiments, the linker is a furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, the linker of the present disclosure is a furin-Gly-Ser-Gly-T2A fusion polypeptide.

[0058] Generally, a multicistronic system can express any number of genes or portions thereof using any number or combination of linkers (e.g., an engineered nucleic acid can encode a first, second, and third chimeric protein, each separated by a linker, such that separate polypeptides encoded by the first, second, and third chimeric proteins are produced).

[0059] Generally, the exogenous polynucleotides encoding 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 a multicistronic system in the order of (i) membrane-cleavable chimeric protein, (ii) bivalent aCAR, and (iii) iCAR from 5' to 3'. In another example, the membrane-cleavable chimeric protein, iCAR, and bivalent aCAR can be encoded by a multicistronic system in the order of (i) membrane-cleavable chimeric protein, (ii) iCAR, and (iii) bivalent aCAR from 5' to 3'.

[0060] Membrane-cleavable chimeric proteins The multicistronic system herein encodes a membrane-cleavable chimeric protein having the formula SC-MT or MT-CS, oriented N- to C-terminally and expressed as a single polypeptide. S refers to a secreted effector molecule. C refers to a protease cleavage site. MT refers to a cell membrane-tethering domain. The membrane-cleavable chimeric protein is engineered so that secretion of the effector molecule can be regulated in a protease-dependent manner. Specifically, the membrane-cleavable chimeric protein is engineered so that secretion of the effector molecule can be regulated as part of a "membrane-cleavable" system, where incorporation of a protease cleavage site ("C") and a cell membrane-tethering domain ("MT") allows for regulated secretion of the effector molecule in a protease-dependent manner. Without wishing to be bound by theory, the components of the membrane-cleavable system present in the membrane-cleavable chimeric protein generally regulate secretion through the following cellular processes: - MT: Cell membrane anchoring domain comprises a transmembrane domain (or transmembrane-intracellular domain) that directs cellular transport of the chimeric protein so that the protein is inserted into or associated with (anchored to) the cell membrane. - C: Following expression and localization of the chimeric protein in the cell membrane, the protease cleavage site directs 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 desired kinetics of payload cleavage and release (e.g., ratio of membrane-bound to secreted chimeric protein levels).

[0061] Figure 17 illustrates a schematic diagram of a membrane-cleavable system described herein, in which a desired payload is expressed as a chimeric protein in which a protease cleavage site is inserted between the payload and the membrane-tethering domain. The left panel illustrates a schematic diagram of a membrane-cleavable system using a transmembrane structure (e.g., the chimeric protein includes a transmembrane domain). The right panel illustrates a schematic diagram of a membrane-cleavable system using a membrane-associated structure (e.g., the chimeric protein includes a post-translational modification tag that enables association with the cell membrane). Figure 18 illustrates a representative membrane-cleavable system for regulated secretion of IL-15, in which IL-15 is expressed as a chimeric protein with a cleavage site capable of cleavage by TACE inserted between the IL-15 payload and the membrane-tethering domain. The left panel illustrates a type I transmembrane structure (e.g., SC-MT) through the use of type I transmembrane domains, such as PDGFR-beta and CD8. The right panel shows type II transmembrane structures (eg, MT-CS) through the use of type II transmembrane domains such as NKG2D and TNFR2.

[0062] In some aspects, provided herein are membrane-cleavable chimeric proteins (or engineered nucleic acids encoding membrane-cleavable chimeric proteins) comprising a protein of interest (e.g., any of the effector molecules described herein), a protease cleavage site, and a cell membrane anchoring domain.

[0063] 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 molecule to which it binds. For example, an effector molecule may act as a ligand to increase or decrease enzymatic activity, gene expression, or cell signaling. Thus, in some embodiments, an effector molecule modulates (activates or inhibits) a different immune regulatory mechanism. By directly binding to and modulating a molecule, an effector molecule may also indirectly modulate a second, downstream molecule.

[0064] In certain embodiments described herein (e.g., generally for all membrane-cleavable chimeric proteins described herein), the effector molecule is a secreted effector molecule (e.g., referred to as "S" in the formula SC-MT or MT-CS for membrane-cleavable chimeric proteins described herein). Non-limiting examples of effector molecules include cytokines, chemokines, enzymes that regulate metabolite levels, growth factors, co-activation molecules, tumor microenvironment modifiers, ligands, peptides, enzymes, antibodies, cytokines, homing molecules, and / or antibodies or decoy molecules that regulate integrins.

[0065] The term "modulate" encompasses maintaining biological activity, inhibiting biological activity (partially or completely), and stimulating / activating biological activity (partially or completely). The term also encompasses decreasing or increasing (e.g., enhancing) biological activity. Two different effector molecules are considered to "modulate different tumor-mediated immunosuppressive mechanisms" if one effector molecule modulates a different tumor-mediated immunosuppressive mechanism (e.g., stimulating T cell signaling) than the tumor-mediated immunosuppressive mechanism modulated by the other effector molecule (e.g., stimulating antigen presentation and / or processing).

[0066] Modulation by an effector molecule can be direct or indirect. Direct modulation occurs when an effector molecule binds to another molecule and modulates the activity of that molecule. Indirect modulation occurs when an effector molecule binds to another molecule and modulates the activity of that molecule, which in turn modulates the activity of yet another molecule (to which the effector molecule is not bound).

[0067] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in an increase in immunostimulatory and / or anti-tumor immune response (e.g., systemically or in the tumor microenvironment) by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). For example, modulation of tumor-mediated immunosuppressive mechanisms can result in an increase in immunostimulatory and / or anti-tumor immune response by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in a 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% increase in immunostimulatory and / or anti-tumor immune responses. It should be understood that an "increase" in an immunostimulatory and / or anti-tumor immune response relates to an immunostimulatory and / or anti-tumor immune response that would otherwise occur in the absence of the effector molecule, e.g., systemically or in the tumor microenvironment.

[0068] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in an at least 2-fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100-fold) increase in immunostimulatory and / or anti-tumor immune response (e.g., systemically or in the tumor microenvironment). For example, modulation of tumor-mediated immunosuppressive mechanisms can result in an 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 increase in immunostimulatory and / or anti-tumor immune response. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in a 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100-fold increase in immunostimulatory and / or anti-tumor immune response.

[0069] Non-limiting examples of immunostimulatory and / or anti-tumor immune mechanisms include T cell signaling, activity, and / or recruitment, antigen presentation and / or processing, natural killer cell-mediated cytotoxic signaling, activity, and / or recruitment, dendritic cell differentiation and / or maturation, immune cell recruitment, proinflammatory macrophage signaling, activity, and / or recruitment, stromal degradation, production of immunostimulatory metabolites, stimulator of interferon genes (STING) signaling (which increases the secretion of IFN and Th1 polarization, promoting anti-tumor immune responses), and / or type I interferon signaling. Effector molecules can stimulate at least one (or more) of the aforementioned immunostimulatory mechanisms, thus resulting in an increase in the immunostimulatory response. Changes in the aforementioned immunostimulatory 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, expression assays (e.g., of specific markers), and / or cellular secretion assays (e.g., of cytokines).

[0070] In some embodiments, modulation of the tumor-mediated immunosuppressive mechanism by at least one effector molecule results in a decrease in the immunosuppressive response (e.g., systemically or in the tumor microenvironment) by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). For example, modulation of the tumor-mediated immunosuppressive mechanism can result in a decrease in the 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 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% reduction in the immunosuppressive response. It should be understood that a "reduction" in the immunosuppressive response is relative to the immunosuppressive response that would otherwise occur in the absence of effector molecules, e.g., systemically or in the tumor microenvironment.

[0071] In some embodiments, modulation of the tumor-mediated immunosuppressive mechanism by at least one effector molecule results in at least a 2-fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100-fold) decrease in the immunosuppressive response (e.g., systemically or in the tumor microenvironment). For example, modulation of the tumor-mediated immunosuppressive mechanism can result in at least a 3-fold, at least a 5-fold, at least a 10-fold, at least a 20-fold, at least a 50-fold, or at least a 100-fold decrease in the immunosuppressive response. In some embodiments, modulation of the tumor-mediated immunosuppressive mechanism results in a 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100-fold decrease in the immunosuppressive response.

[0072] 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, production / maintenance of tumor checkpoint molecules, myeloid-derived suppressor cell signaling, activity, and / or recruitment, immunosuppressive factor / metabolite production, and / or vascular endothelial growth factor signaling. Effector molecules can inhibit at least one (or more) of the aforementioned immunosuppressive mechanisms, thus resulting in a decrease in the immunosuppressive response. Alterations in the aforementioned immunosuppressive mechanisms can result, for example, in an increase in T cell proliferation and / or an increase in IFNγ production (negative costimulatory signaling, T reg cell signaling, and / or MDSCs); Annexin V / PI flow staining (pro-apoptotic signaling); flow staining for expression, e.g., PDL1 expression (production / maintenance of tumor checkpoint molecules); RNA via ELISA, LUMINEX®, qPCR, enzymatic assays, e.g., IDO tryptophan catabolism (immunosuppressant / metabolite production); and assays for phosphorylation of PI3K, Akt, p38 (VEGF signaling).

[0073] In some embodiments, effector molecules function additively: the effect of two effector molecules can, for example, be equal to the sum of the effect of the two effector molecules functioning separately. In other embodiments, effector molecules function synergistically: the effect of two effector molecules can, for example, be greater than the combined function of the two effector molecules.

[0074] Effector molecules that regulate tumor-mediated immunosuppressive mechanisms and / or modify the tumor microenvironment can be, for example, secreted factors (e.g., cytokines, chemokines, antibodies, and / or decoy receptors that regulate extracellular mechanisms involved in the immune system), inhibitors (e.g., antibodies, antibody fragments, ligands TRAP, and / or small blocking peptides), intracellular factors that control cellular states (e.g., microRNAs and / or transcription factors that regulate cellular states to enhance pro-inflammatory properties), factors packaged in exosomes (e.g., microRNAs, cytosolic 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).

[0075] In some embodiments, at least one of the effector molecules stimulates immune stimulatory mechanisms in the tumor microenvironment and / or inhibits immune suppressive mechanisms in the tumor microenvironment.

[0076] In some embodiments, at least one of the effector molecules (a) stimulates T cell signaling, activity, and / or recruitment; (b) stimulates antigen presentation and / or processing; (c) stimulates natural killer cell-mediated cytotoxicity 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 stromal degradation; (h) stimulates immunostimulatory metabolite production; (i) stimulates type I interferon signaling; (j) inhibits negative costimulatory signaling; (k) inhibits pro-apoptotic signaling of anti-tumor immune cells; (l) inhibits regulatory T (T reg(m) inhibit cell signaling, activity, and / or recruitment; (n) inhibit tumor checkpoint molecules; (o) 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.

[0077] In some embodiments, effector molecules can be selected from the following non-limiting classes of molecules: cytokines, antibodies, chemokines, nucleotides, peptides, and enzymes. Non-limiting examples of effector molecules from the foregoing classes are listed in Table 1, and specific sequences encoding exemplary effector molecules are listed in Table 2. Effector molecules can be human, such as those listed in Table 1 or Table 2, or the human equivalent of a mouse effector molecule listed in Table 1 or Table 2. Effector molecules can be human-derived, but are, for example, endogenous human effector molecules or effector molecules that have been modified and / or optimized for function, such as optimized for improved expression, modified for improved stability, or codon-modified in their signal sequences (see below). Various programs and algorithms for optimizing function are known to those of skill in the art and can be selected based on the desired improvement, such as codon optimization for a particular species (e.g., human, mouse, bacterial, etc.).

[0078] In some embodiments, the effector molecule comprises interleukin-12 (IL-12), e.g., p35 and p40 as a dimer, 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.

[0079] 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 IL-15 receptor alpha (IL-15Rα), such as the sushi domain as 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 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (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, the IL-15 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 the sequence AATTGGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAACAGCCTGTCCAGCAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACACCTCA (SEQ ID NO: 225).

[0080] 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 the sequence (SEQ ID NO: 227).

[0081] Table 1. Exemplary effector molecules TIFF2025513427000002.tif116159TIFF2025513427000003.tif217159

[0082] Table 2: Exemplary effector molecule sequences TIFF2025513427000004.tif91152TIFF2025513427000005.tif241152TIFF2025513427000006.tif240152TIFF2025513427000007.tif241152 TIFF2025513427000008.tif241152TIFF2025513427000009.tif241152TIFF2025513427000010.tif232152TIFF2025513427000011.tif239152 TIFF2025513427000012.tif236152TIFF2025513427000013.tif238152TIFF2025513427000014.tif238152TIFF2025513427000015.tif240152 TIFF2025513427000016.tif238152TIFF2025513427000017.tif233152TIFF2025513427000018.tif238152TIFF2025513427000019.tif151152

[0083] Secretion signal and signal anchor One or more effector molecules of the chimeric proteins provided herein can be secretory 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 for SC-MT) that directs newly synthesized proteins destined for secretion or membrane localization (also referred to as membrane insertion) into the appropriate protein processing pathway. For chimeric proteins having the formula MT-CS, the membrane-tethering domain generally has a signal anchor sequence (e.g., the signal anchor sequence of a type II transmembrane protein) that directs newly synthesized proteins destined for membrane localization into the appropriate protein processing pathway. For chimeric proteins having the formula SC-MT, a membrane-tethering domain having a reverse signal anchor sequence (e.g., the signal anchor sequence of a particular type III transmembrane protein) can be used, generally without a separate secretory signal peptide, thereby directing newly synthesized proteins destined for membrane localization into the appropriate protein processing pathway.

[0084] Generally, for all membrane-cleavable chimeric proteins described herein, one or more effector molecules are secretable effector molecules (referred to as "S" in the formula SC-MT or MT-CS). In embodiments involving two or more chimeric proteins, each chimeric protein may contain a secretion signal. In embodiments involving two or more chimeric proteins, each chimeric protein may contain a secretion signal such that each effector molecule is capable of being secreted from the engineered cell after cleavage of the protease cleavage site.

[0085] The secretory signal peptide operably associated with the effector molecule can be a native secretory signal peptide (e.g., a secretory signal peptide typically endogenously associated with a given effector molecule). The secretory signal peptide operably associated with the effector molecule can be a non-native secretory signal peptide, a native secretory signal peptide. The non-native secretory signal peptide can facilitate improved expression and function, such as sustained secretion, in a particular environment, such as a tumor microenvironment. Non-limiting examples of non-native secretory signal peptides are shown in Table 3.

[0086] The secretory signal peptide can be an IgE signal peptide. The IgE signal peptide can comprise the amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 228). The IgE signal peptide can be encoded by a polynucleotide sequence comprising 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).

[0087] The secretory 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 comprise 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 comprising 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 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).

[0088] Table 3. Exemplary signal secretion peptides TIFF2025513427000020.tif203155TIFF2025513427000021.tif214155TIFF2025513427000022.tif93155

[0089] Protease cleavage site 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 SC-MT or MT-CS for the membrane-cleavable chimeric proteins described herein). Generally, a protease cleavage site can be any amino acid sequence motif capable of being cleaved by a protease. Examples of protease cleavage sites include 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, an ADAM40 protease cleavage site, an ADAM42 protease cleavage site, an ADAM43 protease cleavage site, an ADAM44 protease cleavage site, an ADAM45 protease cleavage site, an ADAM46 protease cleavage site, an ADAM47 protease cleavage site, an ADAM48 protease cleavage site, an ADAM49 ... Protease cleavage sites include, but are not limited to, a BACE1 protease cleavage site, a BACE2 protease cleavage site, a SIP protease cleavage site, an MT1-MMP protease cleavage site, an MT3-MMP protease cleavage site, an MT5-MMP protease cleavage site, a furin protease cleavage site, a PCSK7 protease cleavage site, a matriptase protease cleavage site, a matriptase-2 protease cleavage site, an MMP9 protease cleavage site, or an NS3 protease cleavage site.

[0090] One example of a protease cleavage site is the hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease cleavage site, including, but not limited to, the NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, or NS5A / NS5B cleavage site. For a description of representative sequences of NS3 proteases and their cleavage sites for various strains of HCV, see, for example, Hepatitis C Viruses: Genomes and Molecular Biology (SLTan ed., Taylor & Francis, 2006), Chapter 6, pp. 163-206; the entire contents of which are incorporated herein by reference. For example, the 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, e.g., NCBI entries: Accession Numbers: YP_001491553, YP_001469631, YP_001469632, NP_803144, NP_671491, YP_001469634, YP_001469630, YP_001469633, ADA68311, ADA68307, AFP99000, AFP98987, ADA68322, AFP99033, ADA68330, AFP99056, AFP99041, CBF60982, CBF60817, A See HH29575, AIZ00747, AIZ00744, ABI36969, ABN05226, KF516075, KF516074, KF516056, AB826684, AB826683, JX171009, JX171008, JX171000, EU847455, EF154714, GU085487, JX171065, JX171063; all of the sequences (as entered by the filing date of this application) are incorporated herein by reference.

[0091] Another example of a protease cleavage site is an ADAM17-specific protease (also referred to as tumor necrosis factor alpha-converting enzyme [TACE]) cleavage site. The ADAM17-specific protease cleavage site can be an endogenous sequence of a substrate naturally cleaved by ADAM17. The ADAM17-specific protease cleavage site can be an engineered sequence capable of being cleaved by ADAM17. The engineered ADAM17-specific protease cleavage site can be engineered for specific desired properties, including, but not limited to, optimal expression of the chimeric protein, specificity for ADAM17, cleavage rate by ADAM17, the ratio of secreted and membrane-bound chimeric protein levels, and cleavage in different cellular conditions. The protease cleavage site can be selected for specific cleavage by ADAM17. For example, a specific protease cleavage site capable of being cleaved by ADAM17 can also be cleaved by additional ADAM family proteases, such as ADAM10. Thus, an ADAM17-specific protease cleavage site can be selected and / or engineered to reduce or eliminate cleavage by other proteases, such as ADAM10. The protease cleavage site can be selected for the rate of cleavage by ADAM17. For example, it may be desirable to select a protease cleavage site that exhibits a particular cleavage rate by ADAM17, such as reduced cleavage kinetics with respect to the endogenous sequence of a substrate that is naturally cleaved by ADAM17. In such cases, a particular cleavage rate can generally be selected to regulate the processing rate of the chimeric protein, which in turn regulates the release / secretion rate of the payload effector molecule. Thus, an ADAM17-specific protease cleavage site can be selected and / or engineered so that the sequence exhibits a desired cleavage rate by ADAM17. The protease cleavage site can be selected for both specific cleavage by ADAM17 and the rate of cleavage by ADAM17. Exemplary ADAM17-specific protease cleavage sites, including those exhibiting particular specificity and cleavage rate kinetics, are shown in Table 4A below, with reference to the cleavage site (P5-P1: N-terminus; P1'-P5': C-terminus).Further details of ADAM17 and ADAM10, including expression and protease cleavage sites, are described in Sharma, et al. (J Immunol October 15, 2017, 199(8)2865-2872), Pham et al. (Anticancer Res. 2017 Oct;37(10):5507-5513), Caescu et al. (Biochem J. 2009 Oct 23;424(1):79-88), and Tucher et al. (J. Proteome Res. 2014, 13,4,2205-2214), each of which is incorporated herein by reference for the purpose.

[0092] Table 4A: Various ADAM17 protease cleavage site sequences TIFF2025513427000023.tif99135

[0093] In some embodiments, the protease cleavage site comprises a first region having the amino acid sequence of PRAE (SEQ ID NO: 176). In some embodiments, the protease cleavage site comprises a second region having the amino acid sequence of KGG (SEQ ID NO: 177). In some embodiments, the first region is located N-terminal to the second region. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 178), wherein 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 the 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 the amino acid sequence of PRAEAVKGG (SEQ ID NO: 179). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEALKGG (SEQ ID NO: 180). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEYSKGG (SEQ ID NO: 181). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEPIKGG (SEQ ID NO: 182). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAYKGG (SEQ ID NO: 183). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAESSKGG (SEQ ID NO: 184). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEFTKGG (SEQ ID NO: 185). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAAKGG (SEQ ID NO: 186).

