Protein payload release

Abstract

Described herein are chimeric proteins, particularly membrane-cleavable chimeric systems. Nucleic acids, cells, and methods related thereto are also described herein. TIFF2023548586000047.tif78170

Description

Background Art

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Application No. 63 / 193,004, filed May 25, 2021, and No. 63 / 109,812, filed November 4, 2020, each of which is hereby incorporated by reference in its entirety for all purposes.

[0002] Sequence listing This application includes a sequence listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. The ASCII copy was created in 20XX, named XXX - XXX_sequencelisting.txt, and is , bytes in size.

[0003] Background Cell - based therapeutic platforms offer promising means for treating various diseases. One such promising platform is CAR - T - based therapy in cancer treatment. Assuming their promise, improvements in cell - based therapies are needed. An active area of exploration is manipulating cell - based therapies to produce and / or secrete effector molecules, such as cytokines, in a process referred to as armoring, which enhances cell - based therapies. For example, non - armored CAR - T therapies have insufficient efficacy in solid tumors, and armoring can affect the entire cancer immune cycle and enhance the activity of CAR - Ts. However, controlled or uncontrolled armoring strategies can have negative effects on the therapy, such as off - target effects and toxicity in the subject. Thus, additional methods for controlling and regulating the armoring of cell - based therapies, such as modulating the production and / or secretion of payload effector molecules, are needed.

Summary of the Invention

[0004] Summary Provided herein are cell-based therapeutic platforms, which in some embodiments include regulated armoring for cell-based therapies, such as regulated secretion of payload effector molecules. Also provided herein are combination cell-based immunotherapies, which in some embodiments include regulated armoring for targeted therapies of cancers, such as ovarian cancer, breast cancer, colon cancer, lung cancer, and pancreatic cancer.

[0005] Therapies provided herein, however, may limit the systemic toxicity of armoring. For example, immunotherapies provided herein may be tumor-specific and effective while limiting systemic toxicity and / or other off-target effects attributable to armoring. These therapies involve delivering the target protein, such as an immunomodulatory effector molecule, in a regulated manner, including the modulation of secretory dynamics, cellular state specificity, and cell or tissue specificity. The design of the delivery medium is optimized to improve the overall function in cell-based therapies, such as cancer therapy, and includes, but is not limited to, optimization of membrane cleavage sites, promoters, linkers, signal peptides, delivery methods, combinations, modulation, and sequence of immunomodulatory effector molecules.

[0006] Non-limiting examples of effector molecules included in this disclosure include cytokines, antibodies, chemokines, nucleotides, peptides, enzymes, and oncolytic viruses. For example, cells can be manipulated to express and secrete at least one, two, three, or more of the following effector molecules in a controlled manner: IL-12, IL-16, IFN-β, IFN-γ, IL-2, IL-15, IL-7, IL-36γ, IL-18, IL-1β, IL-21, OX40 ligand, CD40L, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-TGFβ antibody, anti-TNFR2, MIP1α(CCL3), MIP1β(CCL5), CCL21, CpG oligodeoxynucleotide, and antitumor peptides (e.g., antimicrobial peptides with antitumor activity). For example, Gaspar, D. et al. Front Microbiol. 2013; 4: 294; Chu, H. et al. PLoS One. 2015; 10(5): See e0126390 and the website: aps.unmc.edu / AP / main.php.

[0007] Provided herein is an engineered nucleic acid comprising an expression cassette comprising an exogenous polynucleotide sequence oriented N-terminal to C-terminal, comprising a promoter and a membrane-cleavable chimeric protein, having the formula:SC-MT or MT-CS, wherein S comprises a secreted effector molecule, C comprises a protease cleavage site, and MT comprises a cell membrane anchoring domain, wherein the promoter is operably ligated to the exogenous polynucleotide sequence, and wherein SC-MT or MT-CS is configured to be expressed as a single polypeptide.

[0008] Furthermore, provided herein are membrane-cleavable chimeric proteins oriented from the N-terminus to the C-terminus, having the formula SC-MT or MT-CS, where S comprises a secreted effector molecule, 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.

[0009] Also provided herein are isolated cells comprising an engineered nucleic acid, wherein the engineered nucleic acid comprises an expression cassette comprising an exogenous polynucleotide sequence oriented from the N-terminus to the C-terminus, comprising a promoter and a membrane-cleavable chimeric protein, having the formula: SC-MT or MT-CS, wherein S comprises a secreted effector molecule, C comprises a protease cleavage site, and MT comprises a cell membrane anchoring domain, wherein the promoter is operably ligated to the exogenous polynucleotide sequence, and wherein SC-MT or MT-CS is configured to be expressed as a single polypeptide.

[0010] Also provided herein are isolated cells containing a membrane-cleavable chimeric protein, wherein the membrane-cleavable chimeric protein, oriented from the N-terminus to the C-terminus, has the formula SC-MT or MT-CS, where S comprises a secreted effector molecule, C comprises a protease cleavage site, and MT comprises a cell membrane anchoring domain, and where SC-MT or MT-CS is configured to be expressed as a single polypeptide.