[0094] 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.

[0095] In some embodiments, the protease cleavage site may comprise an N-terminal peptide linker such as SGGGGSGGGGSG (SEQ ID NO: 230). In some embodiments, the protease cleavage site may comprise a C-terminal peptide linker such as GGGSGGGGSGGGSLQ (SEQ ID NO: 231). In some embodiments, the protease cleavage site may comprise an N-terminal peptide linker and a C-terminal peptide linker such as both (SEQ ID NO: 230) and GGGSGGGGSGGGSLQ (SEQ ID NO: 231).

[0096] In some embodiments, the protease cleavage site may comprise 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 comprise the TACE-specific cleavage site VTPEPIFSLI (SEQ ID NO: 191), an N-terminal peptide linker, and a C-terminal peptide linker. In some embodiments, the protease cleavage site may comprise the TACE-specific cleavage site, an N-terminal peptide linker, and a C-terminal peptide linker, and have the amino acid sequence SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ (SEQ ID NO: 250). In some embodiments, the protease cleavage site may comprise a TACE-specific cleavage site, an N-terminal peptide linker, and a C-terminal peptide linker encoded by a polynucleotide sequence comprising the sequence TCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGTGACCCCTGAGCCTATCTTCAGCCTGATCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAA (SEQ ID NO: 251). In some embodiments, the protease cleavage site may comprise a TACE-specific cleavage site, an N-terminal peptide linker, and a C-terminal peptide linker encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to TCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGTGACCCCTGAGCCTATCTTCAGCCTGATCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAA (SEQ ID NO: 251).

[0097] In some embodiments, the protease cleavage site comprises the amino acid sequence ITQGLAVSTISSFF (SEQ ID NO: 198), which is a cleavage site native to CD16 and cleavable by ADAM17.

[0098] The protease cleavage site can be at the C-terminus of the secreted effector molecule. The protease cleavage site can be at the N-terminus of the secreted effector molecule. Generally, for all membrane-cleavable chimeric proteins described herein, the protease cleavage site is either: (1) at the C-terminus of the secreted effector molecule and the N-terminus of the cell membrane-tethering domain (in other words, the protease cleavage site is between the secreted effector molecule and the cell membrane-tethering domain); or (2) at the N-terminus of the secreted effector molecule and the C-terminus of the cell membrane-tethering domain (also between the secreted effector molecule and the cell membrane-tethering domain with an inverted domain orientation). The protease cleavage site can be connected to the secreted effector molecule by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the effector molecule or the protease cleavage site. The protease cleavage site can be connected to the cell membrane anchoring domain by a polypeptide linker, i.e., a polypeptide sequence not generally considered part of the cell membrane anchoring domain or protease cleavage site. The polypeptide linker can be any amino acid sequence that connects a first polypeptide sequence and a 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]GG [SEQ ID NO: 182]), A(EAAAK)A (SEQ ID NO: 183), and a Whitlow linker (e.g., a "KEGS" linker, e.g., the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), an eGK linker, e.g., the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 185), and linkers described in more detail in issued U.S. Patent No. 5,990,275, which is incorporated herein by reference). Additional exemplary polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, and SEQ ID NO: 197. Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition, etc.) and are known to those of skill in the art.

[0099] In membrane-cleavable systems, following expression and localization of the chimeric protein in the cell membrane, the protease cleavage site directs cleavage of the chimeric protein, allowing the effector molecule to be released (secreted) into the extracellular space of the cell.

[0100] Generally, a protease that cleaves a protease cleavage site is a protease specific for that particular protease cleavage site. For example, in the case of a disintegrin and metalloproteinase (ADAM) family proteases, proteases that cleave a particular ADAM protease cleavage site are generally limited to ADAM proteases that specifically recognize a particular ADAM protease cleavage site motif. The protease cleavage site can be selected and / or engineered to reduce or eliminate cleavage by undesired proteases. The protease can be membrane-bound or membrane-associated. The protease can be, for example, secreted in a particular cellular environment, such as the tumor microenvironment ("TME").

[0101] The protease that cleaves the protease cleavage site of the chimeric protein can be expressed in the same cell that expresses the chimeric protein. The protease that cleaves the protease cleavage site of the chimeric protein can be endogenous to the cell that expresses the chimeric protein. In other words, cells engineered to express the chimeric protein can endogenously express a protease specific for the protease cleavage site present in the chimeric protein. Endogenous expression of a protease generally refers to both expression under homeostatic conditions (e.g., cells generally considered healthy) and differential expression under non-homeostatic conditions (e.g., upregulated expression in tumor cells). The protease cleavage site can be selected based on known proteases endogenously expressed by the desired cell population. In such cases, cleavage of the protease cleavage site (and thus release / secretion of the payload) can generally be restricted to only the cells of interest due to the cell-restricted protease needing to contact the protease cleavage site of the chimeric protein expressed in the same cells. For example, without wishing to be bound by theory, it is believed that ADAM17 is restricted in its endogenous expression to NK cells and T cells. Thus, selection of an ADAM17-specific protease cleavage site can restrict cleavage of the protease cleavage site to NK cells and T cells that co-express the chimeric protein. In another example, the protease cleavage site can be selected for a particular tumor-associated protease known to be expressed in a particular tumor population of interest (e.g., in particular tumor cells engineered to express the chimeric protein).Suitable protease cleavage sites are selected using protease and / or expression databases, for example, by consulting Oncomine (www.oncomine.org), the European Bioinformatics Institute (www.ebi.ac.uk), in particular (www.ebi.ac.uk / gxa), PMAP (www.proteolysis.org), ExPASy Peptide Cutter (ca.expasy.org / tools / peptide cutter), and PMAP.Cut DB (cutdb.burnham.org), each of which is incorporated by reference for all purposes, to select suitable protease cleavage sites that are cleaved by tumor-associated proteases.

[0102] The protease that cleaves the protease cleavage site of the chimeric protein can be heterologous to the cell expressing the chimeric protein. For example, a cell engineered to express a chimeric protein can be engineered to express a protease not generally expressed by the cell that is specific for the protease cleavage site present in the chimeric protein. Cells engineered to express both a chimeric protein and a protease can be engineered to express each from separate engineered nucleic acids or from a multicistronic system (multicistronic and multipromoter systems are described in more detail herein in the section entitled "Multicistronic and Multipromoter Systems"). Heterologous proteases and their corresponding protease cleavage sites can be selected as described above with reference to endogenous proteases.

[0103] The protease that cleaves the protease cleavage site of the chimeric protein can be expressed on a different cell than the cell that expresses the chimeric protein. For example, the protease can be generally expressed in a specific cellular environment, such as the tumor microenvironment. In such cases, cleavage of the protease cleavage site can generally be restricted to only the cellular environment of interest (e.g., the tumor microenvironment) due to the environmentally restricted protease that must contact the protease cleavage site. In embodiments with a membrane-cleavable chimeric protein, secretion of the effector molecule can generally be restricted to only the cellular environment of interest (e.g., the tumor microenvironment) due to the environmentally restricted protease that must contact the protease cleavage site. The protease that cleaves the protease cleavage site of the chimeric protein can be endogenous to the different cells. The protease that cleaves the protease cleavage site of the chimeric protein can be heterologous to the different cells. For example, the different cells can be engineered to express a protease that is not generally expressed by the different cells.

[0104] Proteases include, but are not limited to, type 1 transmembrane proteases, type II transmembrane proteases, GPI-anchored proteases, 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 protease, PCSK7 protease, matriptase protease, matriptase 2 protease, and MMP9 protease. The protease may be an NS3 protease. The protease may be an ADAM17 protease.

[0105] The protease can be a tumor-associated protease, such as a cathepsin, a cysteine ​​protease, an aspartyl protease, a serine protease, or a metalloprotease. Specific examples of tumor-associated proteases include cathepsin B, cathepsin L, cathepsin S, cathepsin D, cathepsin E, cathepsin A, cathepsin G, thrombin, plasmin, urokinase, tissue plasminogen activator, metalloproteinase 1 (MMP1), MMP2, MMP3, MMP4, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP20, MMP21, MMP23, MMP24, MMP25, MMP26, MMP28, ADAM, ADAMTS, CD10 (CALLA), or prostate-specific antigen. Proteases also include, but are not limited to, the proteases listed in Table 4B below. Exemplary cognate protease cleavage sites for particular proteases are also listed in Table 4B.

[0106] Table 4B: Exemplary proteases and inhibitors with cognate cleavage sites TIFF2025513427000024.tif127164TIFF2025513427000025.tif246164TIFF20255134270 00026.tif223164TIFF2025513427000027.tif204164TIFF2025513427000028.tif242164 TIFF2025513427000029.tif222164TIFF2025513427000030.tif227164TIFF20255134270 00031.tif242164TIFF2025513427000032.tif223164TIFF2025513427000033.tif139164

[0107] The protease can be any of the following human proteases (MEROPS peptidase database numbers provided in parentheses; Rawlings ND, Morton FR, Kok, CY, Kong, J. & Barrett AJ (2008) MEROPS: the peptidase database. Nucleic Acids Res. 36 Database issue, D320-325; incorporated herein by reference for all purposes): pepsin A (MER000885), gastricin (MER000894), memapsin-2 (MER005870), renin (MER000917), cathepsin D (MER000911), cathepsin E (MER000944), memapsin-1 (MER005534), napsin A (MER004981), Mername-AA034 peptidase (MER014038), pepsin A4 (MER037290), pepsin A5 (Homo sapiens sapiens) (MER037291), hCG1733572 (Homo sapiens) type putative peptidase (MER107386), napsin B pseudogene (MER004982), CYMP gp (Homo sapiens) (MER002929), subfamily A1A unassigned 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 putative RTVL-H type peptidase (MER047206), putative RTVL-H type peptidase (MER047253), putative RTVL-H type peptidase (MER047260), putative RTVL-H type peptidase (MER047291), putative RTVL-H type peptidase (MER047418), putative RTVL-H type peptidase (MER047440), putative RTVL-H type peptidase (MER047479), putative RTVL-H type peptidase (MER047559), putative RTVL-H type 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) (MER002094), endogenous retroviral retropepsin pseudogene 7 (Homo sapiens chromosome 6) (MER029776 ...12) (MER029664), endogenous retroviral retropepsin pseudogene 6 (Homo sapiens chromosome 12) (MER029664), endogen Viral 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 14) (MER003344), endogenous retroviral retropepsin pseudogene 14 (Homo sapiens chromosome 15) (MER003344), endogenous retroviral retropepsin pseudogene 15 (Homo sapiens chromosome 16) (MER003344), endogenous retroviral retropepsin pseudogene 16 (Homo sapiens chromosome 17) (MER004378), endogenous retroviral retropepsin pseudogene 17 (Homo sapiens chromosome 18) (MER003344), endogenous retroviral retropepsin pseudogene 18 (Homo sapiens chromosome 19) (MER029680), endogenous retroviral retropepsin pseudogene 19 (Homo sapiens chromosome 19) (MER029680), endogenous retroviral retropepsin pseudogene 20 (Homo sapiens chromosome 21) (MER003344), endogenous retroviral retropepsin pseudogene 21 (Homo sapiens chromosome 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 retropepsin pseudogene 16 (MER047200), endogenous retroviral retropepsin pseudogene 16 (MER047315), endogenous retroviral retropepsin pseudogene 16 (MER047405), endogenous retroviral retropepsin pseudogene 16 (MER030292), endogenous retroviral retropepsin pseudogene 17 (Homo sapiens chromosome 8) (MER005305), endogenous retroviral retropepsin pseudogene 18 (Homo sapiens chromosome 4) (MER030288), endogenous retroviral retropepsin 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) (MER 047477), 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 (MER047076), subfamily A2A nonpeptidase homolog (MER047080), subfamily A2A nonpeptidase peptidase homolog (MER047088), subfamily A2A nonpeptidase homolog (MER047089), subfamily A2A nonpeptidase homolog (MER047091), subfamily A2A nonpeptidase homolog (MER047092), subfamily A2A nonpeptidase homolog (MER047093), subfamily A2A nonpeptidase homolog (MER047094), subfamily A2A nonpeptidase homolog (MER047097), subfamily A2A nonpeptidase homolog (MER047099),Subfamily A2A nonpeptidase homologue MER047101), subfamily A2A nonpeptidase homologue (MER047102), subfamily A2A nonpeptidase homologue (MER047107), subfamily A2A nonpeptidase homologue (MER047108), subfamily A2A nonpeptidase homologue (MER047109), subfamily A2A nonpeptidase homologue (MER047110), subfamily A2A nonpeptidase homologue MER047111), subfamily A2A nonpeptidase homologue (MER0 47114), subfamily A2A nonpeptidase homolog (MER047118), subfamily A2A nonpeptidase homolog (MER047121), subfamily A2A nonpeptidase homolog (MER047122), subfamily A2A nonpeptidase homolog (MER047126), subfamily A2A nonpeptidase homolog (MER047129), subfamily A2A nonpeptidase homolog (MER047130), subfamily A2A nonpeptidase homolog (MER047134), subfamily A2A nonpeptidase Homologue (MER047135), Subfamily A2A nonpeptidase homologue (MER047137), Subfamily A2A nonpeptidase homologue (MER047140), Subfamily A2A nonpeptidase homologue (MER047141), Subfamily A2A nonpeptidase homologue (MER047142), Subfamily A2A nonpeptidase homologue (MER047148), Subfamily A2A nonpeptidase homologue (MER047149), Subfamily A2A nonpeptidase homologue (MER047151), Subfamily A 2A nonpeptidase homolog (MER047154), subfamily A2A nonpeptidase homolog (MER047155), subfamily A2A nonpeptidase homolog (MER047156), subfamily A2A nonpeptidase homolog (MER047157), subfamily A2A nonpeptidase homolog (MER047159), subfamily A2A nonpeptidase homolog (MER047161), subfamily A2A nonpeptidase homolog (MER047163), subfamily A2A nonpeptidase homolog (MER047166),Subfamily A2A nonpeptidase homolog (MER047171), Subfamily A2A nonpeptidase homolog (MER047173), Subfamily A2A nonpeptidase homolog (MER047174), Subfamily A2A nonpeptidase homolog (MER047179), Subfamily A2A nonpeptidase homolog (MER047183), Subfamily A2A nonpeptidase homolog (MER047186), Subfamily A2A nonpeptidase homolog (MER047190), Subfamily A2A nonpeptidase homolog (MER 047191), subfamily A2A nonpeptidase homolog (MER047196), subfamily A2A nonpeptidase homolog (MER047198), subfamily A2A nonpeptidase homolog (MER047199), subfamily A2A nonpeptidase homolog (MER047201), subfamily A2A nonpeptidase homolog (MER047202), subfamily A2A nonpeptidase homolog (MER047203), subfamily A2A nonpeptidase homolog (MER047204), subfamily A2A nonpeptidase Subfamily A2A nonpeptidase homolog (MER047205), subfamily A2A nonpeptidase homolog (MER047207), subfamily A2A nonpeptidase homolog (MER047208), subfamily A2A nonpeptidase homolog (MER047210), subfamily A2A nonpeptidase homolog (MER047211), subfamily A2A nonpeptidase homolog (MER047212), subfamily A2A nonpeptidase homolog (MER047213), subfamily A2A nonpeptidase homolog (MER047215), subfamily A 2A nonpeptidase homolog (MER047216), subfamily A2A nonpeptidase homolog (MER047218), subfamily A2A nonpeptidase homolog (MER047219), subfamily A2A nonpeptidase homolog (MER047221), subfamily A2A nonpeptidase homolog (MER047224), subfamily A2A nonpeptidase homolog (MER047225), subfamily A2A nonpeptidase homolog (MER047226), subfamily A2A nonpeptidase homolog (MER047227),Subfamily A2A nonpeptidase homolog (MER047230), Subfamily A2A nonpeptidase homolog (MER047232), Subfamily A2A nonpeptidase homolog (MER047233), Subfamily A2A nonpeptidase homolog (MER047234), Subfamily A2A nonpeptidase homolog (MER047236), Subfamily A2A nonpeptidase homolog (, MER047238), subfamily A2A nonpeptidase homolog (MER047239), subfamily A2A nonpeptidase homolog (MER047240), subfamily A2A nonpeptidase homolog (MER047242), subfamily A2A nonpeptidase homolog (MER047243), subfamily A2A nonpeptidase homolog (MER047249), subfamily A2A nonpeptidase homolog (MER047251), subfamily A2A nonpeptidase homolog (MER047252), subfamily A2A nonpeptidase homolog (MER047254), subfamily A2A nonpeptidase homolog (MER047255), subfamily A2A nonpeptidase homolog (MER047263), subfamily A2A nonpeptidase homolog (MER047265), subfamily A2A nonpeptidase homolog (MER047266), subfamily A2A nonpeptidase homolog (MER047267), subfamily A2A nonpeptidase homolog (MER047268), subfamily A2A nonpeptidase homolog (MER047269). 7269), subfamily A2A nonpeptidase 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 peptidase homolog (MER047281), subfamily A2A nonpeptidase homolog (MER047282), subfamily A2A nonpeptidase homolog (MER047284), subfamily A2A nonpeptidase homolog (MER047285), subfamily A2A nonpeptidase homolog (MER047289), subfamily A2A nonpeptidase homolog (MER047290), subfamily A2A nonpeptidase homolog (MER047294), subfamily A2A nonpeptidase homolog (MER047295),Subfamily A2A nonpeptidase homolog (MER047298), Subfamily A2A nonpeptidase homolog (MER047300), Subfamily A2A nonpeptidase homolog (MER047302), Subfamily A2A nonpeptidase homolog (MER047304), Subfamily A2A nonpeptidase homolog (MER047305), Subfamily A2A nonpeptidase homolog (MER047306), Subfamily A2A nonpeptidase homolog (MER047307), Subfamily A2A nonpeptidase homolog (MER 047310), subfamily A2A nonpeptidase homolog (MER047311), subfamily A2A nonpeptidase homolog (MER047314), subfamily A2A nonpeptidase homolog (MER047318), subfamily A2A nonpeptidase homolog (MER047320), subfamily A2A nonpeptidase homolog (MER047321), subfamily A2A nonpeptidase homolog (MER047322), subfamily A2A nonpeptidase homolog (MER047326), subfamily A2A nonpeptidase Subfamily A2A nonpeptidase homolog (MER047327), subfamily A2A nonpeptidase homolog (MER047330), subfamily A2A nonpeptidase homolog (MER047333), subfamily A2A nonpeptidase homolog (MER047362), subfamily A2A nonpeptidase homolog (MER047366), subfamily A2A nonpeptidase homolog (MER047369), subfamily A2A nonpeptidase homolog (MER047370), subfamily A2A nonpeptidase homolog (MER047371), subfamily A 2A nonpeptidase homolog (MER047375), subfamily A2A nonpeptidase homolog (MER047376), subfamily A2A nonpeptidase homolog (MER047381), subfamily A2A nonpeptidase 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 (MER047403), Subfamily A2A nonpeptidase homolog (MER047406), Subfamily A2A nonpeptidase homolog (MER 047407), Subfamily A2A nonpeptidase homolog (MER047410), Subfamily A2A nonpeptidase homolog (MER047411), Subfamily A2A nonpeptidase homolog (MER047413), Subfamily A2A nonpeptidase homolog (MER047414), Subfamily A2A nonpeptidase homolog (MER047416), Subfamily A2A nonpeptidase homolog (MER047417), Subfamily A2A nonpeptidase homolog (MER047420), Subfamily A2A nonpeptidase Subfamily A2A nonpeptidase homolog (MER047423), subfamily A2A nonpeptidase homolog (MER047424), subfamily A2A nonpeptidase homolog (MER047428), subfamily A2A nonpeptidase homolog (MER047429), subfamily A2A nonpeptidase homolog (MER047431), subfamily A2A nonpeptidase homolog (MER047434), subfamily A2A nonpeptidase homolog (MER047439), subfamily A2A nonpeptidase homolog (MER047442), subfamily A 2A nonpeptidase homolog (MER047445), subfamily A2A nonpeptidase homolog (MER047449), subfamily A2A nonpeptidase homolog (MER047450), subfamily A2A nonpeptidase homolog (MER047452), subfamily A2A nonpeptidase homolog (MER047455), subfamily A2A nonpeptidase homolog (MER047457), subfamily A2A nonpeptidase homolog (MER047458), subfamily A2A nonpeptidase homolog (MER047459),Subfamily A2A nonpeptidase homolog (MER047463), Subfamily A2A nonpeptidase homolog (MER047468), Subfamily A2A nonpeptidase homolog (MER047469), Subfamily A2A nonpeptidase homolog (MER047470), Subfamily A2A nonpeptidase homolog (MER047476), Subfamily A2A nonpeptidase homolog (MER047478), Subfamily A2A nonpeptidase homolog (MER047483), Subfamily A2A nonpeptidase homolog (MER 047488), 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 Subfamily A2A nonpeptidase homolog (MER047499), subfamily A2A nonpeptidase homolog (MER047502), subfamily A2A nonpeptidase homolog (MER047504), subfamily A2A nonpeptidase homolog (MER047511), subfamily A2A nonpeptidase homolog (MER047513), subfamily A2A nonpeptidase homolog (MER047514), subfamily A2A nonpeptidase homolog (MER047515), subfamily A2A nonpeptidase homolog (MER047516), subfamily A 2A nonpeptidase homolog (MER047520), subfamily A2A nonpeptidase homolog (MER047533), subfamily A2A nonpeptidase 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 nonpeptidase homolog (MER047662), Subfamily A2A nonpeptidase homolog (ME R048004), subfamily A2A nonpeptidase homolog (MER048018), subfamily A2A nonpeptidase homolog (MER048019), subfamily A2A nonpeptidase homolog (MER048023), subfamily A2A nonpeptidase homolog (MER048037), subfamily A2A unassigned peptidase (MER047164), subfamily A2A unassigned peptidase (MER047231), subfamily A2A unassigned peptidase (MER047386), skin aspartate protease tease (MER057097), presenilin 1 (MER005221), presenilin 2 (MER005223), impas1 peptidase (MER019701), impas1 peptidase (MER184722), impas4 peptidase (MER019715), impas2 peptidase (MER019708), impas5 peptidase (MER019712), impas3 peptidase (MER019711), possible family A22 pseudogene (Homo sapiens chromosome 18) (MER029974), possible A 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) (MER02945, 7), 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 (MER020139 ), calpain-14 (MER029744), Mername-AA253 peptidase (MER005537), carpamodulin (MER000718), hypothetical protein 940251 (MER003201), ubiquitinyl hydrolase-L1 (MER000832), ubiquitinyl hydrolase-L3 (MER000836), ubiquitinyl hydrolase-BAP1 (MER003989), ubiquitinyl 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 9 X (MER005877), ubiquitin-specific peptidase 10 (MER004439), ubiquitin-specific peptidase 1 (MER004978), 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 (MER01403 3), ubiquitin-specific peptidase 32 (MER014290), ubiquitin-specific peptidase 26 (Homo sapiens type) (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 (MER014634), ubiquitin-specific peptidase 47 (MER014636), ubiquitin-specific peptidase 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 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 [misread] (MER064621), Mername-AA090 non-peptidase homolog (MER014739), ubiquitin-specific peptidase 43 [misread] (MER030140), ubiquitin-specific peptidase 52 [misread] (MER030317), NEK2 pseudogene (MER 014736), C19 pseudogene (Homo sapiens: chromosome 5) (MER029972), Mername-AA088 peptidase (MER014750), autophagin-2 (MER013564), autophagin-1 (MER013561), autophagin-3 (MER014316), autophagin-4 (MER064622), Cezanne deubiquitinating peptidase (MER02904 2), 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 idase (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), ataxin-3 (MER099998), ATXN3L putative peptidase (MER115261), Josephin domain-containing 1 (Homo sapiens) (MER125334), Josephin domain-containing 2 (Homo sapiens) (MER124068), YOD1 peptidase (MER116559), legumain (plant alpha type) (MER044591), legumain (MER001800), glycosylphosphatidylinositol:protein transamidase (MER002479),legumain pseudogene (Homo sapiens) (MER029741), family C13 unassigned peptidase (MER175813), caspase-1 (MER000850), caspase-3 (MER000853), caspase-7 (MER002705), caspase-6 (MER002708), caspase-2 (MER001644), caspase-4 (MER001938), caspase-5 (MER002240), caspase-8 (MER002849), caspase-9 (MER002707), caspase-10 (MER001644), R002579), caspase-14 (MER012083), paracaspase (MER019325), Mername-AA143 peptidase (MER021304), Mername-AA186 peptidase (MER020516), putative caspase (Homo sapiens) (MER021463), FLIP protein (MER003026), Mername-AA142 protein (MER021316), caspase-12 pseudogene (Homo sapiens) (MER019698), Mername-AA093 caspase pseudogene child (MER014766), subfamily C14A nonpeptidase homolog (MER185329), subfamily C14A nonpeptidase homolog (MER179956), separase (Homo sapiens type) (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 (chordate) (MER011032), Mername-AA073 peptidase (MER029978), sonic hedgehog protein (MER002539), Indian hedgehog protein (MER002538), desert hedgehog protein (MER012170), dipeptidyl-peptidase III (MER004252),Mername-AA164 protein (MER020410), LOC138971 gp (Homo sapiens) (MER020074), Atp23 peptidase (MER060642), prenylpeptidase 1 (MER004246), aminopeptidase N (MER000997), aminopeptidase A (MER001012), leukotriene A4 hydrolase (MER001013), pyroglutamyl-peptidase II (MER012221), cytosolic alanyl aminopeptidase (MER002746), cystinyl aminopeptidase (MER002060), aminopeptidase B (M ER001494), aminopeptidase PILS (MER005331), arginyl aminopeptidase-like 1 (MER012271), leukocyte-derived arginine aminopeptidase (MER002968), aminopeptidase Q (MER052595), aminopeptidase O (MER019730), Tata binding protein-related factor (MER026493), angiotensin-converting enzyme peptidase unit 1 (MER004967), angiotensin-converting enzyme peptidase unit 2 (MER001019), and Geotensin-converting enzyme-2 (MER011061), Mername-AA153 protein (MER020514), Thimet oligopeptidase (MER001737), Neurolysin (MER010991), Mitochondrial intermediate peptidase (MER003665), Mername-AA154 protein (MER021317), Reishimanolysin-2 (MER014492), Reishimanolysin-3 (MER180031), Matrix metallopeptidase-1 (MER001063), Matrix metallo Peptidase-8 (MER001084), matrix metallopeptidase-2 (MER001080), matrix metallopeptidase-9 (MER001085), matrix metallopeptidase-3 (MER001068), matrix metallopeptidase-10 (Homo sapiens type) (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 (MER0030, 21), matrix metallopeptidase-19 (MER002076), matrix metallopeptidase-23B (MER004766), membrane-type matrix metallopeptidase-5 (MER005638), membrane-type matrix metallopeptidase-6 (MER012071), matrix metallopeptidase-21 (MER006101), matrix metallopeptidase-22 (MER014098), matrix metallopeptidase-26 (MER012072), matrix metallopeptidase-28 (MER013587), matrix metallopeptidase-29 (MER013588), matrix metallopeptidase-30 (MER013589), matrix metallopeptidase-40 (MER013589), matrix metallopeptidase-50 (MER014098), matrix metallopeptidase-60 (MER012071), matrix metallopeptidase-21 (MER006101), matrix metallopeptidase-22 (MER014098), matrix metallopeptidase-26 (MER012072), matrix metallopeptidase-28 (MER013587), matrix metallopeptidase-29 (MER013589), matrix metallopeptidase-30 (MER013589), matrix metallopeptidase-40 (MER014098 ... 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 M10A nonpeptidase homolog (MER187997), Subfamily M10A nonpeptidase homolog (MER187998), Subfamily M10A nonpeptidase idase homolog (MER180000), meprin alpha subunit (MER001111), meprin 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 (MER01 5689), 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 type) (MER005495), ADAMTS5 peptidase (MER005548), ADAMTS8 peptidase (MER005545), ADAMTS6 peptidase (MER005893), ADAMTS7 Peptidase (MER005894), ADAM30 peptidase (MER006268), ADAM21 peptidase (Homo sapiens type) (MER004726), ADAMTS10 peptidase (MER014331), ADAMTS12 peptidase (MER014337), ADAMTS13 peptidase (MER015450), ADAM33 peptidase (MER015143), ovastacin (MER029996), ADAMTS20 peptidase (Homo sapiens type) (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) (MER005199), 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), ADAM3A gp (Homo sapiens) (MER005200), ADAM1 gp (Homo sapiens) (MER003912), subfamily M12B nonpeptidase homolog (MER188210), subfamily M12B nonpeptidase homolog (MER188211), subfamily M12B nonpeptidase homolog (MER188212), subfamily M12B nonpeptidase homolog (MER188220), neprilysin (MER001050), endothelin-converting enzyme 1 (MER001057), endothelin-converting enzyme 2 (MER004776), DINE-P peptidase (MER005197), neprilysin-2 (MER013406), Kell blood group protein (MER001054), PHEX peptidase (MER002062), i-AAA peptidase (MER001246), i-AAA peptidase (MER005755), paraplegin (MER004454), Afg3-like protein 2 (MER005496), Afg3-like protein 1A (MER014306), papalysin-1 (MER002217), papalysin-2 (MER01452 1), farnesyl protein convertase 1 (MER002646), metalloprotease-related protein-1 (MER030873), aminopeptidase AMZ2 (MER011907), aminopeptidase AMZ1 (MER058242), carboxypeptidase A1 (MER001190), carboxypeptidase A2 (MER001608), carboxypeptidase B (MER001194), carboxypeptidase N (MER001198), carboxypeptidase E (MER0011 99), 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 (MER013421), carboxypeptidase A6 (MER013456),Carboxypeptidase A5 (MER017121), metallocarboxypeptidase O (MER016044), cytosolic carboxypeptidase-like protein 5 (MER033174), cytosolic carboxypeptidase 3 (MER033176), cytosolic carboxypeptidase 6 (MER033178), cytosolic carboxypeptidase 1 (MER033179), cytosolic 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), cytosolic carboxypeptidase (MER026952), family M14 non-peptidase homolog (MER199530), insulysin (MER00121 4), mitochondrial processing peptidase beta subunit (MER004497), nardilysin (MER003883), eupitolilidine (MER004877), mitochondrial processing peptidase non-peptidase alpha subunit (MER001413), ubiquinol-cytochrome c reductase core protein I (MER003543), ubiquinol-cytochrome c reductase core protein II (MER003543), R003544), ubiquinol-cytochrome c reductase core protein domain 2 (MER043998), insulysin unit 2 (MER046821), nardilysin unit 2 (MER046874), insulysin unit 3 (MER078753), mitochondrial processing peptidase subunit alpha unit 2 (MER124489), nardilysin unit 3 (MER142856), LOC133083 gp (Homo sapiens) (MER021876), subfamily M16B non-peptidase homolog (MER188757), leucyl aminopeptidase (animal) (MER003100), Mername-AA040 peptidase (MER003919), leucyl aminopeptidase-1 (Cenorhabditis type) (MER013416), methionyl aminopeptidase 1 (MER001342),Methionyl aminopeptidase 2 (MER001728), aminopeptidase P2 (MER004498), Xaa-Pro dipeptidase (eukaryotic) (MER001248), aminopeptidase P1 (MER004321), mitochondrial intermediate cleavage peptidase 55 kDa (MER013463), mitochondrial methionyl aminopeptidase (MER014055), Mername-AA020 peptidase homolog (MER010972), proliferation-associated protein 1 (MER005497), chromatin-specific transcription elongation factor 140 kDa subunit (MER026495), proliferation-associated protein 1-like (human chromosome X) (MER029983), Mername-AA22 6 peptidase homolog (Homo sapiens) (MER056262), Mername-AA227 peptidase homolog (Homo sapiens) (MER047299), 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 (MER0 05152), 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, Zease-related protein-5 (MER030136), hypothetical protein like 5730457F11RIK (MER033184), 1300019j08rik protein (MER033186), guanine aminohydrolase (MER037714), Kae1 putative peptidase (MER001577), OSGEPL1-like protein (MER013498), S2P peptidase (MER004458), subfamily M23B non-peptidase homolog (MER199845), subfamily M23B non-peptidase homolog (MER199845), 9846), Subfamily M23B nonpeptidase homolog (MER199847), Subfamily M23B nonpeptidase homolog (MER137320), Subfamily M23B nonpeptidase homolog (MER201557), Subfamily M23B nonpeptidase homolog (MER199417), Subfamily M23B nonpeptidase homolog (MER199418), Subfamily M23B nonpeptidase homolog (MER199419), Subfamily M23B nonpeptidase homolog (MER199420), Family 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 (homolog Sapiens chromosome 2) (MER029970), Mername-AA168 protein (MER021886), 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 unassigned peptidase (MER191144),Granzyme B (Homo sapiens type) (MER000168), testisin (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 Se, serine 4 (MER011104), intestinal serine peptidase (rodent) (MER016130), adrenal secretory serine peptidase (MER003734), tryptase delta 1 (Homo sapiens) (MER005948), matriptase-3 (MER029902), marapsin (MER006119), tryptase-6 (MER006118), ovochymase-1 domain 1 (MER099182), transmembrane peptidase, serine 3 (MER005926), kallikrein-related peptidase 15 (MER000064), Merna me-AA031 peptidase (MER014054), TMPRSS13 peptidase (MER014226), Mername-AA038 peptidase (MER062848), Mername-AA204 peptidase (MER029980), cationic trypsin (Homo sapiens type) (MER000020), elastase-2 (MER000118), mannan-binding lectin-associated serine peptidase-3 (MER031968), cathepsin G (MER000082), myeloblastin (MER000170), granzyme A (MER001379), Granzyme M (MER001541), Chymase (Homo sapiens type) (MER000123), Tryptase alpha (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),Prostasin (MER002460), kallikrein 1 (MER000093), kallikrein-related peptidase 2 (MER000094), kallikrein-related peptidase 3 (MER000115), mesotrypsin (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 peptide 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 (MER R000078), hepsin (MER000156), hepatocyte growth factor activator (MER000186), mannan-binding lectin-associated 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), epitheliacin (MER003736), kallikrein-related peptidase 4 (MER005266), prosemin (MER004214), chymopasin (MER001503), kallikrein-related peptidase 11 (MER004861), kallikrein-related peptidase 11 (MER216142), trypsin type 2A (MER000021), HtrA1 peptidase (Homo sapiens type) (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 C (MER021898), kallikrein-related peptidase 7 (MER002001), matriptase (MER003735), kallikrein-related peptidase 13 (MER005269), kallikrein-related peptidase 9 (MER005270), matriptase -2 (MER005278), umbilical vein peptidase (MER005421), LCLP peptidase (MER001900), spinesin (MER014385), marapsin-2 (MER021929), complement factor D-like putative peptidase (MER056164), ovochymase-2 (MER022410), HAT-like 4 peptidase (MER044589), ovochymase 1 domain 1 (MER022412), epidermis-specific SP-like putative peptidase (MER029900), testicular serine peptidase 5 (MER029901), Mername-A A258 peptidase (MER000285), polyserase-IA unit 1 (MER030879), polyserase-IA unit 2 (MER030880), testicular serine peptidase 2 (human type) (MER033187), hypothetical acrosin-like peptidase (Homo sapiens) (MER033253), HAT-like 5 peptidase (MER028215), polyserase-3 unit 1 (MER061763), polyserase-3 unit 2 (MER061748), tryptophan / serine protease-like peptidase (MER0562 63), 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 (human) (MER098873), hCG2041108 (Homo sapiens) (MER099173), polymerase-2 unit 2 (MER061760), polymerase-2 unit 3 (MER065694),Mername-AA201 (peptidase homolog) MER099175, secreted trypsin-like serine peptidase homolog (MER030000), polyserase-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), LOC13 6242 protein (MER016132), plasma kallikrein-like protein 4 (MER016346), PRSS35 protein (MER016350), 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 unassigned peptidase (MER216982), subfamily S1A unassigned peptidase (MER216148), amidophosphoribosyltransferase precursor (MER003314), glutamine-fructose-6-phosphate transaminase 1 (MER003322), glutamine:fructose-6-phosphate amidotransferase (MER012158), Mername-AA144 protein (MER021319), asparagine synthetase (MER033254), family C44 non-peptidase homolog (MER159286), family C 44 unassigned peptidase (MER185625), family C44 unassigned peptidase (MER185626), cecernin 1 (MER045376), cecernin 2 (MER064573), cecernin 3 (MER064582), acid ceramidase precursor (MER100794), N-acylethanolamino acid amidase precursor (MER141667), 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 alpha 6 (, MER000557), proteasome subunit alpha 2 (MER000550), proteasome subunit alpha 4 (MER000554), proteasome subunit alpha 7 (MER033250), proteasome subunit alpha 5 (MER000558), proteasome subunit alpha 1 (MER000549), proteasome subunit alpha 3 (MER000553), proteasome subunit XAPC7 (MER004372), proteasome subunit beta 3 (MER0017 10), proteasome subunit beta 2 (MER002676), proteasome subunit beta 1 (MER000551), proteasome subunit beta 4 (MER001711), Mername-AA230 peptidase homolog (Homo sapiens) (MER047329), Mername-AA231 pseudogene (Homo sapiens) (MER047172), Mername-AA232 pseudogene (Homo sapiens) (MER047316), glycosylasparaginase precursor (MER003299), isoasparaginase Rutile dipeptidase (threonine type) (MER031622), Taspase-1 (MER016969), gamma-glutamyltransferase 5 (mammalian type) (MER001977), gamma-glutamyltransferase 1 (mammalian type) (MER001629), gamma-glutamyltransferase 2 (Homo sapiens) (MER001976), gamma-glutamyltransferase-like protein 4 (MER002721), gamma-glutamyltransferase-like protein 3 (MER016970), gamma-glutamyltransferase Similar to gamma-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 (MER172554), gamma-glutamyl hydrolase (MER002963), guanine 5''-monophosphate synthase (MER043387), carbamoylphosphate synthase (Homo sapiens type) (MER078640), dihydroorotase (N-terminal unit) (Homo sapiens type) (MER060647), DJ-1 putative peptidase (MER003390), Mername-AA100 putative peptidase (MER014802), Mername-AA101 non-peptidase homolog (MER014803), KIAA0361 protein (Homo sapiens type) (MER042827), F1134283 protein (Homo sapiens) (MER044553), non-peptidase peptidase homolog chromosome 21 open reading frame 33 (Homo sapiens) (MER160094), family C56 nonpeptidase homolog (MER177016), family C56 nonpeptidase homolog (MER176613), family C56 nonpeptidase homolog (MER176918), EGF-like module containing mucin-like hormone receptor-like 2 (MER037230), CD97 antigen (human) (MER037286), EGF-like module containing mucin-like hormone receptor-like 3 (MER037288), EGF-like module containing mucin-like hormone receptor-like 1 (MER037278), EGF-like module containing mucin-like hormone receptor-like 4 (MER037294), cadherin EGF LAG7 pathway G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (house mouse) type protein (MER123205), GPR56 (Homo sapiens) type protein (MER122057), Latrophilin 2 (MER122199), Latrophilin-1 (MER126380), Latrophilin 3 (MER124612), Protocadherin Flamingo 2 (MER124239), ETL protein (MER126267), G protein-coupled receptor 112 (MER126114), seven-transmembrane helix 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), unassigned homolog (MER166269), GPR113 protein (MER159352), brain-specific angiogenesis inhibitor 2 (MER159746), PIDD autoprocessing protein unit 1 (MER020001), PIDD autoprocessing protein unit 2 (MER063690), MUC1 autocleaving mucin (MER074260), dystroglycan (MER054741), proprotein convertase 9 (MER022416), site 1 peptidase (MER001948), furin (MER000375), proprotein convertase Element 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 S8 A unassigned peptidase (MER191611), subfamily S8A unassigned peptidase (MER191612), subfamily S8A unassigned peptidase (MER191614), tripeptidyl peptidase I (MER003575), prolyl oligopeptidase (MER000393), dipeptidyl peptidase IV (eukaryotic) (MER000401), acylaminoacyl 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 (MER0332 45), 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 DPP10 (MER005988), protein similar to Mus musculus chromosome 20 open reading frame 135 (MER 037845), kynurenine formamidase (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 (MER033274), neuroligin 1 (MER033280), neuroligin 2 (MER033283), family S9 non-peptidase homolog (MER212939),Family S9 non-peptidase homolog (MER211490), subfamily S9C unassigned peptidase (MER192341), family S9 unassigned peptidase (MER209181), family S9 unassigned peptidase (MER200434), family S9 unassigned peptidase (MER209507), family S9 unassigned peptidase (MER209142), serine carboxypeptidase A (MER000430), vitellogenic carboxypeptidase-like protein (MER000430). 05492), RISC peptidase (MER010960), family S15 unassigned peptidase (MER199442), family S15 unassigned peptidase (MER200437), family S15 unassigned peptidase (MER212825), lysosomal Pro-Xaa carboxypeptidase (MER000446), dipeptidyl peptidase II (MER004952), thymus-specific serine peptidase (MER005538), epoxide hydrolase-like putative peptidase (MER0316 14), Loc328574-like protein (MER033246), Abhydrolase domain-containing protein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm-specific transcription protein (MER199890), mesoderm-specific transcription protein (MER017123), cytosolic epoxide hydrolase (MER029997), cytoplasmic epoxide hydrolase (MER213866) similar to hypothetical protein FLJ22408 (MER031608), CGI-58 putative peptidase (M ER030163), 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 hydrolase (MER033259), Ccg1 interacting factor b (MER210738), glycosylasparaginase precursor (MER003299),Isoaspartyl dipeptidase (threonine type) (MER031622). Taspase-1 (MER016969), gamma-glutamyltransferase 5 (mammalian type) (MER001977), gamma-glutamyltransferase 1 (mammalian type) (MER001629), gamma-glutamyltransferase 2 (Homo sapiens) (MER001976), gamma-glutamyltransferase-like protein 4 (MER002721). gamma-glutamyltransferase, -like protein 3 (MER016970). Similar to gamma-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). Polycystin-1-like 3 (MER172554). gamma-glutamylhydrolase (MER002963). Guanine 5" monophosphate synthetase (MER043387). Carbamoylphosphate synthase (Homo sapiens type) (MER078640). Dihydroorotase (N-terminal unit) (Homo sapiens type) (MER060647). DJ-1 putative peptidase (MER003390). Mername-AA100 putative peptidase (MER014802). Mername-AA101 non-peptidase homolog (MER014803). KIAA0361 protein (Homo sapiens type) (MER042827). F1134283 protein (Homo sapiens) (MER044553). Non-peptidase homolog chromosome 21 open reading frame Team 33 (Homo sapiens) (MER160094). Family C56 nonpeptidase homolog (MER177016), Family C56 nonpeptidase homolog (MER176613). Family C56 nonpeptidase homolog (MER176918). EGF-like module containing mucin-like hormone receptor-like 2 (MER037230). CD97 antigen (human) (MER037286). EGF-like module containing mucin-like hormone receptor-like 3 (MER037288). EGF-like module containing mucin-like hormone receptor-like 1 (MER037278). EGF-like module containing mucin-like hormone receptor-like 4 (MER037294). Seven-transmembrane G-type receptor 2 precursor of cadherin EGF LAG (Homo sapiens) (MER045397).Gpr64 (Mus musculus) type protein (MER123205), GPR56 (Homo sapiens) type protein (MER122057), Latrophilin 2 (MER122199), Latrophilin-1 (MER126380), Latrophilin 3 (MER124612), Protocadherin Flamingo 2 (MER124239), ETL protein (MER126267), G protein-coupled receptor 112 (MER126114), 7-transmembrane helix receptor (MER125448), Gpr114 protein (MER159320), GPR126 angiogenic 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), unassigned 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 (MER07426 0), 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 non-peptidase homolog (MER201339), Subfamily S8A non-peptidase homolog (MER191613), Subfamily S8A unassigned peptidase (MER191611), Subfamily S8A unassigned peptidase (MER191612), Subfamily S8A unassigned 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), hypothetical esterase / lipase / thioesterase (MER047309), protein bat 5 (MER037840), hypothetical protein fl j40219 (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 formamidase (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 (MER033274), neuroligin 1 (MER033280), neuroligin 2 (MER033283), family S9 nonpeptidase homolog (MER212939), family S9 nonpeptidase homolog (MER211490), subfamily S9C unassigned peptidase (MER192341), family S9 unassigned peptidase (MER209181), family S9 unassigned peptidase (MER200434), family S9 unassigned peptidase (MER2095 07), family S9 unassigned peptidase (MER209142), serine carboxypeptidase A (MER000430), vitellogenic carboxypeptidase-like protein (MER005492), RISC peptidase (MER010960), family S15 unassigned peptidase (MER199442), family S15 unassigned peptidase (MER200437), family S15 unassigned 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), Abhydrolase domain-containing protein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm-specific transcription protein (MER199890), mesoderm-specific transcription protein (MER017123), cytosolic epoxide hydrolase (MER029997),Cytosolic 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 flj22408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), hypothetical protein (MER033249), valacyclovir hydrolase (MER033259), Ccg1 interacting factor b (MER210738).