[0011] Also provided herein is a method for inducing the release of a membrane-bound effector molecule, comprising: a) providing a cell comprising a membrane-bound protease and a membrane-cleavable chimeric protein oriented from N-terminus to C-terminus, having the formula: SC-MT or MT-CS, wherein S comprises a secreted effector molecule and C comprises a homogeneous protease cleavage site of the membrane-bound protease, wherein SC-MT or MT-CS is configured to be expressed as a single polypeptide; and b) culturing the cell under conditions suitable for the expression of the membrane-bound protease and the membrane-cleavable chimeric protein, wherein, upon expression, the membrane-cleavable chimeric protein is anchored to the cell membrane, wherein, upon expression, the membrane-bound protease cleaves the homogeneous membrane-bound protease cleavage site of the membrane-cleavable chimeric protein, thereby releasing the secreted effector molecule from the cell membrane.

[0012] In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is selected from the group consisting of: CAG, HLP, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter includes a minimal promoter and response elements selected from the group consisting of: 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, inducer molecule response promoter, and their tandem repeats.

[0013] In some embodiments, the promoter is a synthetic promoter. In some embodiments, the synthetic promoter comprises an activation-conditional control polypeptide-(ACP-) binding domain sequence and a promoter sequence. In some embodiments, the promoter sequence is derived from a promoter selected from the group consisting of: 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-responsive element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducer molecule-responsive promoter, and their tandem repeats. In some embodiments, the ACP-binding domain comprises one or more zinc finger binding sites. In some embodiments, the synthetic promoter is moduloable by an activation-conditional control polypeptide (ACP) that binds to the ACP-binding domain of the synthetic promoter.

[0014] In some embodiments, ACP is a transcription modulator. In some embodiments, ACP is a transcription repressor. In some embodiments, ACP is a transcription activator. In some embodiments, ACP further comprises an inhibitory protease and one or more homologous cleavage sites of an inhibitory protease. In some embodiments, ACP further comprises the hormone-binding domain (ERT2 domain) of the estrogen receptor.

[0015] In some embodiments, ACP is a transcription factor. In some embodiments, the transcription factor is a zinc finger-containing transcription factor. In some embodiments, ACP comprises a DNA-binding zinc finger protein domain (ZF protein domain) and a transcription effector domain. In some embodiments, the ZF protein domain is a modular design and consists of a zinc finger array (ZFA). In some embodiments, the ZF protein domain contains 1 to 10 ZFAs.

[0016] In some embodiments, the effector domain is selected from the group consisting of: herpes simplex virus protein 16 (VP16) activation domain; activation domain containing four tandem copies of VP16; VP64 activation domain; p65 activation domain of NFκB; Epstein-Barr virus R transactivator (Rta) activation domain; tripartite activator containing VP64, p65, and Rta activation domains (VPR activation domain); histone acetyltransferase (HAT) core domain of human E1A-related protein p300 (p300 HAT core activation domain); Kruppel-related box (KRAB) repressor domain; repressor element silencing transcription factor (REST) ​​repressor domain; WRPW of hairy-related basic helix-loop-helix repressor protein. (Sequence ID 224) The motif, this motif is WRPW (Sequence ID 224) Known repressive domains include the DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repressive domain and the HP1 alpha-chromoshadow repressive domain.

[0017] In some embodiments, one or more homogeneous cleavage sites of the inhibitory protease are localized between the ZF protein domain and the effector domain. In some embodiments, the inhibitory protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3). In some embodiments, the homogeneous cleavage sites include NS3 protease cleavage sites. In some embodiments, the NS3 protease cleavage sites include NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, or NS5A / NS5B junction cleavage sites. In some embodiments, the NS3 protease can be inhibited by a protease inhibitor. In some embodiments, the protease inhibitor is selected from the group consisting of: simeprevir, danoprevir, asunaprevir, silprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and boxilaprevir.

[0018] In some embodiments, ACP can undergo nuclear localization upon binding of the ERT2 domain to tamoxifen or its metabolites. In some embodiments, the tamoxifen metabolites are selected from the group consisting of: 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0019] In some embodiments, the ACP further comprises a degron domain, in which the degron domain is operably linked to the ACP. In some embodiments, the degron domain comprises HCV NS4 degron, PEST (two copies of human IκBα residues 277-307), GRR (human p105 residues 352-408), DRR (yeast Cdc34 residues 210-295), SNS (tandem repeat of SP2 and NB (SP2-NB-SP2 of influenza A or influenza B), RPB (four copies of yeast RPB residues 1688-1702), SPmix (tandem repeat of SP1 and SP2) Peat (SP2-SP1-SP2-SP1-SP2 of influenza A virus M2 protein), NS2 (three copies of residues 79-93 of influenza A virus NS protein), ODC (residues 106-142 of ornithine decarboxylase), Nek2A, mouse ODC (residues 422-461), mouse ODC_DA (residues 422-461 of mODC containing point mutations D433A and D434A), APC / C The group is selected from degron, COP1 E3 ligase-conjugated degron motif, CRL4-Cdt2-conjugated PIP degron, actinphylline-conjugated degron, KEAP1-conjugated degron, KLHL2 and KLHL3-conjugated degron, MDM2-conjugated motif, N degron, hydroxyproline modification in hypoxia signaling, plant hormone-dependent SCF-LRR-conjugated degron, SCF ubiquitin ligase-conjugated phosphodegron, plant hormone-dependent SCF-LRR-conjugated degron, SCF ubiquitin ligase-conjugated phosphodegron, plant hormone-dependent SCF-LRR-conjugated degron, DSGxxS phosphate-dependent degron, Siah-conjugated motif, SPOP SBC-docking motif, and PCNA-conjugated PIP box. In some embodiments, the degron domain includes a cerebron (CRBN) polypeptide substrate domain capable of binding to CRBN in response to an immunomodulatory drug (IMiD), thereby promoting ubiquitin-mediated degradation of ACP.In some embodiments, the CRBN polypeptide substrate domain is selected from the group consisting of: IKZF1, IKZF3, CK1a, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827 or a drug-inducible binding fragment of CRBN. In some embodiments, the CRBN polypeptide substrate domain is a chimeric fusion product of a natural CRBN polypeptide sequence. In some embodiments, the CRBN polypeptide substrate domain is an IKZF3 / ZFP91 / IKZF3 chimeric fusion product having the amino acid sequence FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 175). In some embodiments, IMiD is an FDA-approved drug. In some embodiments, IMiD is selected from the group consisting of: thalidomide, lenalidomide, and pomalidomide. In some embodiments, the degron domain is the N-terminus of an inhibitory protease, the C-terminus of an inhibitory protease, the N-terminus of a ZF protein domain, the C-terminus of a ZF protein domain, the N-terminus of an effector domain, or the C-terminus of an effector domain.

[0020] In some embodiments, the promoter is a tissue-specific promoter.

[0021] In some embodiments, the secreted effector molecule includes a signal peptide or a signal anchor sequence. In some embodiments, the signal peptide includes a native signal peptide that is native to the secreted effector molecule. In some embodiments, the signal peptide includes a non-native signal peptide, or the signal anchor sequence includes a non-native signal anchor sequence that is non-native to the secreted effector molecule. In some embodiments, the non-native signal peptide or non-native signal anchor sequence is selected from the group consisting of: IL-12, IL-2, optimized IL-2, trypsinogen-2, Gaussian alciferase, CD5, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azulocidine preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpine E1, GRO-alpha, CXCL12, IL21, CD8, NKG2D, TNFR2, and GMCSF.

[0022] In some embodiments, the secreted effector molecule is selected from a therapeutic class, where the therapeutic class is selected from the group consisting of: cytokines, chemokines, homing molecules, growth factors, coactivating molecules, tumor microenvironment modifiers, ligands, antibodies, peptides, and enzymes. In some embodiments, the cytokine is selected from the group consisting of: IL-1-beta, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-12p70 fusion protein, IL-15, IL-17A, IL-18, IL-21, IL-22, type I interferon, interferon-gamma, and TNF-alpha. In some embodiments, the secreted effector molecule comprises IL-15, IL-12, or IL-12p70 fusion protein. In some embodiments, the secreted effector molecule comprises IL-15. In some embodiments, the secreted effector molecule comprises IL-15 having the amino acid sequence of SEQ ID NO: 199. In some embodiments, the secreted effector molecule contains IL-15 and IL-15Rαsushi domains. In some embodiments, the secreted effector molecule contains an IL-15 / IL-15Rαsushi domain fusion protein having the amino acid sequence of SEQ ID NO: 202. In some embodiments, the secreted effector molecule consists of IL-15 and IL-15Rαsushi domains. In some embodiments, the secreted effector molecule contains IL-12. In some embodiments, the secreted effector molecule contains an IL-12p70 fusion protein. In some embodiments, the secreted effector molecule contains an IL-12p70 fusion protein having the amino acid sequence of SEQ ID NO: 203. In some embodiments, the secreted effector molecule consists of IL-12. In some embodiments, the secreted effector molecule consists of an IL-12p70 fusion protein. In some embodiments, the secreted effector molecule is IL-15. In some embodiments, the chemokine is selected from the group consisting of: CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1. In some embodiments, the homing molecule is selected from the group consisting of: anti-integrin alpha 4, beta 7; anti-MAdCAM; SDF1; and MMP-2.In some embodiments, the growth factor is selected from the group consisting of: FLT3L and GM-CSF. In some embodiments, the co-activating molecule is selected from the group consisting of: 4-1BBL and CD40L. In some embodiments, the tumor microenvironment modifier is selected from the group consisting of: adenosine deaminase, TGF beta inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2. In some embodiments, the TGF beta inhibitor is selected from the group consisting of: anti-TGF beta peptides, anti-TGF beta antibodies, TGFb-TRAP, and combinations thereof. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of: anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIM-3 antibody, anti-TIGIT antibody, anti-VISTA antibody, anti-KIR antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-GAL9 antibody, anti-A2AR antibody, anti-phosphatidylserine antibody, anti-CD27 antibody, anti-TNFα antibody, anti-TREM1 antibody, and anti-TREM2 antibody. In some embodiments, the VEGF inhibitor includes an anti-VEGF antibody, an anti-VEGF peptide, or a combination thereof. In some embodiments, the secreted effector molecule is a human-derived effector molecule.