[0108] Protease enzyme activity can be modulated. For example, certain proteases can be inactivated by the presence or absence of certain drugs (e.g., drugs that bind to the protease, such as specific small molecule inhibitors). Such proteases can be referred to as "inhibitory proteases." Exemplary inhibitors for certain proteases are listed in Table 4B. For example, NS3 protease can be inhibited by protease inhibitors, including, but not limited to, simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxilaprevir. In another example, protease activity can be regulated through regulating the expression of the protease itself, such as by engineering cells to express the protease using an inducible promoter system (e.g., the Tet On / Off system) or a cell-specific promoter (promoters that can be used to express heterologous proteases are described in more detail herein in the section entitled "Promoters"). The protease can also include a degron, such as any of the degrons described herein, and can be regulated using any of the degron systems described herein.

[0109] Protease enzyme activity can also be modulated through the selection of a particular protease cleavage site. For example, a protease cleavage site can be selected and / or engineered so that the sequence exhibits a desired cleavage rate by a desired protease, such as a reduced cleavage rate relative to the endogenous sequence of a substrate naturally cleaved by the desired protease. As another example, a protease cleavage site can be selected and / or engineered so that the sequence exhibits a desired cleavage rate in a cellular state-specific manner. For example, various cellular states (e.g., following cell signaling, such as immune cell activation) can affect the expression and / or localization of particular proteases. As an illustrative example, ADAM17 protein levels and localization are known to be affected by signal transduction, for example, through the protein kinase C (PKC) signaling pathway (e.g., activation by the PKC activator phorbol-12-myristat-13-acetate [PMA]). Thus, protease cleavage sites can be selected and / or engineered such that cleavage of the protease cleavage site and subsequent release of the effector molecule is increased or decreased, as desired, depending on the protease characteristics (e.g., expression and / or localization) of a particular cellular state. As another example, protease cleavage sites (especially in combination with particular membrane-tethering domains) can be selected and / or engineered for optimal protein expression of the chimeric protein.

[0110] Plasma membrane anchoring domain The membrane-cleavable chimeric proteins provided herein comprise a plasma membrane-tethering domain (referred to as "MT" in the formula SC-MT or MT-CS). Generally, the plasma membrane-tethering domain can be any amino acid sequence motif capable of directing the chimeric protein to localize to (e.g., be inserted into) or otherwise associate with the plasma membrane of a cell expressing the chimeric protein. The plasma membrane-tethering domain can be a transmembrane-intracellular domain. The plasma membrane-tethering domain can be a transmembrane domain. The plasma membrane-tethering domain can be an endogenous protein domain (e.g., a transmembrane domain). The plasma membrane-tethering domain can be derived from a type I, type II, or type III transmembrane protein. The plasma membrane-tethering domain can comprise a post-translational modification tag or a motif capable of post-translational modification for modifying the chimeric protein to include a post-translational modification tag, where the post-translational modification tag enables association with the plasma 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 tethering domains include, but are not limited to, transmembrane-intracellular domains and / or transmembrane domains derived from PDGFR-beta, CD8, CD28, CD3 zeta chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, or BTLA. The cell membrane tethering domain may comprise a cell surface receptor or a cell membrane-bound portion thereof.

[0111] In some embodiments, the cell membrane anchoring domain comprises a transmembrane domain derived from a B7-1 polypeptide. In some embodiments, the B7-1 transmembrane domain comprises the sequence LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 204). In some embodiments, the B7-1 transmembrane domain is encoded by a polynucleotide sequence having the sequence TTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 252). In some embodiments, the B7-1 transmembrane domain is encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to TTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 252).

[0112] 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 LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 204). In some embodiments, the B7-1 transmembrane domain of the membrane-cleavable chimeric protein (e.g., IL-15) is encoded by a polynucleotide sequence having the sequence TTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 252). In some embodiments, the B7-1 transmembrane domain of a 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).

[0113] In some embodiments, the cell membrane-tethering domain comprises a transmembrane domain derived from a CD8 polypeptide. Any suitable CD8 polypeptide may be used. Exemplary CD8 polypeptides include, 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 anchoring domain comprises a hinge and transmembrane domain derived from CD8. In some embodiments, the CD8 hinge comprises the sequence TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 271). In some embodiments, the CD8 hinge comprises the sequence AAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 209).

[0114] Generally, for all membrane-cleavable chimeric proteins described herein, the plasma membrane-tethering domain is either: (1) C-terminal to the protease cleavage site, if present, and N-terminal to any intracellular domain (in other words, the plasma 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, with inverted domain orientation). In embodiments featuring a degron associated with the chimeric protein, the degron domain is specifically a terminal cytoplasm-directing domain with respect to the plasma membrane tethering (in other words, the plasma membrane-tethering domain is between the protease cleavage site and the degron). The plasma membrane-tethering domain can be connected to the protease cleavage site by a polypeptide linker, i.e., a polypeptide sequence not generally considered part of the plasma membrane-tethering domain or the protease cleavage site. When present, the cell membrane anchoring domain can be connected to the intracellular domain by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane anchoring domain or the intracellular domain. When present, the cell membrane anchoring domain can be connected to the degron by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane anchoring 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]GG [SEQ ID NO: 182]), A(EAAAK)A (SEQ ID NO: 183), and a Whitlow linker (e.g., a "KEGS" linker, e.g., amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), an eGK linker, e.g., amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 185), and linkers described in more detail in issued U.S. Patent No. 5,990,275, which is incorporated herein by reference).Additional polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, and SEQ ID NO: 197. Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition, etc.) and are known to those of skill in the art.

[0115] Generally, the cell membrane anchoring domain is oriented such that the secreted effector molecule and the protease cleavage site are exposed extracellularly after insertion into or association with the cell membrane, allowing the protease cleavage site to be cleaved by its respective protease to release (secrete) the effector molecule into the extracellular space.

[0116] Degron Systems and Domains In some embodiments, any of the proteins described herein can contain a degron domain, including, but not limited to, a protease, a transcription factor, a promoter or component of a promoter system (e.g., ACP), and / or any of the membrane-cleavable chimeric proteins described herein. Generally, a degron domain can be any amino acid sequence motif capable of directing regulated degradation, such as regulated degradation through a ubiquitin-mediated pathway. In the presence of an immunomodulatory drug (IMiD), the degron domain directs ubiquitin-mediated degradation of the degron fusion protein.

[0117] The degron domain can be a cereblon (CRBN) polypeptide substrate domain capable of binding CRBN in response to an immunomodulatory drug (IMiD), including, but not limited to, IKZF1, IKZF3, CKla, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827, and fragments thereof capable of drug-induced binding of 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 A1, which is incorporated herein by reference for all purposes.Other examples of degron domains include, but are not limited to, HCV NS4 degron, PEST (two copies of residues 277-307 of human 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) nucleotide (SP2-SP1-SP2-SP1-SP2 of the influenza A virus M2 protein; SEQ ID NO: 166), NS2 (three copies of residues 79-93 of the influenza A virus NS protein; SEQ ID NO: 167), ODC (residues 106-142 of ornithine decarboxylase; SEQ ID NO: 168), Nek2A, mouse ODC (residues 422-461, SEQ ID NO: 169), mouse ODC_DA (residues 422-461 of mODC containing D433A and D434A point mutations), APC / C degron, COP1 These include E3 ligase-binding degron motifs, CRL4-Cdt2-binding PIP degrons, actinfilin-binding degrons, KEAP1-binding degrons, KLHL2- and KLHL3-binding degrons, MDM2-binding motifs, N-degrons, hydroxyproline modification in hypoxia signaling, plant hormone-dependent SCF-LRR-binding degrons, SCF ubiquitin ligase-binding phosphodegrons, plant hormone-dependent SCF-LRR-binding degrons, DSGxxS phosphate-dependent degrons, Siah-binding motifs, SPOP SBC docking motifs, and PCNA-binding PIP boxes.

[0118] The regulated degradation can be drug-induced. Drugs capable of mediating / regulating degradation can be small molecule compounds. Drugs capable of mediating / regulating degradation can include "immunomodulatory drugs" (IMiDs). Generally, as used herein, IMiD refers to a class of small molecule immunomodulatory drugs containing an imide group. Cereblon (CRBN) is a known target of IMiDs, and binding of an IMiD to CRBN or a CRBN polypeptide substrate domain alters the substrate specificity of the CRBN E3 ubiquitin ligase complex, leading to the degradation of proteins having a CRBN polypeptide substrate domain (e.g., secreted effector molecules described herein or other proteins of interest). For degron domains having a CRBN polypeptide substrate domain, examples of imide-containing IMiDs include, but are not limited to, thalidomide, lenalidomide, or pomalidomide. The IMiD can be an FDA-approved drug.

[0119] The chimeric proteins described herein can include a degron domain (e.g., referred to as "D" in the formula SC-MT-D or D-MT-CS for the membrane-cleavable chimeric proteins described herein). In the absence of an IMiD, degron / ubiquitin-mediated degradation of the chimeric protein does not occur. Following expression and localization of the chimeric protein in the cell membrane, the protease cleavage site directs cleavage of the chimeric protein, resulting in the release (secretion) of effector molecules into the extracellular space. In the presence of an immunomodulatory drug (IMiD), the degron domain directs ubiquitin-mediated degradation of the chimeric protein, resulting in reduced or eliminated secretion of the effector molecules. Generally, for membrane-cleavable chimeric proteins fused to a degron domain, the degron domain is a terminal cytoplasm-directing domain, specifically relative to a cell membrane-tethering domain, e.g., the most C-terminal domain in the formula SC-MT-D or the most N-terminal domain in the formula D-MT-CS. The degron domain can be connected to the cell membrane-tethering domain, i.e., a polypeptide sequence not generally considered part of a cell membrane-tethering domain or a degron domain, by a polypeptide linker. The polypeptide linker can be any amino acid sequence that connects a first polypeptide sequence and a 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]GG [SEQ ID NO: 182]), A(EAAAK)A (SEQ ID NO: 183), and a Whitlow linker (e.g., a "KEGS" linker, e.g., the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), an eGK linker, e.g., the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 185), and linkers described in more detail in issued U.S. Patent No. 5,990,275, which is incorporated herein by reference). Additional polypeptide linkers include SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, and SEQ ID NO:197.Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition, etc.) and are known to those of skill in the art. Generally, the degron is oriented in relation to the cell membrane-tethering domain, such that the degron is exposed to the cytosol after localization to the cell membrane, and the degron domain is capable of mediating degradation (e.g., exposure to the cytosol and cytosol) and is capable of mediating ubiquitin-mediated degradation.

[0120] For degron fusion proteins, the degron domain can be at the N-terminus or C-terminus of the protein of interest, e.g., an effector molecule. The degron domain can be connected to the protein of interest by a polypeptide linker, i.e., a polypeptide sequence not generally considered part of the protein of interest or the degron domain. The polypeptide linker can be any amino acid sequence that connects the first and second polypeptide sequences. 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]GG [SEQ ID NO: 182]), A(EAAAK)A (SEQ ID NO: 183), and a Whitlow linker (e.g., a "KEGS" linker, e.g., the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), an eGK linker, e.g., the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 185), and linkers described in more detail in issued U.S. Patent No. 5,990,275, incorporated herein by reference). Additional polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, and SEQ ID NO: 197. Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition, etc.) and are known to those of skill in the art. The polypeptide linker may be cleavable, e.g., any of the protease cleavage sites described herein.

[0121] homing molecules The "tumor microenvironment" is the cellular environment in which a tumor resides, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM) (see, e.g., Pattabiraman, D.R. & Weinberg, R.Nature Reviews Drug Discovery 13, 497-512 (2014); Balkwill, F.R. et al. J. Cell Sci. 125, 5591-5596, 2012; and Li, H. et al. J. Cell Biochem. 101(4), 805-15, 2007).

[0122] In some embodiments, the engineered nucleic acid is configured to produce at least one homing molecule. For example, in a membrane-cleavable chimeric protein described herein that includes a secreted effector molecule, the secreted effector molecule can be a homing molecule. "Homing" refers to the active navigation (migration) of a cell to a target site (e.g., a cell, tissue (e.g., a tumor), or organ). A "homing molecule" refers to a molecule that directs a cell to a target site. In some embodiments, a homing molecule functions to recognize and / or initiate interaction of the engineered cell with the target site. Non-limiting examples of homing molecules include CXCR1, CCR9, CXCR2, CXCR3, CXCR4, CCR2, CCR4, FPR2, VEGFR, IL6R, CXCR1, CSCR7, and PDGFR.

[0123] In some embodiments, the homing molecule is a chemokine receptor (a cell surface molecule that binds to a chemokine). Chemokines are small cytokines or signaling proteins secreted by cells that can induce directional chemotaxis in cells. Chemokines can be classified into four major subfamilies: CXC, CC, CX3C, and XC, all of which exert their biological effects by selectively binding to chemokine receptors located on the surface of target cells. In some embodiments, the engineered nucleic acid is configured to produce a chemokine receptor that enables the engineered cell to home along a chemokine gradient toward CXCR4, stromal cell-derived factor 1 (also known as SDF1, CXC motif chemokine 12, and CXCL12)-expressing cells, tissues, or tumors. Non-limiting examples of chemokine receptors that can be encoded by the engineered nucleic acids of the present disclosure include: C-X-C chemokine receptors (e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7), C-C chemokine receptors (CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, and CCR11), C-X-C chemokine receptors (e.g., CX3CR1 that binds CX3CL1), and C-C chemokine receptors (e.g., XCR1). In some embodiments, the chemokine receptor is a G protein-linked transmembrane receptor or a member of the tumor necrosis factor (TNF) receptor superfamily (including, but not limited to, TNFRSF1A, TNFRSF1B). In some embodiments, the engineered nucleic acid is configured to produce CXCL8, CXCL9, and / or CXCL10 (promotes T cell recruitment), CCL3 and / or CXCL5, CCL21 (Th1 recruitment and polarization).

[0124] In some embodiments, the engineered nucleic acid is configured to produce a G protein-coupled receptor (GPCR) that detects N-formylation-containing oligopeptides (including but not limited to, FPR2 and FPRL1).

[0125] In some embodiments, the engineered nucleic acid is configured to produce a receptor that detects interleukins (including but not limited to, IL6R).