[0023] In some embodiments, the protease cleavage site is selected from the group consisting of: type 1 transmembrane protease cleavage site, type 2 transmembrane protease cleavage site, GPI-anchored protease cleavage site, ADAM8 protease cleavage site, ADAM9 protease cleavage site, ADAM10 protease cleavage site, ADAM12 protease cleavage site, ADAM15 protease cleavage site, ADAM17 protease cleavage site, ADAM19 protease cleavage site, ADAM20 protease cleavage site, ADAM21 protease cleavage site, ADAM2 8 protease cleavage sites, ADAM30 protease cleavage sites, ADAM33 protease cleavage sites, BACE1 protease cleavage sites, BACE2 protease cleavage sites, SIP protease cleavage sites, MT1-MMP protease cleavage sites, MT3-MMP protease cleavage sites, MT5-MMP protease cleavage sites, furin protease cleavage sites, PCSK7 protease cleavage sites, matryptase protease cleavage sites, matryptase 2 protease cleavage sites, MMP9 protease cleavage sites, and NS3 protease cleavage sites. In some embodiments, the protease cleavage site can be cleaved by a protease selected from the group consisting of: type 1 transmembrane protease, type 2 transmembrane protease, GPI-anchored protease, ADAM8 protease, ADAM9 protease, ADAM10 protease, ADAM12 protease, ADAM15 protease, ADAM17 protease, ADAM19 protease, ADAM20 protease, ADAM21 protease, ADAM28 protease, ADAM30 protease, ADAM33 protease, BACE1 protease, BACE2 protease, SIP protease, MT1-MMP protease, MT3-MMP protease, MT5-MMP protease, furin protease, PCSK7 Proteases, matryptase proteases, matryptase 2 proteases, MMP9 proteases, and NS3 proteases.

[0024] In some embodiments, the protease cleavage site is cleavable by ADAM17 protease. In some embodiments, the protease cleavage site includes a first region having the amino acid sequence of PRAE (SEQ ID NO: 176). In some embodiments, the protease cleavage site includes a second region having the amino acid sequence of KGG (SEQ ID NO: 177). In some embodiments, the first region is located at the N-terminus of the second region. In some embodiments, the protease cleavage site includes the amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 177). 219) and 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). 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). In some embodiments, the protease cleavage site comprises the amino acid sequence of ITQGLAVSTISSFF (SEQ ID NO: 198).

[0025] In some embodiments, the cell membrane anchoring domain includes a transmembrane-intracellular domain or a transmembrane domain. In some embodiments, the transmembrane-intracellular domain and / or the transmembrane domain is 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. In some embodiments, the cell membrane anchoring domain includes a cell surface receptor or its cell membrane binding portion.

[0026] In some embodiments, the cell membrane anchoring domain includes a post-translational modification tag, or a post-translational modifiable motif for modifying a chimeric protein to include a post-translational modification tag, wherein the post-translational modification tag is capable of associating with the cell membrane. In some embodiments, the post-translational modification tag includes a lipid anchoring domain, which may be selected from the group consisting of: GPI lipid anchors, myristoylation tags, and palmitoylation tags.

[0027] In some embodiments, when expressed in a cell, the secreted effector molecule is tethered to the cell's cell membrane. In some embodiments, when expressed in a cell that expresses a protease capable of cleaving a protease cleavage site, the secreted effector molecule is released from the cell membrane. In some embodiments, the protease expressed on the cell membrane is endogenous to the cell. In some embodiments, the protease is selected from the group consisting of: type I transmembrane protease, type II transmembrane protease, GPI-anchored protease, ADAM8 protease, ADAM9 protease, ADAM10 protease, ADAM12 protease, ADAM15 protease, ADAM17 protease, ADAM19 protease, ADAM20 protease, ADAM21 protease, ADAM28 protease, ADAM30 protease, ADAM33 protease, BACE1 protease, BACE2 protease, SIP protease, MT1-MMP protease, MT3-MMP protease, MT5-MMP protease, furin protease, PCSK7 protease, matriptase protease, matriptase-2 protease, and MMP9 protease. In some embodiments, the protease is ADAM17 protease.

[0028] In some embodiments, the protease expressed on the cell membrane is heterogeneous to the cell. In some embodiments, the protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3). In some embodiments, the protease cleavage site includes an NS3 protease cleavage site. In some embodiments, the NS3 protease cleavage site includes an NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, or NS5A / NS5B junction cleavage site. In some embodiments, the protease can be suppressed by a protease inhibitor. In some embodiments, the protease inhibitor is selected from the group consisting of: simeprevir, danoprevir, asunaprevir, silprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and boxilaprevir. In some embodiments, the expression and / or localization of the protease is moduloable. In some embodiments, expression and / or localization are regulated by the cellular state of the cell.

[0029] In some embodiments, the manipulated nucleic acid is a single-stranded or double-stranded nucleic acid selected from the group consisting of: DNA, cDNA, RNA, mRNA, and naked plasmid.

[0030] In some embodiments, isolated cells are selected from the following groups: T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells. In some embodiments, isolated cells are natural killer (NK) cells.

[0031] In some embodiments, the isolated cells are autologous. In some embodiments, the isolated cells are homogeneous. In some embodiments, the isolated cells are tumor cells selected from the group consisting of: bladder tumor cells, brain tumor cells, breast tumor cells, cervical tumor cells, colorectal tumor cells, esophageal tumor cells, glioma cells, kidney tumor cells, liver tumor cells, lung tumor cells, melanoma cells, ovarian tumor cells, pancreatic tumor cells, prostate tumor cells, skin tumor cells, thyroid tumor cells, and uterine tumor cells.

[0032] In some embodiments, isolated cells were manipulated via transduction using oncolytic viruses.

[0033] In some embodiments, the isolated cells further contain a protease capable of cleaving protease cleavage sites. In some embodiments, the protease contains an endogenous protease. In some embodiments, the endogenous protease is selected from the group consisting of: type 1 transmembrane proteases, type 2 transmembrane proteases, GPI-anchored proteases, ADAM8 proteases, ADAM9 proteases, ADAM10 proteases, ADAM12 proteases, ADAM15 proteases, ADAM17 proteases, ADAM19 proteases, ADAM20 proteases, ADAM21 proteases. Theases include 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, matryptase protease, matryptase 2 protease, and MMP9 protease. In some embodiments, the endogenous protease is ADAM17 protease. In some embodiments, the protease is a heterologous protease. In some embodiments, the heterologous protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3). In some embodiments, the protease is expressed on the cell membrane of isolated cells. In some embodiments, the protease is capable of cleaving protease cleavage sites. In some embodiments, cleavage of the protease cleavage site releases secretory effector molecules from the cell membrane of isolated cells.

[0034] In some embodiments, the protease cleavage site includes a first region having the amino acid sequence of PRAE (SEQ ID NO: 176). In some embodiments, the protease cleavage site includes a second region having the amino acid sequence of KGG (SEQ ID NO: 177). In some embodiments, the first region is located at the N-terminus of the second region. In some embodiments, the protease cleavage site includes the amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 177). 219) includes, where X1 is A, Y, P, S, or F, and where X2 is V, L, S, I, Y, T, or A. In some embodiments, the protease cleavage site includes the amino acid sequence PRAEX1X2KGG (SEQ ID NO: 178), where X1 is A, Y, P, S, or F, and where X2 is V, L, S, I, Y, or T. In some embodiments, the protease cleavage site includes the amino acid sequence PRAEAVKGG (SEQ ID NO: 179). In some embodiments, the protease cleavage site includes the amino acid sequence PRAEALKGG (SEQ ID NO: 180). In some embodiments, the protease cleavage site includes the amino acid sequence PRAEYSKGG (SEQ ID NO: 181). In some embodiments, the protease cleavage site includes the amino acid sequence PRAEPIKGG (SEQ ID NO: 182). In some embodiments, the protease cleavage site includes the amino acid sequence PRAEAYKGG (SEQ ID NO: 183). In some embodiments, the protease cleavage site includes the amino acid sequence PRAESSKGG (SEQ ID NO: 184). In some embodiments, the protease cleavage site includes the amino acid sequence PRAEFTKGG (SEQ ID NO: 185). In some embodiments, the protease cleavage site includes the amino acid sequence PRAEAAKGG (SEQ ID NO: 186). In some embodiments, the protease cleavage site includes the amino acid sequence DEPHYSQRR (SEQ ID NO: 187). In some embodiments, the protease cleavage site includes the amino acid sequence PPLGPIFNPG (SEQ ID NO: 188). In some embodiments, the protease cleavage site includes the amino acid sequence PLAQAYRSS (SEQ ID NO: 189). In some embodiments, the protease cleavage site includes the amino acid sequence TPIDSSFNPD (SEQ ID NO: 190). In some embodiments, the protease cleavage site includes the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191). In some embodiments, the protease cleavage site includes the amino acid sequence of ITQGLAVSTISSFF (SEQ ID NO: 198).