[0126] In some embodiments, the engineered nucleic acid is configured to produce a receptor (including but not limited to, FGFR, PDGFR, EGFR, and receptors of the VEGF family, including but not limited to, VEGF-C and VEGF-D) that detects growth factors secreted by other cells, tissues, or tumors.

[0127] In some embodiments, the homing molecule is an integrin. Integrins are transmembrane receptors that promote extracellular matrix (ECM) adhesion. Integrins are essential heterodimers having two subunits: α (alpha) and β (beta). Integrin α subunits can be, but are not limited to: ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, IGTA7, ITGA8, ITGA9, IGTA10, IGTA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGA2B, or ITGAX. Integrin β subunits can be, but are 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.

[0128] In some embodiments, the homing molecule is a matrix metalloproteinase (MMP). MMPs are enzymes that cleave components of the basement membrane underlying endothelial cell walls. Non-limiting examples of MMPs include MMP-2, MMP-9, and MMP-1. In some embodiments, the engineered nucleic acid is configured to produce an inhibitor of a molecule (e.g., a protein) that inhibits an MMP. For example, the engineered nucleic acid can be configured to express an inhibitor (e.g., an RNAi molecule) of membrane type 1 MMP (MT1-MMP) or TIMP metallopeptidase inhibitor 1 (TIMP-1).

[0129] In some embodiments, the homing molecule is a ligand that binds to a selectin, for example, on 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).

[0130] The term "homing molecule" also encompasses transcription factors that regulate the production of molecules that improve / enhance homing of cells.

[0131] In some embodiments, the homing molecule comprises an antibody, such as anti-integrin alpha 4, beta 7, or anti-MAdCAM.

[0132] Chimeric antigen receptor (CAR) Certain aspects of the present disclosure relate to chimeric receptors having any one of the antigen-specific antigen-binding domains described herein (e.g., EMCN-specific, FLT3-specific, and / or CD33-specific) and capable of specifically binding to a protein, antigen-derived antigen, or antigen-derived epitope.

[0133] In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). Generally, a CAR is a chimeric protein comprising an antigen-binding domain and a polypeptide molecule heterologous to the antigen-binding domain, such as a peptide heterologous to the 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 region, one or more peptide linkers, or a combination thereof.

[0134] In some embodiments, a CAR is an engineered receptor that transfers or confers the specificity of a target of interest (e.g., EMCN, FLT3, or CD33) onto an immune effector cell. In certain embodiments, a CAR can be used to transfer the specificity of an antibody onto an immunoresponsive cell, such as a T cell. In some embodiments, a CAR of the present disclosure comprises an extracellular antigen-binding domain (e.g., an scFv) fused to a transmembrane domain, which is fused to one or more intracellular signaling domains.

[0135] In some embodiments, the chimeric antigen receptor is an activating chimeric antigen receptor (aCAR, and also commonly referred to as a CAR unless otherwise specified). In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce activation of an immunoresponsive cell. In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce stimulation of an immunoresponsive cell. In some embodiments, activation of an immunoresponsive cell results in killing of a target cell. In some embodiments, activation of an immunoresponsive cell results in cytokine or chemokine expression and / or secretion by the immunoresponsive cell. In some embodiments, stimulation of an immunoresponsive cell results in cytokine or chemokine expression and / or secretion by the immunoresponsive cell. In some embodiments, stimulation of an immunoresponsive cell induces differentiation of the immunoresponsive cell. In some embodiments, stimulation of an immunoresponsive cell induces proliferation of the immunoresponsive cell. In some embodiments, activation and / or stimulation of an immunoresponsive cell can be a combination of the above responses.

[0136] The number of ABDs in a binding molecule, such as the chimeric proteins described herein, defines the "valency" of the binding molecule. A binding molecule with a single ABD is "monovalent." A binding molecule with multiple ABDs is said to be "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 of the 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 multivalent embodiments in which the ABDs collectively have two recognition specificities, the binding molecule is "bispecific." In multivalent embodiments in which the ABDs collectively have three recognition specificities, the binding molecule is "trispecific." In multivalent embodiments in which the ABDs collectively have multiple recognition specificities for different epitopes present on the same antigen, the binding molecule is "multiparatopic." Multivalent embodiments in which the ABDs collectively recognize two epitopes on the same antigen are "dualparatopic."

[0137] In various multivalent embodiments, the multivalent binding molecule improves the avidity of the binding molecule for a specific target. As described herein, "avidity" refers to the overall strength of the interaction between two or more molecules, e.g., multivalent binding molecules, for a specific target, and avidity is the cumulative strength of the interaction provided by the affinity of multiple ABDs. Avidity can be measured by the same methods used to determine affinity, as described above. In certain embodiments, the avidity of a binding molecule for a specific target is measured when the interaction is a specific binding interaction, and the avidity between two molecules is 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10In certain embodiments, the avidity of a binding molecule for a specific target has a KD value such that the interaction is a specific binding interaction, and the affinity of one or more of the individual ABDs does not have a KD value that recognizes specific binding to their own respective antigen or epitope. In certain embodiments, the avidity is the cumulative strength of the interaction provided by the affinity of multiple ABDs for distinct antigens on a shared specific target or complex, such as distinct antigens found on individual cells. In certain embodiments, the avidity is the cumulative strength of the interaction provided by the affinity of multiple ABDs for distinct epitopes on a shared individual antigen.

[0138] In some embodiments, the aCAR can be a bivalent, bispecific CAR. In some embodiments, the aCAR can be a bivalent, bispecific CAR expressed on a cell (e.g., an immunoresponsive cell) of the present disclosure as an OR logic gate to increase the potential targets of an activating chimeric receptor (e.g., an OR logic gate targeting multiple tumor targets). The bivalent, bispecific aCAR can comprise an antigen-binding domain specific for FLT3 and an antigen-binding domain specific for CD33. The bivalent, bispecific aCAR can comprise: (i) an antigen-binding domain specific for FLT3; (ii) an antigen-binding domain specific for CD33; (iii) one or more intracellular signaling domains that stimulate an immune response; and (iv) one or more polypeptides, including, but not limited to, a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

[0139] The CARs of the present disclosure can be first-, second-, or third-generation CARs. "First-generation" CARs contain a single intracellular signaling domain, generally derived from a T cell receptor chain. "First-generation" CARs generally have an intracellular signaling domain from the CD3-zeta (CD3ζ) chain, which is the primary transmitter of signals from endogenous TCRs. "First-generation" CARs confer de novo antigen recognition and transduce CD4 T cells via the CD3ζ chain signaling domain within a single fusion molecule, independent of HLA-mediated antigen presentation. + and CD8 + "Second-generation" CARs add a second intracellular signaling domain from one of a variety of costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provide an additional signal to the T cell. "Second-generation" CARs provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). Preclinical studies have shown that "second-generation" CARs can enhance the anti-tumor activity of immunoresponsive cells such as T cells. "Third-generation" CARs have multiple intracellular costimulatory signaling domains (e.g., CD28 and 4-1BB) and an intracellular activation signaling domain (CD3ζ).

[0140] 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 derived from a tissue selected from the group consisting of brain, nervous tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, urinary bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

[0141] In some embodiments, a chimeric inhibitory receptor (e.g., an EMCN-specific chimeric inhibitory receptor) can be used in conjunction with one or more activating chimeric receptors (e.g., activating chimeric TCRs or CARs, such as FLT3 and / or CD33 aCARs) expressed on a cell (e.g., an immunoresponsive cell) of the present disclosure, e.g., as a NOT logic gate to control, regulate, or otherwise inhibit one or more activities of one or more activating chimeric receptors. For example, if a healthy cell expresses both an antigen recognized by a tumor-targeting chimeric receptor and an antigen recognized by an inhibitory chimeric receptor, the immunoresponsive cell expressing the tumor antigen can bind to the healthy cell. In such a case, the inhibitory chimeric antigen also binds to its cognate ligand on the healthy cell, and the inhibitory function of the inhibitory chimeric receptor decreases, reduces, prevents, or inhibits activation of the immunoresponsive cell via the tumor-targeting chimeric receptor ("NOT logic gating"). In some embodiments, the inhibitory chimeric receptor of the present disclosure can inhibit one or more activities of a cell (e.g., an immunoresponsive cell) of the present disclosure. In some embodiments, immunoresponsive cells may comprise one or more tumor-targeting chimeric receptors and one or more inhibitory chimeric receptors that target antigens that are not expressed or not commonly considered to be expressed on tumors (e.g., EMCN). A combination of tumor-targeting chimeric receptors and inhibitory chimeric receptors on the same immunoresponsive cell can be used to reduce extratumoral toxicity on the target.

[0142] In some embodiments, the extracellular antigen binding domain of a CAR of the disclosure comprises about 2×10 -7 M or less, approximately 1×10 -7 M or less, approximately 9 x 10 -8 M or less, approximately 1×10 -8 M or less, approximately 9 x 10 -9 M or less, about 5 x 10 -9 M or less, approximately 4 x 10 -9 M or less, about 3 x 10 -9 M or less, approximately 2×10 -9 M or less, or about 1 x 10 -9 The dissociation constant (K d ) binds to one or more antigens (e.g., EMCN, FLT3, or CD33). In some embodiments, Kd is about 2 x 10 -7 M ~ approx. 1×10 -9 It is in the range of M.

[0143] Binding of the extracellular antigen-binding domain of a CAR of the present disclosure can be determined, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), biolayer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., Biacore®), or Western blot assay. Each of these assays generally detects the presence of a protein-antibody complex of particular interest by using a labeled reagent (e.g., antibody or scFv) specific for the complex of interest. For example, an scFv can be radiolabeled and used in an RIA assay. The radioisotope can be detected by means such as the use of a gamma counter or scintillation counter, or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a secondary antibody specific for the extracellular antigen-binding domain, and the secondary antibody is labeled (e.g., with a radioactive or fluorescent marker).

[0144] In some embodiments, a CAR of the present disclosure comprises an extracellular antigen-binding domain that binds to EMCN, FLT3, or CD33. In some embodiments, a CAR of the present disclosure comprises an extracellular antigen-binding domain that binds to an EMCN protein, an EMCN-derived antigen, or an EMCN-derived epitope. In some embodiments, a CAR of the present disclosure comprises an extracellular antigen-binding domain that binds to an FLT3 protein, an FLT3-derived antigen, or an FLT3-derived epitope. In some embodiments, a CAR of the present disclosure comprises an extracellular antigen-binding domain that binds to a CD33 protein, a CD33-derived antigen, or a CD33-derived epitope. A CAR of the present disclosure comprises an extracellular antigen-binding domain, a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the extracellular antigen-binding domain comprises a Fab fragment that can be crosslinked. In certain embodiments, the extracellular binding domain is a F(ab)2 fragment.

[0145] Extracellular antigen-binding domain Antigen-binding domains of the present disclosure can include 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 (sdAbs), such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid-derived nanobodies (VHH), as well as any domain associated with an alternative scaffold known in the art to function as an antigen-binding domain, such as recombinant fibronectin domains, T cell receptors (TCR), affinity-enhanced recombinant TCRs, or fragments thereof, e.g., single-chain TCRs. In some cases, it is beneficial for the antigen-binding domain to be derived from the same species in which the CAR will ultimately be used.

[0146] 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.

[0147] In some embodiments, the extracellular antigen-binding domain comprises an F(ab) fragment. In certain embodiments, the extracellular antigen-binding domain comprises an F(ab') fragment.

[0148] In some embodiments, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the extracellular antigen-binding domain comprises two single-chain variable fragments (scFvs). In some embodiments, each of the two scFvs binds to a distinct epitope on the same antigen. In some embodiments, the extracellular antigen-binding domain comprises a first scFv and a second scFv. In some embodiments, the first scFv and the second scFv bind to a distinct epitope 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).

[0149] In certain embodiments, the VH and VL are separated by a peptide linker, hi certain embodiments, the peptide linker comprises any of the amino acid sequences shown in Table 6.

[0150] In certain embodiments, an scFv comprises the structure VH-L-VL or VL-L-VH, where VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain. In some embodiments, each of the one or more scFvs comprises the structure VH-L-VL or VL-L-VH, where 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 to each other, each scFv can be linked to the next scFv linked by a peptide. In some embodiments, each of the one or more scFvs is separated by a peptide linker.

[0151] In some embodiments, the peptide linker comprises the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 244). In some embodiments, the peptide linker between the antigen binding domains of iCAR comprises the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 244). In some embodiments, the peptide linker is encoded by a polynucleotide sequence comprising the sequence GGAGGCGGAGGATCTGGTGCGGAGGAAGTGGCGGAGGCGGTTCT (SEQ ID NO: 253). 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 GGAGGCGGAGGATCTTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCT (SEQ ID NO: 253).

[0152] In some embodiments, the peptide linker comprises the amino acid sequence GGGGS (SEQ ID NO: 242). In some embodiments, the peptide linker between the antigen binding domains of the aCAR comprises the amino acid sequence GGGGS (SEQ ID NO: 242). In some embodiments, the peptide linker is encoded by a polynucleotide sequence comprising the sequence GCGGCGGTGGCTCT (SEQ ID NO: 254) or GGTGGCGGCGGATCC (SEQ ID NO: 255). 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 GCGGCGGTGGCTCT (SEQ ID NO: 254) or GGTGGCGGCGGATCC (SEQ ID NO: 255).

[0153] In some embodiments, the peptide linker comprises 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).

[0154] In some embodiments, the peptide linker comprises the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 247). In some embodiments, the peptide linker between the antigen binding domains of the aCAR comprises 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 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 GGCTCTACATCTGGCTCTGGCAAACCTGGAAGCGGCGAGGGATCTACCAAGGGC (SEQ ID NO: 249).

[0155] Table 6: Peptide linkers TIFF2025513427000034.tif133128

[0156] In some embodiments, the immune effector cell comprises a first chimeric receptor and a second chimeric receptor. The antigen-binding domain of the first chimeric receptor and the antigen-binding domain of the second chimeric receptor can be any suitable antigen-binding domain described herein or known in the art. For example, the first or second antigen-binding domain can be one or more antibodies, antigen-binding fragments of antibodies, F(ab) fragments, F(ab') fragments, single-chain variable fragments (scFvs), or single-domain antibodies (sdAbs). In some embodiments, the antigen-binding domain of the first chimeric receptor and / or the second chimeric receptor comprises two single-chain variable fragments (scFvs). In some embodiments, each of the two scFvs binds to a distinct epitope on the same antigen. In some embodiments, the antigen-binding domain of the first chimeric receptor can be specific for EMCN, and the chimeric receptor can be specific for a second distinct antigen, such as a cancer antigen (e.g., an antigen expressed on myeloid cells, such as AML cells).

[0157] 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.

[0158] In some embodiments, a CAR of the present disclosure may comprise two or more antigen-binding domains, three or more antigen-binding domains, four or more antigen-binding domains, five or more antigen-binding domains, six or more antigen-binding domains, seven or more antigen-binding domains, eight or more antigen-binding domains, nine or more antigen-binding domains, or ten or more antigen-binding domains. In some embodiments, each of the two or more antigen-binding domains binds to the same antigen. In some embodiments, each of the two or more antigen-binding domains binds to a different epitope of the same antigen. In some embodiments, each of the two or more antigen-binding domains binds to a different antigen.

[0159] In some embodiments, the CAR comprises two antigen-binding domains. In some embodiments, the two antigen-binding domains are linked to each other via a flexible linker. In some embodiments, each of the two antigen-binding domains may be independently selected from an antibody, an antigen-binding fragment of an antibody, an scFv, an sdAb, a recombinant fibronectin domain, a T cell receptor (TCR), an affinity-enhanced recombinant TCR, and a single-chain TCR. In some embodiments, the CAR comprising two antigen-binding domains is a bispecific CAR or a tandem CAR (tanCAR).

[0160] In certain embodiments, a bispecific CAR or tanCAR comprises antigen-binding domains comprising a bispecific antibody or antibody fragment (e.g., scFv). In some embodiments, within each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, the VH can be upstream or downstream of the VL. In some embodiments, an upstream antibody or antibody fragment (e.g., scFv) is positioned with its VH (VH1) upstream of its VL (VL1), and a downstream antibody or antibody fragment (e.g., scFv) is positioned with its VL (VL2) upstream of its VH (VH2), such that the overall bispecific antibody molecule has the configuration VH1-VL1-VL2-VH2. In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL1) upstream of its VH (VH1), and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH2) upstream of its VL (VL2), with the overall bispecific antibody molecule having the arrangement VL1VH1-VH2-VL2. In some embodiments, a linker is placed between the two antibodies or antibody fragments (e.g., scFvs), for example, between VL1 and VL2 when the construct is arranged as VH1-VL1-VL2-VH2, or between VH1 and VH2 when the construct is arranged as VL1-VH1-VH2-VL2. The linker can be a linker described herein, e.g., a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6. Generally, the linker between two scFvs must be long enough to avoid mispairing between the domains of the two scFvs. In some embodiments, the linker is disposed between the VL and VH of a first scFv. In some embodiments, the linker is disposed between the VL and VH of a second scFv. In constructs with multiple linkers, any two or more linkers may be the same or different. Thus, in some embodiments, a bispecific CAR or tanCAR comprises a VL, a VH, and may further comprise one or more linkers in an arrangement described herein.

[0161] In an illustrative, non-limiting example, a CD33 / FLT3 bispecific bivalent aCAR can have antigen-binding domains encoded in the following order: (FLT3-VH)-L1-(CD33-VH)-L2-(CD33-VL)-L3-(FLT3-VL), where L1, L2, and L3 are the first peptide linker, the second peptide linker, and the third peptide linker, respectively. L1, L2, and L3 can be the same peptide linker. L1, L2, and L3 can be the same peptide linker encoded by the same polynucleotide sequence. L1, L2, and L3 can be the same peptide linker encoded by different polynucleotide sequences. L1 and L3 can be the same peptide linker, and L2 can be a different peptide linker. L1 and L3 can be the same peptide linker encoded by the same polynucleotide sequence. L1 and L3 can be the same peptide linker encoded by different polynucleotide sequences. L1, L2, and L3 can each be a different peptide linker.

[0162] CAR transmembrane domain In some embodiments, the transmembrane domain of a CAR of the present disclosure (e.g., an EMCN-specific, FLT3-specific, and / or CD33-specific CAR described herein) comprises a hydrophobic alpha helix that spans at least a portion of the cell membrane. It has been shown that different transmembrane domains can confer different receptor stability. After antigen recognition, receptors cluster and a signal is transmitted to the cell. In some embodiments, the transmembrane domain of a CAR of the present disclosure can comprise the transmembrane domain of a CD8 polypeptide, a CD28 polypeptide, a CD3-zeta polypeptide, a CD4 polypeptide, a 4-1BB 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, an LIR-1 (LILRB1) polypeptide, or can be a synthetic peptide, or any combination thereof.

[0163] In some embodiments, the transmembrane domain is derived from a CD8 polypeptide. Any suitable CD8 polypeptide may 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 may 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-zeta polypeptide. Any suitable CD3-zeta polypeptide may be used. Exemplary CD3-zeta 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 may 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 may 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 may 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 may be used. Exemplary LIR-1 (LILRB1) polypeptides include, but are not limited to, NCBI reference numbers NP_001075106.2 and NP_001075107.2.

[0164] In some embodiments, the transmembrane domain is selected from the group consisting of 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_00500 The present invention also includes polypeptides comprising an amino acid sequence, or a fragment thereof, 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 NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2. In some embodiments, homology can be determined using standard software such as BLAST or FASTA. In some embodiments, the polypeptide may contain 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 is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, or at least 240 amino acids in length (NCBI Reference Nos. NP_001139345, AAA92533.1, NP_006130.1, NP_00333.2, NP_00333.3, NP_00333.4, NP_00333.5, NP_00333.6, NP_00333.7, NP_00333.8, NP_00333.9 ... 1668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2, NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_005009, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2.