[0035] In some embodiments, isolated cells further contain antigen-recognizing receptors. In some embodiments, the antigen-recognizing receptors recognize antigens selected from the group consisting of: 5T4, ADAM9, AFP, AXL, B7-H3, B7-H4, B7-H6, C4.4, CA6, Cadherin 3, Cadherin 6, CCR4, CD123, CD133, CD138, CD142, CD166, CD25, CD30, CD352, CD37, CD38, CD44, CD56, CD66e, CD70, CD71, CD74, CD79b, CD80, CEA, CEACAM5, Claudin 18.2, cMet, CSPG4, CTLA, DLK1, DLL3, D R5, EGFR, ENPP3, EpCAM, EphA2, EphrinA4, ETBR, FGFR2, FGFR3, FR alpha, FRb, GCC, GD2, GFRa4, gpA33, GPC3, gpNBM, GPRC5, HER2, IL-13R, IL-13Ra, IL-13Ra 2, IL-8, IL-15, IL1RAP, integrin aV, KIT, L1CAM, LAMP1, Lewis Y, LeY, LIV-1, LRRC, LY6E, MCSP, mesothelin, MUC1, MUC16, MUC1C, NaPi2B, Nectin 4, NKG2D, NOTCH3, NY ESO 1, Obalin, P-cadherin, pan-Erb2, PSCA, PSMA, PTK7, ROR1, S Aures, SCT, SLAMF7, SLITRK6, SSTR2, STEAP1, Survivin, TDGF1, TIM1, TROP2, and WT1. In some embodiments, the antigen-recognizing receptor includes an antigen-binding domain. In some embodiments, the antigen-binding domain includes an antibody, an antigen-binding fragment of an antibody, an F(ab) fragment, an F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb). In some embodiments, the antigen-binding domain includes a single-chain variable fragment (scFv). In some embodiments, the scFv includes a heavy-chain variable domain (VH) and a light-chain variable domain (VL). In some embodiments, VH and VL are separated by a peptide linker. In some embodiments, 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, the antigen-recognizing receptor is a chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

[0036] In some embodiments, the antigen-recognition receptor is a CAR. In some embodiments, the CAR comprises one or more intracellular signaling domains, one or more of which are selected from the group consisting of: a CD3 zeta chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, and a MyD88 intracellular signaling domain. In some embodiments, the CAR includes a transmembrane domain, which is selected from the group consisting of: CD8 transmembrane domain, CD28 transmembrane domain, CD3 zeta chain transmembrane domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane domain, 2B4 transmembrane domain, and BTLA transmembrane domain. In some embodiments, the CAR includes a spacer region between the antigen-binding domain and the transmembrane domain.

[0037] Furthermore, provided herein are compositions comprising any of the isolated cells described herein and a pharmaceutically acceptable carrier.

[0038] Also provided herein are methods for treating a subject in need thereof, the method comprising administering either a therapeutically effective dose of isolated cells or a composition described herein. In some embodiments, the isolated cells are derived from the subject. In some embodiments, the isolated cells are homogeneous with respect to the subject. In some embodiments, the method further comprises administering a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is selected from the group consisting of: anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIM-3 antibody, anti-TIGIT antibody, anti-VISTA antibody, anti-KIR antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-GAL9 antibody, anti-A2AR antibody, anti-phosphatidylserine antibody, anti-CD27 antibody, anti-TNFα antibody, anti-TREM1 antibody, and anti-TREM2 antibody. In some embodiments, the method further includes administering an anti-CD40 antibody.

[0039] Also provided herein are lipid-based structures comprising any of the engineered nucleic acids described herein, any of the expression vectors described herein, or any of the membrane-cleavable chimeric proteins described herein. In some embodiments, the lipid-based structures comprise extracellular vesicles, lipid nanoparticles, micelles, or liposomes. In some embodiments, the extracellular vesicles are selected from the group consisting of: nanovesicles and exosomes. In some embodiments, the lipid-based structures comprise lipid nanoparticles or micelles. In some embodiments, the lipid-based structures comprise liposomes.

[0040] Furthermore, provided herein are compositions comprising any of the lipid-based structures described herein and a pharmaceutically acceptable carrier.

[0041] Also provided herein are methods for treating a subject in need thereof, the method comprising administering any of the lipid-based structures or compositions described herein in a therapeutically effective dose. In some embodiments, the administration comprises systemic administration. In some embodiments, the lipid-based structures are capable of manipulating cells in the subject. In some embodiments, the method further comprises administering a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is selected from the group consisting of: anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIM-3 antibody, anti-TIGIT antibody, anti-VISTA antibody, anti-KIR antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-GAL9 antibody, anti-A2AR antibody, anti-phosphatidylserine antibody, anti-CD27 antibody, anti-TNFα antibody, anti-TREM1 antibody, and anti-TREM2 antibody. In some embodiments, the method further includes administering an anti-CD40 antibody.

[0042] Furthermore, provided herein are nanoparticles comprising any of the manipulated nucleic acids described herein or any of the membrane-cleavable chimeric proteins described herein. In some embodiments, the nanoparticles include inorganic materials.

[0043] Furthermore, the materials provided herein include compositions comprising any of the nanoparticles described herein.

[0044] Also provided herein are methods for treating a subject in need thereof, the method comprising administering any of the therapeutically effective doses of nanoparticles described herein or any of the compositions described herein. In some embodiments, the administration comprises systemic administration. In some embodiments, the nanoparticles are capable of modifying cells in the subject. In some embodiments, the method further comprises administering a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is selected from the group consisting of: anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIM-3 antibody, anti-TIGIT antibody, anti-VISTA antibody, anti-KIR antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-GAL9 antibody, anti-A2AR antibody, anti-phosphatidylserine antibody, anti-CD27 antibody, anti-TNFα antibody, anti-TREM1 antibody, and anti-TREM2 antibody. In some embodiments, the method further includes administering an anti-CD40 antibody.

[0045] Furthermore, provided herein are viruses engineered to include any of the engineered nucleic acids described herein or any of the expression vectors described herein. In some embodiments, the viruses are selected from the group consisting of: lentiviruses, retroviruses, oncolytic viruses, adenoviruses, adeno-associated viruses (AAVs), and virus-like particles (VLPs).

[0046] Furthermore, provided herein are pharmaceutical compositions comprising any of the manipulated cells described herein and a pharmaceutically acceptable carrier.

[0047] Also provided herein are methods for treating a subject in need thereof, the method comprising administering a therapeutically effective dose of any of the engineered viruses described herein or any of the compositions described herein. In some embodiments, the administration comprises systemic administration. In some embodiments, the engineered virus infects cells in the subject and expresses an expression cassette. In some embodiments, the method further comprises administering a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is selected from the group consisting of: anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIM-3 antibody, anti-TIGIT antibody, anti-VISTA antibody, anti-KIR antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-GAL9 antibody, anti-A2AR antibody, anti-phosphatidylserine antibody, anti-CD27 antibody, anti-TNFα antibody, anti-TREM1 antibody, and anti-TREM2 antibody. In some embodiments, the method further includes administering an anti-CD40 antibody. [Invention 1001] A membrane-cleavable chimeric protein oriented from the N-terminus to the C-terminus, with formula: SC-MT or MT-CS It has, During the ceremony, S contains secretory effector molecules, C includes the protease cleavage site, MT includes a cell membrane anchoring domain, Here, SC-MT or MT-CS is configured to be expressed as a single polypeptide. A chimeric protein capable of membrane cleavage. [Invention 1002] The secretory effector molecule comprises a signal peptide or a signal anchor sequence, and optionally the signal peptide comprises a natural signal peptide that is natural to the secretory effector molecule, or the signal peptide comprises a non-natural signal peptide, or the signal anchor sequence comprises a non-natural signal anchor sequence that is non-natural to the secretory effector molecule, and optionally the non-natural signal peptide or the non-natural signal anchor sequence comprises IL-12, IL-2, and optimization. A membrane-cleavable chimeric protein according to the present invention 1001, selected from the group consisting of IL-2, trypsinogen-2, Gaussial alciferase, CD5, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azulocidine preprotein, osteonectin, CD33, IL-6, IL-8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpine E1, GRO-alpha, CXCL12, IL-21, CD8, NKG2D, TNFR2, and GMCSF. [Invention 1003] The secreted effector molecule is selected from a therapeutic class, the therapeutic class is selected from the group consisting of cytokines, chemokines, homing molecules, growth factors, coactivating molecules, tumor microenvironment modifiers, ligands, antibodies, peptides, and enzymes, and optionally the cytokine is IL-1-beta, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-12p70 fusion protein, IL-15, IL-17A, IL-18, IL-21, IL-22, type I interferon, interferon-gamma, and T The chemokine is selected from the group consisting of NF-alpha, and optionally the chemokine is selected from the group consisting of CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1, and optionally the homing molecule is selected from the group consisting of anti-integrin alpha 4, beta 7; anti-MAdCAM; SDF1; and MMP-2, and optionally the growth factor is selected from the group consisting of FLT3L and GM-CSF, and optionally the co-activating molecule is 4-1B The group consisting of BL and CD40L is selected, and optionally the tumor microenvironment modifier is selected from the group consisting of adenosine deaminase, TGF beta inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2, and optionally the TGF beta inhibitor is selected from the group consisting of anti-TGF beta peptides, anti-TGF beta antibodies, TGFb-TRAP, and combinations thereof, and optionally the immune checkpoint inhibitor is anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-L A membrane-cleavable chimeric protein according to Invention 1001 or Invention 1002, selected from the group consisting of AG-3 antibody, anti-TIM-3 antibody, anti-TIGIT antibody, anti-VISTA antibody, anti-KIR antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-GAL9 antibody, anti-A2AR antibody, anti-phosphatidylserine antibody, anti-CD27 antibody, anti-TNFa antibody, anti-TREM1 antibody, and anti-TREM2 antibody, or optionally, the VEGF inhibitor comprises an anti-VEGF antibody, an anti-VEGF peptide, or a combination thereof. [Invention 1004] The secreted effector molecule is a membrane-cleavable chimeric protein according to any of the inventions 1001 to 1003, comprising IL-15, IL-12, or IL-12p70 fusion protein. [Invention 1005] The aforementioned protease cleavage sites include type 1 transmembrane protease, type 2 transmembrane protease, GPI-anchored protease, ADAM8 protease, ADAM9 protease, ADAM10 protease, ADAM12 protease, ADAM15 protease, ADAM17 protease, ADAM19 protease, ADAM20 protease, ADAM21 