[0165] Further examples of suitable polypeptides from which the transmembrane domain may be derived include T cell receptor, CD27, CD3 epsilon, 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, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, These include, but are not limited to, the transmembrane regions of the alpha, beta, or zeta chains of ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, 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.

[0166] 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).

[0167] In some embodiments, the iCAR transmembrane domain comprises a LIR1 transmembrane domain. In some embodiments, the iCAR transmembrane domain comprises a LIR1 transmembrane domain having the sequence VIGILVAVILLLLLLLLLFLI (SEQ ID NO: 259). In some embodiments, the iCAR transmembrane domain comprises a LIR1 transmembrane domain encoded by a polynucleotide sequence having the sequence GTGATCGGCATTCTGGTCGCCGTGATCCTGCTCCTGTTGCTCCTGCTGCTTCTGTTCCTGATC (SEQ ID NO: 260). In some embodiments, the iCAR transmembrane domain comprises a LIR1 transmembrane domain encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to GTGATCGGCATTCTGGTCGCCGTGATCCTGCTCCTGTTGCTCCTGCTGCTTCTGTTCCTGATC (SEQ ID NO: 260).

[0168] In some embodiments, the aCAR transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the aCAR transmembrane domain comprises a CD8 transmembrane domain having the sequence IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 206). In some embodiments, the aCAR transmembrane domain comprises a CD8 transmembrane domain encoded by a polynucleotide sequence having the sequence ATCTATATCTGGGCCCCTCTGGCTGGCACATGCGGAGTTCTGCTGCTCAGCCTGGTCATCACCCTGTACTGCAACCACAGA (SEQ ID NO: 261). In some embodiments, the aCAR transmembrane domain comprises a CD8 transmembrane domain encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to ATCTATATCTGGGCCCCTCTGGCTGGCACATGCGGAGTTCTGCTGCTCAGCCTGGTCATCACCCTGTACTGCAACCACAGA (SEQ ID NO: 261).

[0169] Spacer region In some embodiments, a CAR of the present disclosure (e.g., an EMCN-specific, FLT3-specific, and / or CD33-specific CAR described herein) may also include a spacer region linking the extracellular antigen-binding domain to the transmembrane domain. The spacer region may be sufficiently flexible to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition. In some embodiments, the spacer region may be a hinge derived from a human protein. For example, the hinge may be a human Ig (immunoglobulin) hinge, including, but not limited to, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge. In some embodiments, the spacer region may comprise an IgG4 hinge, an IgG2 hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, an LNGFR hinge, or a PDGFR-beta extracellular linker. In some aspects, the spacer region is localized between the antigen-binding domain and the transmembrane domain. In some embodiments, a spacer region may comprise any of the amino acid sequences listed in Table 7, or an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the amino acid sequences listed in Table 7. In some embodiments, a nucleic acid encoding any of the spacer regions of the present disclosure may comprise any of the nucleic acid sequences listed in Table 8, or a nucleic acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the nucleic acid sequences listed in Table 8.

[0170] In some embodiments, the aCAR hinge domain comprises a CD8 hinge. In some embodiments, the aCAR hinge domain comprises a CD8 hinge having the amino acid sequence ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 272). In some embodiments, the aCAR hinge domain comprises a CD8 hinge encoded by a polynucleotide sequence having the sequence GCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTGCCCGCCAAGCCTACAACAACCCCTGCTCCTAGACCACCTACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCCGAAGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCATACAAGAGGACTGGATTTTGCCTGCGAC (SEQ ID NO: 284). In some embodiments, the aCAR hinge domain comprises a CD8 hinge encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to GCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTGCCCGCCAAGCCTACAACAACCCCTGCTCCTAGACCACCTACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCCGAAGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCATACAAGAGGACTGGATTTTGCCTGCGAC (SEQ ID NO: 284).

[0171] In some embodiments, the iCAR hinge domain comprises a CD8 hinge. In some embodiments, the iCAR hinge domain comprises a CD8 hinge having the amino acid sequence TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 271). In some embodiments, the iCAR hinge domain comprises a CD8 hinge encoded by a polynucleotide sequence having the sequence ACAACAACACCCGCACCTCGGCCTCCAACTCCAGCTCCAACAATTGCACTGCAACCCCTGAGTCTGAGGCCCGAGGCCTGTAGGCCAGCAGCTGGCGGAGCTGTTCACACTAGAGGCCTGGACTTTGCCTGTGAC (SEQ ID NO: 283). In some embodiments, the iCAR hinge domain comprises a CD8 hinge encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to ACAACAACACCCGCACCTCGGCCTCCAACTCCAGCTCCAACAATTGCACTGCAACCCCTGAGTCTGAGGCCCGAGGCCTGTAGGCCAGCAGCTGGCGGAGCTGTTCACACTAGAGGCCTGGACTTTGCCTGTGAC (SEQ ID NO: 283).

[0172] In some embodiments, the iCAR hinge domain comprises an LIR1 hinge. In some embodiments, the iCAR hinge domain comprises an LIR1 hinge having the amino acid sequence HPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV (SEQ ID NO: 417). In some embodiments, the iCAR hinge domain comprises an 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 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 CACCCATCCGATCCTCTCGAGCTGGTGGTTTCTGGACCTTCTGGCGGCCCTAGCAGCCCTACAACAGGACCTACAAGCACAAGCGGCCCTGAGGACCAACCTCTGACACCAACAGGCAGCGATCCTCAGTCTGGACTGGGGAGACATCTGGGCGTT (SEQ ID NO: 418).

[0173] Table 7: Spacer amino acid sequences TIFF2025513427000035.tif146157

[0174] Table 8: Spacer nucleic acid sequences TIFF2025513427000036.tif231157

[0175] In some embodiments, a CAR of the present disclosure may further comprise a short oligopeptide or polypeptide linker between 2 and 10 amino acid residues in length that can form a bond between the transmembrane domain and the cytoplasmic region of the CAR. A non-limiting example of a suitable linker is a glycine-serine duplex. In some embodiments, the linker comprises the amino acid sequence GGCKJSGGCKJS (SEQ ID NO: 208).

[0176] In some aspects, the transmembrane domain further comprises at least a portion of the extracellular domain of the same protein.

[0177] Intracellular signaling domains In some embodiments, a CAR of the present disclosure (e.g., an EMCN-specific, FLT3-specific, and / or CD33-specific CAR described herein) comprises one or more cytoplasmic domains or regions. The cytoplasmic domains or regions of a CAR may comprise an intracellular signaling domain.

[0178] Examples of suitable intracellular signaling domains that can be used in the CARs of the present disclosure include, but are not limited to, the cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act cooperatively to regulate signal transduction after antigen receptor engagement, as well as any derivatives or variants of these sequences, and any recombinant sequences that have the same functional capabilities.

[0179] Without wishing to be bound by theory, it is believed that signals generated through the TCR alone are insufficient for full activation of T cells, and therefore secondary and / or costimulatory signals are typically also required for full activation. Thus, T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains). Furthermore, T cell signaling and function (e.g., activation signaling cascades) can be negatively regulated by inhibitory receptors present on T cells via intracellular inhibitory co-signaling domains.

[0180] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure can comprise 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 the amino acid and nucleotide sequences, respectively, of exemplary inhibitory intracellular signaling domains. In some embodiments, the inhibitory intracellular signaling domain comprises one or more intracellular inhibitory co-signaling domains (see, e.g., Table 10 and Table 11). In some embodiments, the one or more intracellular inhibitory co-signaling domains are linked to other domains (e.g., transmembrane domains) via peptide linkers (see, e.g., Table 6) or spacer or hinge sequences (see, e.g., Table 7 and Table 8). In some embodiments, when two or more intracellular inhibitory co-signaling domains are present, the two or more intracellular inhibitory co-signaling domains may be linked via peptide linkers (see, e.g., Table 6) or spacer or hinge sequences (see, e.g., Tables 7 and 8). In some embodiments, the intracellular inhibitory co-signaling domain is an inhibitory domain. In some embodiments, the one or more intracellular inhibitory co-signaling domains of the chimeric protein comprise one or more ITIM-containing proteins, or fragments thereof. ITIMs are conserved amino acid sequences 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.

[0181] Table 10. Exemplary inhibitory intracellular signaling domain amino acid sequences TIFF2025513427000037.tif162153TIFF2025513427000038.tif235153TIFF2025513427000039.tif64153

[0182] Table 11. Exemplary inhibitory intracellular signaling domain nucleic acid sequences TIFF2025513427000040.tif164153TIFF2025513427000041.tif236153TIFF2025513427000042.tif239153 TIFF2025513427000043.tif236153TIFF2025513427000044.tif237153TIFF2025513427000045.tif134153

[0183] In some embodiments, the iCAR comprises an LIR1 intracellular inhibitory domain. In some embodiments, the iCAR comprises an LIR1 intracellular inhibitory domain having the amino acid sequence LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 285). In some embodiments, the sequence CTGCGGCACAGAAGGCAGGGCAAGCACTGGACAAGCACCCAGAGAAAGGCCGACTTTCAGCATCCTGCTGGCGCCGTTGGACCTGAGCCTACAGATAGAGGACTGCAGTGGCGGTCTAGCCCTGCCGCT GATGCCCAAGAGGAAAATCTTTACGCCGCCGTGAAGCACACCCAGCCTGAGGATGGCGTGGAAATGGACACCAGATCTCCCCACGATGAGGACCCTCAGGCCGTGACATACGCAGAAGTGAAGCACTCCAGACCTCGGAGAG AGATGGCAAGCCCTCCATCTCCTCTGAGCGGCGAGTTCCTGGACACCAAAGACAGACAGGCCGAAGAGGACAGACAGATGGATACCGAAGCCGCCGCTTCTGAAGCCCCACAGGATGTGACATATGCCCAGCTGCATAGCCTGACACTGCGGAGAGAAGCCACAGAGCCTCCACCTTCTCAAGAAGGCCCATCTCCTGCCGTGCCTTCCATCTATGCCACTCTGGCCATTCAC (SEQ ID NO: 286).In some embodiments, the iCAR is a CTGCGGCACAGAAGGCAGGGCAAGCACTGGACAAGCACCCAGAGAAAGGCCGACTTTCAGCATCCTGCTGGCGCCGTTGGACCTGAGCCTACAGATAGAGGACTGCAGTGGCGGTCTAGCCCTGCCGCTGATGCCCAAGAGGAAAATCTT TACGCCGCCGTGAAGCACACCCAGCCTGAGGATGGCGTGGAAATGGACACCAGATCTCCCCACGATGAGGACCCTCAGGCCGTGACATACGCAGAAGTGAAGCACTCCAGACCTCGGAGAGATGGCAAGCCCTCCATCTCCTCTGAGCGGCGAGTTCCTGGACA and a LIR1 intracellular inhibitory domain encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to CCAAAGACAGACAGGCCGAAGAGGACAGACAGATGGATACCGAAGCCGCCGCTTCTGAAGCCCCACAGGATGTGACATATGCCCAGCTGCATAGCCTGACACTGCGGAGAGAAGCCACAGAGCCTCCACCTTCTCAAGAAGGCCCATCTCCTGCCGTGCCTTCCATCTATGCCACTCTGGCCATTCAC (SEQ ID NO: 286).

[0184] In some embodiments, the one or more intracellular inhibitory co-signaling domains comprise one or more non-ITIM scaffold proteins, or fragments thereof, hi 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.

[0185] The inhibitory intracellular signaling domain can comprise an enzyme inhibitory domain. In some embodiments, the enzyme inhibitory domain comprises an enzyme catalytic domain. In some embodiments, the enzyme catalytic domain is derived from an enzyme selected from the group consisting of CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, and RasGAP. Examples of enzymatic regulation of signal transduction are described in detail by Pavel Otahal et al. (Biochim Biophys Acta. 2011 Feb;1813(2):367-76), Kosugi A., et al. (Involvement of SHP-1 tyrosine phosphatase in TCR-mediated signaling pathways in lipid rafts, Immunity, 2001 Jun;14(6):669-80), and Stanford, et al. (Regulation of TCR signaling by tyrosine phosphatases: from immune homeostasis to autoimmunity, Immunology, 2012 Sep;137(1):1-19), each of which is incorporated herein by reference for all purposes.

[0186] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure can comprise a primary signaling domain that controls primary activation of the TCR complex, either in a stimulatory or inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner can comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). Examples of suitable ITAM-containing primary intracellular signaling domains that can be used in a CAR of the present disclosure include, but are not limited to, those of CD3-zeta, FcR gamma, FcR beta beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, DAP10, DAP12, and CD66d.

[0187] In some embodiments, a CAR of the present disclosure comprises an intracellular signaling domain, such as the 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. In some embodiments, the CD3-zeta polypeptide may contain 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 number 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.

[0188] In other embodiments, the primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain, that has altered (e.g., increased or decreased) activity compared to the native ITAM domain. In one embodiment, the primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and / or truncated ITAM-containing primary intracellular signaling domain. In one embodiment, the primary signaling domain comprises one, two, three, four, or more ITAM motifs.

[0189] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure can comprise a CD3-zeta signaling domain by itself, or it can be combined with any other desired intracellular signaling domain useful in the context of a CAR of the present disclosure. For example, the intracellular signaling domain of a CAR can comprise a portion of a CD3-zeta chain and a costimulatory signaling domain. A costimulatory signaling domain can refer to a portion of a CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule of the present disclosure is a cell surface molecule other than an antigen receptor or its ligand that may be required for an efficient response of lymphocytes to antigens.Examples of suitable costimulatory molecules include 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, a ligand that specifically binds to CD83, an MHC class I molecule, a TNF receptor protein, an immunoglobulin G receptor protein, and the like. Cholesterol-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, IL2R beta, IL2R gamma, IL7R alpha, ITGA4 , VLAl, 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, C These include, but are not limited to, D96 (antennary), 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.

[0190] In some embodiments, the aCAR intracellular signaling domain comprises a CD28 costimulatory domain. In some embodiments, the aCAR intracellular signaling domain comprises a CD28 costimulatory domain having the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 287). In some embodiments, the aCAR intracellular signaling domain comprises 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 comprises a CD28 costimulatory domain encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to AGAAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCCGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGATTTCGCCGCCTACCGGTCC (SEQ ID NO: 288).

[0191] In some embodiments, the aCAR intracellular signaling domain comprises a CD3ζ signaling domain. In some embodiments, the aCAR intracellular signaling domain comprises a CD3ζ signaling domain having the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 289). In some embodiments, the aCAR intracellular signaling domain comprises a CD3 zeta signaling domain encoded by a polynucleotide sequence having: AGAGTGAAGTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 290).In some embodiments, the aCAR intracellular signaling domain is AGAGTGAAGTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGG A CD3ζ signaling domain encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to ATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 290).

[0192] In some embodiments, the aCAR intracellular signaling domain comprises a CD28 costimulatory domain and a CD3ζ signaling domain. In some embodiments, the aCAR intracellular signaling domain comprises a CD28 costimulatory domain having the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 287), and a CD3ζ signaling domain having the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 289).

[0193] In some embodiments, intracellular signaling sequences within the cytoplasmic portion of a CAR of the present disclosure can be linked to each other in a random or specific order. In some embodiments, a short oligopeptide or polypeptide linker, e.g., 2 to 10 amino acids in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), can form the linkage between intracellular signaling sequences. In one embodiment, a glycine-serine duplex can be used as a suitable linker. In one embodiment, a single amino acid, e.g., alanine or glycine, can be used as a suitable linker.

[0194] In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains, e.g., two costimulatory signaling domains, three costimulatory signaling domains, four costimulatory signaling domains, five costimulatory signaling domains, six costimulatory signaling domains, seven costimulatory signaling domains, eight costimulatory signaling domains, nine costimulatory signaling domains, ten costimulatory signaling domains, or more costimulatory signaling domains. In one embodiment, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the two or more costimulatory signaling domains are separated by a linker of the present disclosure (e.g., any of the linkers described in Table 6). In one embodiment, the linker is a glycine residue. In another embodiment, the linker is an alanine residue.

[0195] In some embodiments, the cells of the present disclosure express a CAR comprising an antigen binding domain, a transmembrane domain, a primary signaling domain, and one or more costimulatory signaling domains.

[0196] 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 derived from a protein selected from PD-1, CTLA4, TIGIT, BTLA, and LIR1 (LILRB1), TIM3, KIR3DL1, NKG2A, LAG3, SLAP1, SLAP2, Dok-1, Dok-2, LAIR1, GRB-2, CD200R, SIRPα, HAVR, GITR, PD-L1, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, CD94, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, and SIGLEC-10, respectively.

[0197] 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.

[0198] Natural killer cell receptor (NKR) CAR In some embodiments, a CAR of the present disclosure comprises one or more components of natural killer cell receptors (NKRs), thereby forming an NKR-CAR. The NKR components include KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3S1. The transmembrane domain, hinge domain, or cytoplasmic domain may be from any suitable natural killer cell receptor, including, but not limited to, killer cell immunoglobulin-like receptors (KIRs) such as DPI; natural cytotoxicity receptors (NCRs) such as NKp30, NKp44, and 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, the NKR-CAR may interact with an adapter molecule or an intracellular signaling domain such as DAP12. Exemplary configurations and sequences of CARs containing NKR components are described in International Patent Publication WO2014 / 145252, published September 18, 2014.

[0199] immunoresponsive cells Certain aspects of the present disclosure relate to cells, e.g., immunoresponsive cells, genetically engineered to contain one or more chimeric receptors of the present disclosure or one or more nucleic acids encoding such chimeric receptors, and methods of using such cells to treat myeloid malignancies (e.g., AML).

[0200] 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 neuron, an adipocyte, a liver cell, or a cardiac cell. In some embodiments, the cell is a stem cell. Exemplary stem cells include, but are not limited to, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), adult stem cells, and tissue-specific stem cells, such as hematopoietic stem cells (blood stem cells), mesenchymal stem cells (MSCs), neural stem cells, epithelial stem cells, or skin stem cells. In some embodiments, the cell is a cell derived from or differentiated from a stem cell of the present disclosure. In some embodiments, the cell is an immune cell. The immune cells of the present disclosure can be isolated or differentiated from the stem cells of the present disclosure (e.g., from ESCs or iPSCs). 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, natural killer T cells, alpha beta T cells, and gamma delta T cells), B cells, natural killer (NK) cells, dendritic cells, myeloid cells, macrophages, and monocytes. In some embodiments, the cell is a neuronal cell. Neuronal cells of the present disclosure can be isolated or differentiated from stem cells of the present disclosure (e.g., from ESCs or iPSCs). 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.

[0201] In some embodiments, the cell is an immunoresponsive cell. The immunoresponsive cells of the present disclosure can be isolated or differentiated from stem cells of the present disclosure (e.g., from ESCs or iPSCs). Exemplary immunoresponsive cells of the present disclosure include, but are not limited to, cells of the lymphoid lineage. The lymphoid lineage, including B cells, T cells, and natural killer (NK) cells, is responsible for producing antibodies, regulating the cellular immune system, detecting foreign substances in the blood, detecting cells foreign to the host, and the like. Examples of immunoresponsive cells of the lymphoid lineage 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., from which lymphocytes can be derived or differentiated). T cells mature in the thymus and can be lymphocytes primarily responsible for cell-mediated immunity. T cells participate in the adaptive immune system. In some embodiments, the T cells of the present disclosure include T helper cells, cytotoxic T cells, memory T cells (central memory T cells, stem cell-like memory T cells (or stem-like memory T cells)), and two types of effector memory T cells, e.g., T EM Cells and T EMRA The T cells can be any type of T cell, including, but not limited to, T cells, regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosal-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTLs 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 through the introduction of one or more chimeric receptors, such as chimeric TCRs or CARs.

[0202] Natural killer (NK) cells are lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation to exert their cytotoxic effect on target cells.

[0203] In some embodiments, the immunoresponsive 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 or stem cells.

[0204] In some embodiments, the immunoresponsive cells of the present disclosure are universal T cells with defective TCR-αβ. Methods for generating universal T cells are described in the art, for example, in Valton et al., Molecular Therapy (2015); 23 9, 1507-1518, and Torikai et al., Blood 2012 119:5697-5705.

[0205] In some embodiments, the immunoresponsive cells of the present disclosure are isolated immunoresponsive cells comprising one or more chimeric receptors of the present disclosure, hi some embodiments, the immunoresponsive 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.

[0206] In some embodiments, the immunoresponsive cells are T cells. In some embodiments, the immunoresponsive cells are natural killer (NK) cells.

[0207] In some embodiments, immunoresponsive cells express or are capable of expressing an immunoreceptor. Immunoreceptors generally can induce signal transduction or changes in protein expression in immunoreceptor-expressing cells, resulting in modulation of an immune response upon binding to a cognate ligand (e.g., modulating, activating, initiating, stimulating, increasing, preventing, attenuating, inhibiting, reducing, decreasing, inhibiting, or suppressing an immune response). For example, when CD3 chains present on a TCR / CAR cluster in response to ligand binding, a signal transduction cascade occurs via immunoreceptor tyrosine-based activation motifs (ITAMs). Specifically, in certain embodiments, binding of an endogenous TCR, an exogenous TCR, a chimeric TCR, or a CAR (particularly an activated CAR) to its respective antigen results in the formation of an immune synapse, which involves the clustering of many molecules (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.) near the bound receptor. This clustering of membrane-bound signaling molecules leads to the phosphorylation of ITAM motifs contained within the CD3 chains, which initiates the T cell activation pathway and ultimately activates transcription factors such as NF-κB and AP-1. These transcription factors can induce global gene expression in T cells, increasing IL-2 production for proliferation and expression of master regulator T cell proteins, thereby initiating T cell-mediated immune responses such as cytokine production and / or T cell-mediated killing.

[0208] Cells expressing multiple chimeric receptors In some embodiments, a cell (e.g., an immunoresponsive cell) of the present disclosure comprises two or more chimeric receptors of the present disclosure. In some embodiments, the cell comprises two or more chimeric receptors, and one of the two or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises three or more chimeric receptors, and at least one of the three or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises four or more chimeric receptors, and at least one of the four or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises five or more chimeric receptors, and at least one of the five or more chimeric receptors is a chimeric inhibitory receptor.

[0209] In some embodiments, each of the two or more chimeric receptors comprises a different antigen-binding domain, e.g., antigen-binding domains that bind to the same antigen or different antigens. In some embodiments, each of the antigens bound by the two or more chimeric receptors is expressed on the same cell, such as a myeloid cell type (e.g., the same AML cell type). In some embodiments, each of the antigens bound by the two or more chimeric receptors is an AML-associated antigen (e.g., FLT3, CD33, CD123, CLEC12A, CXCR4, EphA3, etc.).

[0210] In embodiments in which a cell (e.g., an immunoresponsive cell) of the present disclosure expresses two or more distinct chimeric receptors, the antigen-binding domains of each of the different chimeric receptors can be designed so that the antigen-binding domains do not interact with each other. For example, a cell (e.g., an immunoresponsive cell) of the present disclosure that expresses a first chimeric receptor (e.g., an EMCN-specific chimeric receptor) and a second chimeric receptor can include the first chimeric receptor that comprises 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 can comprise an antibody fragment such as an scFv, while the antigen-binding domain of the second chimeric receptor can comprise a VHH.

[0211] Without wishing to be bound by theory, it is believed that in cells bearing multiple chimeric membrane-embedded receptors, each comprising an antigen-binding domain, interactions between the antigen-binding domains of each receptor may be undesirable, as such interactions could inhibit the ability of one or more of the antigen-binding domains to bind to their cognate antigen. Thus, in embodiments in which a cell (e.g., an immunoresponsive cell) of the present disclosure expresses two or more chimeric receptors, the chimeric receptors comprise antigen-binding domains that minimize such inhibitory interactions. In one embodiment, the antigen-binding domain of one chimeric receptor comprises an scFv, and the antigen-binding domain of a second chimeric receptor comprises a single VH domain, e.g., a camel, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence.

[0212] In some embodiments, when present on the surface of a cell, binding of the antigen-binding domain of a first chimeric receptor to its cognate antigen is not substantially reduced by the presence of a second chimeric receptor. In some embodiments, binding of the antigen-binding domain of a 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 less 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 85%, 90%, 95%, 96%, 97%, 98%, or 99% less than when both are scFv antigen-binding domains.

[0213] Chimeric inhibitory receptors In some embodiments, a cell (e.g., an immunoresponsive cell) of the present disclosure comprises one or more chimeric inhibitory receptors of the present disclosure. In some embodiments, each of the one or more chimeric inhibitory receptors comprises an antigen-binding domain that binds to an antigen that is generally expressed on normal cells (e.g., cells generally considered to be healthy) but not expressed on tumor cells, such as AML cells. In some embodiments, the inhibitory chimeric 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).

[0214] 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, nervous tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, urinary bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

[0215] In some embodiments, a chimeric inhibitory receptor (e.g., an EMCN-specific chimeric inhibitory receptor) may be used in conjunction with one or more activating chimeric receptors (e.g., activating chimeric TCRs or CARs) expressed on a cell (e.g., an immunoresponsive cell) of the present disclosure, e.g., as a NOT logic gate to control, modulate, or otherwise inhibit one or more activities of one or more activating chimeric receptors. In some embodiments, an inhibitory chimeric receptor of the present disclosure may inhibit one or more activities of a cell (e.g., an immunoresponsive cell) of the present disclosure.

[0216] In some embodiments, the cells of the present disclosure comprise one or more inhibitory chimeric 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 an AML-associated antigen. 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.

[0217] Costimulatory LigandsIn some embodiments, cells (e.g., immunoresponsive cells) of the present disclosure can further comprise one or more recombinant or exogenous costimulatory ligands. For example, cells can be further transduced with one or more costimulatory ligands such that the cells co-express, or are induced to co-express, one or more chimeric receptors of the present disclosure (e.g., EMCN-specific, FLT3-specific, and / or CD33-specific CARs described herein) 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 may provide a non-antigen-specific signal important for the full activation of cells. Examples of suitable costimulatory ligands include, but are 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 the acute phase response. Its primary role is in regulating immune cells. Members of the TNF superfamily share many common features. Most TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. Examples of suitable TNF superfamily members include, 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 (TNFP) / lymphotoxin-alpha (LTa), lymphotoxin-beta (LTP), CD257 / B cell-activating factor (B AFF) / Bly s / THANK / Tall-1, glucocorticoid-inducible TNF receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins involved in cell recognition, binding, or adhesion processes. These proteins share structural features with immunoglobulins and possess immunoglobulin domains (folds).Examples of suitable immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, both ligands for CD28, and PD-L1 / (B7-H1), a ligand for PD-1. In certain embodiments, the one or more costimulatory ligands are selected from 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof.

[0218] chemokine receptors In some embodiments, cells (e.g., immunoresponsive cells) of the present disclosure comprise one or more chimeric receptors (e.g., EMCN-specific, FLT3-specific, and / or CD33-specific CARs described herein) and may further comprise one or more chemokine receptors. For example, transgenic expression of the chemokine receptors CCR2b or CXCR2 in cells, such as T cells, enhances trafficking to CCL2- or CXCL1-secreting solid tumors (Craddock et al., J Immunother. 2010 Oct;33(8):780-8 and Kershaw et al. Hum Gene Ther. 2002 Nov 1;13(16):1971-80). Without wishing to be bound by theory, it is believed that chemokine receptors expressed on chimeric receptor-expressing cells of the present disclosure may recognize chemokines secreted by tumors and improve targeting of cells to tumors, which may promote tumor infiltration and enhance the anti-tumor effects of cells. The chemokine receptor of the present disclosure may include a naturally occurring chemokine receptor, a recombinant chemokine receptor, or a chemokine-binding fragment thereof. Examples of suitable chemokine receptors that can be expressed on the cells of the present disclosure include, but are not limited to, C-X-C chemokine receptors, such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7; C-C chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11; C-C chemokine receptors, such as CXCR1; and X-C chemokine receptors, such as XCR1; and chemokine-binding fragments thereof. In some embodiments, the chemokine receptors expressed on the cells are selected based on chemokines secreted by tumors.

[0219] Chimeric receptor regulation Some embodiments of the present disclosure relate to regulating one or more chimeric receptor activities of a chimeric receptor-expressing cell of the present disclosure (e.g., an EMCN-specific CAR described herein). There are several ways in which chimeric receptor activity can be regulated. In some embodiments, a regulatable chimeric receptor that can control one or more chimeric receptor activities may be desirable to optimize the safety and / or efficacy of chimeric receptor therapy. For example, inducing apoptosis using a caspase fused to a dimerization domain (see, e.g., Di et al., N Engl. J. Med. 2011 Nov. 3;365(18):1673-1683) can be used as a safety switch in chimeric receptor therapy. In some embodiments, cells expressing a chimeric receptor of the present disclosure can also express inducible caspase-9 (iCaspase-9), which, upon administration of a dimerizing agent such as rimiduside (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)butanoyl]piperidine-2-carbonyl]oxypropyl]phenoxy]acetyl]amino]ethylamino]-2-oxoethoxy]phenyl]propyl](2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate), induces activation of caspase-9, resulting in cellular apoptosis. In some embodiments, iCaspase-9 contains a binding domain that contains a chemical inducer of dimerization (CID) that mediates dimerization in the presence of the CID, resulting in the inducible and selective depletion of chimeric receptor-expressing cells.

[0220] Alternatively, in some embodiments, the chimeric receptors of the present disclosure may be modulated by utilizing small molecules or antibodies that inactivate or inhibit chimeric receptor activity. For example, antibodies may delete chimeric receptor-expressing cells by inducing antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the chimeric receptor-expressing cells of the present disclosure may further express an antigen recognized by a molecule capable of inducing ADCC-mediated cell death or complement-induced cell death. For example, the chimeric receptor-expressing cells of the present disclosure may further express a receptor that can be targeted by an antibody or antibody fragment. Examples of suitable receptors that can be targeted by an antibody or antibody fragment include, but are not limited to, EpCAM, VEGFR, integrins (e.g., ανβ3, α4, αΙ3 / 4β3, α4β7, α5β1, ανβ3, αν), members of the TNF receptor superfamily (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, CD5, 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, and truncated versions thereof.

[0221] 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 (e.g., WO2011 / 056894).

[0222] In some embodiments, the chimeric receptor-expressing cells of the present disclosure further comprise a highly expressed compact marker / suicide gene that combines target epitopes from both the CD32 and CD20 antigens in the chimeric receptor-expressing cells, which binds to an anti-CD20 antibody (e.g., rituximab) and results in selective depletion of the chimeric receptor-expressing cells by ADCC. Other methods for depleting the chimeric receptor-expressing cells of the present disclosure include, but are not limited to, administering a monoclonal anti-CD52 antibody that selectively binds to and targets the chimeric receptor-expressing cells for destruction by inducing ADCC. In some embodiments, the chimeric receptor-expressing cells can be selectively targeted using a chimeric receptor ligand, such as an anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody can induce effector cell activity, such as ADCC or ADC activity. In some embodiments, the chimeric receptor ligand can further be conjugated to an agent that induces cell death, such as a toxin. In some embodiments, the chimeric receptor-expressing cells of the present disclosure can further express a target protein recognized by the cell depletion agent of the present disclosure. In some embodiments, the target protein is CD20 and the cell depletion agent is an anti-CD20 antibody. In such embodiments, the cell depletion agent is administered when it is desired to reduce or eliminate chimeric receptor-expressing cells. In some embodiments, the cell depletion agent is an anti-CD52 antibody.

[0223] 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 on separate polypeptides or members. For example, the set of polypeptides may include a dimerization switch that, in the presence of a dimerization molecule, allows the polypeptides to combine with each other to form a functional chimeric receptor.

[0224] Endomucin-specific antigen-binding domain The present disclosure provides antigen-binding domains (e.g., single-chain variable fragments) that bind to Endomucin (EMCN), chimeric proteins (e.g., any of the iCARs described herein) comprising an antigen-binding domain that binds to EMCN, and nucleic acids encoding such antigen-binding domains and chimeric proteins. Without wishing to be bound by theory, EMCN is a sialoglycoprotein that interferes with the assembly of focal adhesion complexes and inhibits interactions between cells and the extracellular matrix. An EMCN-specific antigen-binding domain binds to human EMCN (e.g., Uniprot Q9ULC0, incorporated herein by reference for all purposes) or an epitope fragment thereof. EMCN can be expressed on cells generally considered healthy, such as healthy hematopoietic stem cells (HSCs), HSPCs, healthy multipotent progenitor cells (MPPs), healthy lymphoid-myeloid primed progenitor cells (LMPPs), and healthy hematopoietic progenitor cells (HPCs). EMCN can be expressed on hematopoietic stem and progenitor cells (HSPCs). EMCN can be expressed on HSCs. EMCN can be expressed on MPPs. EMCN can be expressed on LMPPs. EMCN can be expressed on HPCs. EMCN-specific antibodies have been described, including CBFYE-0213, V.7.C7.1, L4B1, L5F12, L10B5, L3F12, L6H3, L9H8, and L10F12, as described in Samulowitz U. et al., Am. J. Path., 2002 May, 160(5):1669-1681, which is incorporated herein by reference for all purposes.

[0225] The present disclosure provides EMCN-specific antigen-binding domains comprising one or more of the amino acid sequences listed in Table 1.

[0226] (Table 1) TIFF2025513427000046.tif129159TIFF2025513427000047.tif144159

[0227] 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 the EMCN-VH comprises a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 291), a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 296), and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 298), and the EMCN-VL comprises a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of RYDMH (SEQ ID NO: 291), a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of VIWGNGNTHYHSALKS (SEQ ID NO: 296), and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of RIKD (SEQ ID NO: 298). The reference antibody comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SDENTYLN (SEQ ID NO: 299), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of QVSKLDS (SEQ ID NO: 300), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of LQGIHLPWT (SEQ ID NO: 301), and the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the reference antibody are defined based on the Kabat numbering scheme.

[0228] In some embodiments, the antigen-binding domain specific for EMCN comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the EMCN-VH comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQGTMVTVSS (SEQ ID NO: 302), and the EMCN-VL comprises the amino acid sequence DVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQGTKLEIK (SEQ ID NO: 310).

[0229] In some embodiments, an antigen binding domain specific for EMCN comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVSVIWGNGNTHYHSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTLRIKDWGQGTMVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCKSSQSLVASDENTYLNWFQQRPGQSPRRLIYQVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGIHLPWTFGQGTKLEIK (sequence number 311).In some embodiments, the antigen binding domain specific for EMCN is encoded by a polynucleotide sequence comprising the sequence (SEQ ID NO:312).In some embodiments, the antigen binding domain specific for EMCN is encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to (SEQ ID NO: 312).

[0230] FLT3 and CD33 specific antigen binding domains 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.

[0231] In some embodiments, a chimeric receptor comprises one or more of the amino acid sequences listed in Table A1 or Table A2. In some embodiments, an activating chimeric receptor (aCAR) comprises one or more of the amino acid sequences listed in Table A1 or Table A2. In some embodiments, a bispecific, bivalent aCAR comprises one or more of the amino acid sequences listed in Table A1 or Table A2. Table A1 provides the variable domains of antibody heavy or light chains. The CDRs were determined using the Kabat method and are shown in bold italics in Table A1 and in Table A2 for each variable heavy or variable light chain. In some embodiments, a nucleic acid encoding any of the chimeric receptors of the present disclosure comprises one or more of the nucleic acid sequences listed in Table B.

[0232] (Table A1) TIFF2025513427000048.tif39156TIFF2025513427000049.tif223156TIFF2025513427000050.tif54156

[0233] (Table B) TIFF2025513427000051.tif163156TIFF2025513427000052.tif232156TIFF2025513427000053.tif226156TIFF2025513427000054.tif41156

[0234] (Table A2) TIFF2025513427000055.tif198156

[0235] 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 complementarity-determining region 1 (CDR-H1) having the amino acid sequence of DYNMH (SEQ ID NO: 402), a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of YIYPYNGGTGYNQKFKSKA (SEQ ID NO: 403), and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of GRPAMDYWGQ (SEQ ID NO: 404); and the CD33-VL comprises a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of RAS The reference antibody comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of ESVDNYGISFMN (SEQ ID NO: 405), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of AASNQGS (SEQ ID NO: 406), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQSKEVPWT (SEQ ID NO: 407), wherein the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the reference antibody are defined based on the Kabat numbering scheme.

[0236] In some embodiments, an antigen-binding domain specific for CD33 comprises a CD33-VH having the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS (SEQ ID NO: 329), and a CD33-VL having the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 330).

[0237] 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 FLT3-VH comprises a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of GGTFSSYAIS (SEQ ID NO: 360), a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of GIIPIFGTANYAQKFQG (SEQ ID NO: 361), and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of FALFGFREQAFDI (SEQ ID NO: 362), and FLT3-VL comprises a heavy chain variable (VH) region having the amino acid sequence of GGTFSSYAIS (SEQ ID NO: 363), a heavy chain variable (VL) region having the amino acid sequence of GGTFSSYAIS (SEQ ID NO: 364), a heavy chain variable (VL) region having the amino acid sequence of GGTFSSYAIS (SEQ ID NO: 365), a heavy chain variable (VL) region having the amino acid sequence of GGTFSSYAIS (SEQ ID NO: 366), and a light chain variable (VL) region having the amino acid sequence of GGTFSSYAIS (SEQ ID NO: 367). comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of RASQSISSYLN (SEQ ID NO: 363), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of AASSLQS (SEQ ID NO: 364), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQSYSTPFT (SEQ ID NO: 365), wherein the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the reference antibody are defined based on the Kabat numbering scheme.

[0238] In some embodiments, an antigen-binding domain specific for FLT3 comprises an FLT3-VH having the amino acid sequence EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCATFALFGFREQAFDIWGQGTTVTVSS (SEQ ID NO: 315), and an FLT3-VL having the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQSYSTPFTFGPGTKVDIK (SEQ ID NO: 316).

[0239] In some embodiments, the bivalent, bispecific aCAR comprises an antigen binding domain specific for FLT3 and an antigen binding domain specific for CD33.

[0240] In some embodiments, a bivalent, bispecific aCAR specific for FLT3 and CD33 comprises the amino acid sequence (SEQ ID NO: 414).

[0241] In some embodiments, a bivalent, bispecific aCAR specific for FLT3 and CD33 comprises the amino acid sequence (SEQ ID NO: 415).

[0242] Engineered Nucleic Acids Provided herein is a multicistronic expression system comprising an engineered nucleic acid encoding (i) a membrane-cleavable chimeric protein, (ii) a bivalent aCAR, and (iii) an iCAR.

[0243] In certain embodiments described herein, the multicistronic expression system comprises an engineered nucleic acid encoding a promoter and an expression cassette containing exogenous polynucleotide sequences encoding each of (i) a membrane-cleavable chimeric protein, (ii) a bivalent aCAR, and (iii) an iCAR.

[0244] The promoter is operably linked to the multicistronic expression cassette such that the exogenous polynucleotide sequences encoding each of the membrane-cleavable chimeric protein, the bivalent aCAR, and the iCAR are configured to be expressed as a single polypeptide.

[0245] An "engineered nucleic acid" is a nucleic acid that does not occur in nature. However, it should be understood that while an engineered nucleic acid is entirely non-natural, it can contain naturally occurring nucleotide sequences. In some embodiments, an engineered nucleic acid contains nucleotide sequences from different organisms (e.g., from different species). For example, in some embodiments, an engineered nucleic acid contains a murine nucleotide sequence, a bacterial nucleotide sequence, a human nucleotide sequence, and / or a viral nucleotide sequence. The term "engineered nucleic acid" includes recombinant nucleic acids and synthetic nucleic acids. "Recombinant nucleic acid" refers to a molecule constructed by linking nucleic acid molecules and, in some embodiments, capable of replicating in living cells. "Synthetic nucleic acid" refers to a molecule that is amplified or synthesized chemically or by other means. Synthetic nucleic acids include those that are chemically or otherwise modified, but are capable of base-pairing with naturally occurring nucleic acid molecules. Modifications include, but are not limited to, one or more modified internucleotide linkages and non-naturally occurring nucleic acids. Modifications are described in further detail in U.S. Patent No. 6,673,611 and U.S. Patent Application Publication No. 2004 / 0019001, each of which is incorporated by reference in its entirety. The modified internucleotide linkages can be phosphorodithioate or phosphorothioate linkages. The non-natural nucleic acids can be locked nucleic acids (LNAs), peptide nucleic acids (PNAs), glycol nucleic acids (GNAs), phosphorodiamidate morpholino oligomers (PMOs or "morpholinos"), and threose nucleic acids (TNAs). Non-natural nucleic acids are described in further detail in International Application No. 1998 / 039352, U.S. Patent Application Publication No. 2013 / 0156849, and U.S. Patent Nos. 6,670,461, 5,539,082, and 5,185,444, each of which is incorporated by reference in its entirety. Recombinant and synthetic nucleic acids also include molecules resulting from replication of any of the foregoing. The engineered nucleic acids of the present disclosure can be encoded by a single molecule (e.g., contained in the same plasmid or other vector) or by multiple different molecules (e.g., multiple different, independently replicating molecules). The engineered nucleic acid can be an isolated nucleic acid.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.

[0246] 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). In some embodiments, the engineered nucleic acid constructs are produced using GIBSON ASSEMBLY® cloning (see, e.g., Gibson, D.G. et al. Nature Methods, 343-345, 2009, and Gibson, D.G. et al. Nature Methods, 901-903, 2010, each of which is incorporated herein by reference). GIBSON ASSEMBLY® typically uses three enzyme activities in a single-tube reaction: a 5' exonuclease, a Y-extension activity of a DNA polymerase, and a DNA ligase activity. The 5' exonuclease activity bites back the 5'-end sequence, exposing complementary sequences for annealing. The polymerase activity then fills in gaps in the annealed region. DNA ligase then seals the nicks and covalently links the DNA fragments together. The overlapping sequences of adjacent fragments are much longer than those used in Golden Gate Assembly, thus resulting in a higher rate of correct assembly. In some embodiments, engineered nucleic acid constructs are produced using IN-FUSION® Cloning (Clontech).

[0247] promoter Generally, in all embodiments described herein, the engineered nucleic acids encoding the membrane-cleavable chimeric proteins, bivalent aCARs, and iCARs encode expression cassettes comprising a promoter. In some embodiments, the engineered nucleic acids (e.g., engineered nucleic acids comprising expression cassettes) comprise a promoter operably linked to a nucleotide sequence (e.g., an exogenous polynucleotide sequence) encoding at least two different proteins. For example, the engineered nucleic acids may comprise a promoter operably linked 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, the engineered nucleic acids comprise a promoter operably linked to a nucleotide sequence encoding one, two, three, four, five, six, seven, eight, nine, ten, or more different proteins. In some embodiments, the engineered nucleic acids (e.g., engineered nucleic acids comprising expression cassettes) comprise a promoter operably linked to a nucleotide sequence (e.g., an exogenous polynucleotide sequence) encoding at least two membrane-cleavable chimeric proteins. For example, an engineered nucleic acid can comprise a promoter operably linked 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 10 chimeric membrane-cleavable proteins. In some embodiments, an engineered nucleic acid comprises a promoter operably linked to a nucleotide sequence encoding one, two, three, four, five, six, seven, eight, nine, ten, or more chimeric membrane-cleavable proteins.

[0248] A "promoter" refers to a regulatory region of a nucleic acid sequence that controls the initiation and rate of transcription of the remainder of the nucleic acid sequence. A promoter may also contain small regions to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, may bind. A promoter may be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter drives the expression or transcription of a nucleic acid sequence that it regulates. As used herein, a promoter is considered to be "operably linked" when it is in the correct functional location and orientation relative to the nucleic acid sequence that it regulates to control ("drive") transcription initiation and / or expression of that sequence.

[0249] A promoter may be a promoter naturally associated with a given gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter may be referred to as "endogenous." In some embodiments, a coding nucleic acid sequence may be placed under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the coded sequence in its natural environment. Such promoters may include promoters of other genes, promoters isolated from any other cell, and synthetic promoters or enhancers that are not "naturally occurring," such as those containing different elements of transcriptional regulation and / or mutations that alter expression through methods of genetic engineering known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, the sequences may be produced using nucleic acid amplification techniques, including recombinant cloning and / or polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,202 and 5,928,906).

[0250] The promoter of an engineered nucleic acid may be an "inducible promoter," which refers to a promoter characterized by modulating transcriptional activity (e.g., initiating or activating) when in the presence of, affected by, or contacted by a signal. The signal may be an endogenous or usually exogenous condition (e.g., light), compound (e.g., a chemical or non-chemical compound), or protein (e.g., a cytokine) that contacts the inducible promoter in such a way that it is active in modulating transcriptional activity from the inducible promoter. Activation of transcription may involve acting directly on the promoter to drive transcription, or indirectly on the promoter by inactivating a repressor that prevents the promoter from driving transcription. Conversely, inactivation of transcription may involve acting directly on the promoter to prevent transcription, or indirectly on the promoter by activating a repressor that in turn acts on the promoter.

[0251] A promoter is "responsive" to 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, a promoter contains a response element. A "response element" is a short sequence of DNA within the promoter region that binds to specific molecules (e.g., transcription factors) that modulate (regulate) gene expression from the promoter. Response elements that may be used in accordance with the present disclosure include, but are not limited to, phloretin tunable regulatory element (PEACE), zinc finger DNA binding domain (DBD), interferon gamma activating sequence (GAS) (Decker, T. et al. J Interferon Cytokine Res. 1997 Mar;17(3):121-34, incorporated herein by reference), interferon stimulated response element (ISRE) (Han, KJ et al. J Biol Chem. 2004 Apr 9;279(15):15652-61, incorporated herein by reference), NF-kappa B response element (Wang, V. et al. Cell Reports. 2012;2(4):824-839, incorporated herein by reference), and STAT3 response element (Zhang, D. et al. J of Biol Chem. 1996;271:9503-9509, incorporated herein by reference). Other response elements are encompassed herein. Response elements can also contain tandem repeats (e.g., consecutive repeats of the same nucleotide sequence encoding the response element) to generally increase 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.

[0252] Non-limiting examples of responsive promoters (also referred to as "inducible promoters") (e.g., TGF-beta responsive promoters) are listed in Table 5A, which shows the promoter and transcription factor design, and the effects of the transcription factor (TF) and inducer molecule on transgene transcription (T) are shown (B, binding; D, dissociation; nd, undetermined) (A, activation; DA, deactivation; DR, repression) (Horner, M. & Weber, W. FEBS Letters 586 (2012) 20784-2096m, and references cited therein). Other non-limiting examples of inducible promoter components include those listed in Table 5B.

[0253] Table 5A. Examples of responsive promoters TIFF2025513427000056.tif138150TIFF2025513427000057.tif226150TIFF2025513427000058.tif93150

[0254] Table 5B. Exemplary components of inducible promoters TIFF2025513427000059.tif165156

[0255] Other 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).

[0256] Table 5C: Exemplary constitutive promoters TIFF2025513427000060.tif232156TIFF2025513427000061.tif240156TIFF2025513427000062.tif241156TIFF2025513427000063.tif240156TIFF2025513427000064.tif222156

[0257] 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 SC-MT or MT-CS) such that expression is restricted to a particular cell type, organelle, or tissue. Tissue-specific promoters include, but are not limited to, albumin (liver-specific, Pinkert et al., (1987)), lymphocyte-specific promoters (Calame and Eaton, 1988), certain promoters of 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 (opalescent promoter, U.S. Pat. No. 4,873,316 and European Patent Publication No. 264,166), as well as developmentally regulated promoters, such as mouse hox promoters (Kessel and Gruss, Science 249:374-379 (1990)), or alpha-fetoprotein promoters (Campes and Tilghman, Genes Dev. 3:537-546 (1989)), and the like, the contents of each of which are incorporated herein by reference in their entireties. Promoters can be constitutive in each particular cell type, organelle, or tissue.Tissue-specific promoters and / or regulatory elements can also include the liver fatty acid-binding (FAB) protein gene specific for colonic epithelial cells; the insulin gene specific for pancreatic cells; the transfiletin, alpha-1-antitrypsin, plasminogen activator inhibitor type 1 (PAI-I), apolipoprotein AI, and LDL receptor genes specific for hepatocytes; the myelin basic protein (MBP) gene specific for oligodendrocytes; the glial fibrillary acidic protein (GFAP) gene specific for glial cells; a promoter from OPSIN specific for ocular targeting; and the neuron-specific enolase (NSE) promoter specific for neuronal cells. Examples of tissue-specific promoters include, but are not limited to, the promoter for creatine kinase, which has been used to direct expression in muscle and cardiac tissues and the immunoglobulin heavy or light chain promoter for expression in B cells. Other tissue-specific promoters include the human smooth muscle alpha-actin promoter. Exemplary tissue-specific expression elements for the liver include, but are not limited to, 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-alpha hydrolase (CYP-7) promoter, beta-galactosidase alpha-2,6 sialyltransferase promoter, insulin-like growth factor binding protein (IGFBP-I) promoter, aldolase B promoter, human transferrin promoter, and collagen type I promoter. Exemplary tissue-specific expression elements for the prostate include, but are not limited to, the prostatic acid phosphatase (PAP) promoter, the prostate-secreted protein of 94 (PSP94) promoter, the prostate-specific antigen complex promoter, and the human glandular kallikrein gene promoter (hgt-1). Exemplary tissue-specific expression elements for stomach tissue include, but are not limited to, the human H / K-ATPase alpha subunit promoter.Exemplary tissue-specific expression elements for the pancreas include, but are not limited to, the pancreatitis-associated protein promoter (PAP), elastase 1 transcriptional enhancer, pancreas-specific amylase and elastase enhancer promoter, and pancreatic cholesterol esterase gene promoter. Exemplary tissue-specific expression elements for the endometrium include, but are not limited to, the uterine globin 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 gamma-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), lambda 5, 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 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. An example of a tissue-specific expression element for breast cells is, for example, but is not limited to, the human alpha-lactalbumin promoter. An exemplary tissue-specific expression element for lung includes, but is not limited to, the cystic fibrosis transmembrane conductance regulator (CFTR) gene promoter.

[0258] In some embodiments, the promoters of the present disclosure are regulated by signals within the tumor microenvironment. The tumor microenvironment is considered to regulate a promoter if the activity of the promoter is increased or decreased by at least 10% in the presence of the tumor microenvironment compared to the activity of the promoter in the absence of the tumor microenvironment. In some embodiments, the activity of the promoter is increased or decreased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared 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% compared to the activity of the promoter in the absence of a tumor microenvironment.

[0259] In some embodiments, the activity of the promoter is increased or decreased by at least 2-fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100-fold) compared 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-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold compared 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 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100-fold compared to the activity of the promoter in the absence of a tumor microenvironment.

[0260] In some embodiments, the promoters of the present disclosure are activated under hypoxic conditions. "Hypoxia" is a condition in which the body or a region of the body lacks sufficient oxygen supply at the tissue level. Hypoxia can lead to inflammation (e.g., levels of inflammatory cytokines increase under hypoxic conditions). In some embodiments, the promoter activated under hypoxic conditions is operably linked to nucleotides encoding a chimeric protein that reduces the expression of the activity of inflammatory cytokines, thus reducing inflammation caused by hypoxia. In some embodiments, the promoter activated under hypoxic conditions comprises a hypoxia response element (HRE). A "hypoxia response element (HRE)" is a response element that responds to hypoxia-inducible factor (HIF). The HRE, in some embodiments, comprises the consensus motif NCGTG (where N is either A or G).

[0261] Activated conditionally controlled polypeptide (ACP) promoter system In some embodiments, the synthetic promoter is a promoter system comprising an activation-conditional control polypeptide (ACP-) binding domain sequence and a promoter sequence. Such systems are also referred to herein as "ACP-responsive promoters." Generally, an ACP promoter system comprises a first expression cassette encoding an activation-conditional control polypeptide (ACP) and a second expression cassette encoding an ACP-responsive promoter operably linked to an exogenous polynucleotide sequence, such as 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 separate engineered nucleic acids. In other embodiments, the first expression cassette and the second expression cassette are encoded by the same engineered nucleic acid. The ACP-responsive promoter can be operably linked to a nucleotide sequence encoding a single protein of interest or multiple proteins of interest.

[0262] The promoter of the ACP promoter system, e.g., the promoter driving expression of ACP, or any of the promoter sequences of the ACP-responsive promoter, can include any of the promoter sequences 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-responsive promoter, and tandem repeats thereof. In some embodiments, the ACP-responsive promoter comprises a minimal promoter.

[0263] In some embodiments, the ACP binding domain comprises one or more zinc finger binding sites. In some embodiments, the ACP-responsive promoter comprises a minimal promoter, and the ACP binding domain comprises one or more zinc finger binding sites. The ACP binding domain can 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 comprises 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, ZF protein domains are modular in design and comprise zinc finger arrays (ZFAs). A zinc finger array comprises multiple zinc finger protein motifs linked together. Each zinc finger motif binds to a different nucleic acid motif. This results in ZFAs with specificity for any desired nucleic acid sequence, for example, for ACP binding domains with 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, a ZFA is an array, string, or chain of tandemly arranged ZF motifs. A 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 to 10, 1 to 15, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, or 5 to 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 to 10, 1 to 15, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, or 5 to 15 ZFAs. In some embodiments, a ZF protein domain comprises 1 to 10 ZFAs. In some embodiments, a ZF protein domain comprises at least one ZFA. In some embodiments, a ZF protein domain comprises at least two ZFAs. In some embodiments, a ZF protein domain comprises at least three ZFAs. In some embodiments, the ZF protein domain comprises at least 4 ZFAs. In some embodiments, the ZF protein domain comprises at least 5 ZFAs. In some embodiments, the ZF protein domain comprises at least 10 ZFAs.

[0264] In some embodiments, the ACP is a transcriptional modulator. In some embodiments, the ACP is a transcriptional repressor. In some embodiments, the ACP is a transcriptional activator. In some embodiments, the ACP is a transcription factor. In some embodiments, the ACP comprises a DNA-binding domain and a transcriptional effector domain. In some embodiments, the DNA-binding domain comprises a tetracycline (or derivative thereof) repressor (TetR) domain. In some embodiments, the ACP is an antigen-recognition receptor of the present disclosure.

[0265] ACPs can also further comprise an effector domain, such as a transcription effector domain. For example, the transcription effector domain can be the effector domain or activator domain of a transcription factor. Transcription factor activation domains, also known as transactivator domains, act as scaffolding domains of proteins, such as transcriptional coregulators, that act to activate or repress gene transcription. Any suitable transcription effector domain can be used in an ACP, including, but not limited to, the herpes simplex virus protein 16 (VP16) activation domain; the VP64 activation domain, which is an activation domain consisting of four tandem copies of VP16; the p65 activation domain of NFκB; the Epstein-Barr virus R transcription activator (Rta) activation domain; the tripartite activator comprising the VP64, p65, and Rta activation domains (the tripartite activator is known as the VPR activation domain); the histone acetyltransferase (HAT) core domain of human E1A-associated protein p300 (p300 HAT core activation domain); Krüppel-associated box (KRAB) repression domain; repressor element silencing transcription factor (REST) ​​repression domain; WRPW motif of hairy-related basic helix-loop-helix repressor protein (this motif is known as the WRPW repression domain); DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and HP1 alpha chromoshadow repression domain, or any combination thereof.

[0266] In some embodiments, the effector domain is a transcriptional effector domain selected from the following: herpes simplex virus protein 16 (VP16) activation domain; the VP64 activation domain, which is an activation domain consisting of four tandem copies of VP16; the p65 activation domain of NFκB; the Epstein-Barr virus R transcriptional activator (Rta) activation domain; the tripartite activator comprising the VP64, p65, and Rta activation domains (the tripartite activator is known as the VPR activation domain); the histone acetyltransferase (HAT) core domain of the human E1A-associated protein p300 (p300 known as the HAT core activation domain); the Krüppel-associated box (KRAB) repression domain; the repressor element silencing transcription factor (REST) ​​repression domain; the WRPW motif of the hairy-related basic helix-loop-helix repressor protein (this motif is known as the WRPW repression domain); the DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and the HP1 alpha chromoshadow repression domain.

[0267] In some embodiments, the ACP is a small molecule (e.g., drug)-inducible polypeptide. For example, in some embodiments, the ACP can be induced by tetracycline (or a derivative thereof) and comprises a TetR domain and a VP16 effector domain. In some embodiments, the ACP comprises an estrogen receptor variant, such as ERT2, and can be regulated by tamoxifen or its metabolites (e.g., 4-hydroxytamoxifen [4-OHT], N-desmethyltamoxifen, tamoxifen-N-oxide, or endoxifen) through tamoxifen-regulated nuclear localization.

[0268] In some embodiments, the ACP is a small molecule (e.g., drug)-inducible polypeptide that includes an inhibitory protease and one or more cognate cleavage sites for the inhibitory protease. In some embodiments, the inhibitory protease is active (cleaves the cognate cleavage site) in the absence of a specific agent and inactive (does not cleave the cognate cleavage site) in the presence of a specific agent. In some embodiments, the specific agent is a protease inhibitor. In some embodiments, the protease inhibitor specifically inhibits a given inhibitory protease of the present disclosure. The inhibitory protease can be any of the proteases described herein that can be inactivated by the presence or absence of a specific agent (see "Protease Cleavage Sites" above for exemplary inhibitory proteases, cognate cleavage sites, and protease inhibitors).

[0269] In some embodiments, the ACP has a degron domain (see "Degron Systems and Domains" above for exemplary degron sequences). The degron domain can be in any order or location relative to the individual domains of the ACP. For example, the degron domain can be N-terminal to the inhibitory protease, C-terminal to the inhibitory protease, N-terminal to the ZF protein domain, C-terminal to the ZF protein domain, N-terminal to the effector domain, or C-terminal to the effector domain.

[0270] Multicistronic and multiple promoter systems In some embodiments, an engineered nucleic acid (e.g., an engineered nucleic acid comprising an expression cassette) is configured to produce multiple chimeric proteins. For example, the nucleic acid may be configured to produce between 2 and 20 different chimeric proteins.In some embodiments, the nucleic acid is selected from the group consisting of 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10 , 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-1 9, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-1 9, 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, 14 to 19, 14 to 18, 14 to 17, 14 to 16, 14 to 15, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 15 to 16, 16 to 20, 16 to 19, 16 to 18, 16 to 17, 17 to 20, 17 to 19, 17 to 18, 18 to 20, 18 to 19, or 19 to 20 chimeric proteins.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.

[0271] Generally, the engineered nucleic acids described herein are multicistronic, i.e., as described above.

[0272] The engineered nucleic acid can also use multiple promoters to express genes from multiple ORFs, i.e., two or more separate mRNA transcripts can be produced from a single engineered nucleic acid. For example, a first promoter can be operably linked to a polynucleotide sequence encoding a first chimeric protein, and a second promoter can be operably linked to a polynucleotide sequence encoding a second chimeric protein. Generally, any number of promoters can be used to express any number of chimeric proteins. In some embodiments, at least one of the ORFs expressed from the multiple promoters can be multicistronic.

[0273] As used herein, a "linker" can refer to a polypeptide that links a first polypeptide sequence and a second polypeptide sequence, a multicistronic linker as described above, or an additional promoter operably linked to an additional ORF as described above.

[0274] Engineered cells Provided herein are engineered cells and methods for producin...

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