protease, ADAM28 protease, ADAM30 protease, ADAM33 protease, and B A membrane-cleavable chimeric protein according to any of the present invention 1001 to 1004, which can be cleaved by a protease selected from the group consisting of ACE1 protease, BACE2 protease, SIP protease, MT1-MMP protease, MT3-MMP protease, MT5-MMP protease, furin protease, PCSK7 protease, matryptase protease, matryptase 2 protease, MMP9 protease, and NS3 protease. [Invention 1006] A membrane-cleavable chimeric protein according to any of the present invention 1001 to 1005, wherein the protease cleavage site is cleavable by ADAM17 protease, and optionally the protease cleavage site includes a first region having the amino acid sequence of PRAE (SEQ ID NO: 176) and / or a second region having the amino acid sequence of KGG (SEQ ID NO: 177), and optionally the first region is located at the N-terminus of the second region. [Invention 1007] The aforementioned protease cleavage site is PRAEX 1 X 2 It contains the amino acid sequence of KGG (SEQ ID NO: 178), X 1 is A, Y, P, S, or F, and X 2 is V, L, S, I, Y, or T. A membrane-cleavable chimeric protein according to any of the invention's 1001-1006. [Invention 1008] a. The protease cleavage site includes the amino acid sequence of PRAEALKGG (SEQ ID NO: 180), or b. The protease cleavage site includes the amino acid sequence of PRAEALKGG (SEQ ID NO: 180), or c. The protease cleavage site includes the amino acid sequence of PRAEYSKGG (SEQ ID NO: 181), or d. The protease cleavage site includes the amino acid sequence of PRAEPIKGG (SEQ ID NO: 182), or e. The protease cleavage site includes the amino acid sequence PRAEAYKGG (SEQ ID NO: 183), or f. The protease cleavage site includes the amino acid sequence of PRAESSKGG (SEQ ID NO: 184), or g. The protease cleavage site includes the amino acid sequence PRAEFTKGG (SEQ ID NO: 185), or h. The protease cleavage site includes the amino acid sequence of DEPHYSQRR (SEQ ID NO: 187), or i. The protease cleavage site includes the amino acid sequence of PPLGPIFNPG (SEQ ID NO: 188), or j. The protease cleavage site includes the amino acid sequence of PLAQAYRSS (SEQ ID NO: 189), or k. The protease cleavage site includes the amino acid sequence of TPIDSSFNPD (SEQ ID NO: 190), or l. The protease cleavage site includes the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191), or m. The aforementioned protease cleavage site contains the amino acid sequence ITQGLAVSTISSFF (SEQ ID NO: 198), A membrane-cleavable chimeric protein according to any of invention 1001 to 1007. [Invention 1009] A membrane-cleavable chimeric protein according to any of the invention 1001 to 1008, wherein the cell membrane anchoring domain comprises a transmembrane-intracellular domain or a transmembrane domain, and optionally the transmembrane-intracellular domain and / or the transmembrane domain is 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, and optionally the cell membrane anchoring domain comprises a cell surface receptor or its cell membrane binding portion. [Invention 1010] A membrane-cleavable chimeric protein according to any of the invention 1001 to 1009, wherein the cell membrane anchoring domain comprises a post-translational modification tag or a post-translational modification motif for modifying the chimeric protein to include a post-translational modification tag, the post-translational modification tag is capable of associating with a cell membrane, the post-translational modification tag optionally comprises a lipid anchor domain, and the lipid anchor domain optionally comprises a GPI lipid anchor, a myristoylation tag, and a palmitoylation tag. [Invention 1011] a. When expressed in a cell, the secreted effector molecule is anchored to the cell membrane of the cell and / or b. When expressed in cells expressing a protease capable of cleaving the protease cleavage site, the secreted effector molecule is released from the cell membrane and / or c. The protease expressed on the cell membrane is endogenous to the cell, and / or d. The protease is selected from the group consisting of type 1 transmembrane protease, type 2 transmembrane protease, GPI-anchored protease, ADAM8 protease, ADAM9 protease, ADAM10 protease, ADAM12 protease, ADAM15 protease, ADAM17 protease, ADAM19 protease, ADAM20 protease, ADAM21 protease, ADAM28 protease, ADAM30 protease, ADAM33 protease, BACE1 protease, BACE2 protease, SIP protease, MT1-MMP protease, MT3-MMP protease, MT5-MMP protease, furin protease, PCSK7 protease, matryptase protease, matryptase 2 protease, and MMP9 protease. A membrane-cleavable chimeric protein according to any of invention 1001 to 1010. [Invention 1012] An engineered nucleic acid comprising an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a membrane-cleavable chimeric protein of any of the inventions 1001 to 1011, wherein the promoter is optionally selected from the group consisting of a constitutive promoter, an inducible promoter, a tissue-specific promoter, and a synthetic promoter. [Invention 1013] An expression vector comprising the manipulated nucleic acid of the present invention 1012. [Invention 1014] Isolated cells comprising a membrane-cleavable chimeric protein according to any of Invention 1001-1011, an engineered nucleic acid according to Invention 1012, or an expression vector according to Invention 1013. [Invention 1015] Isolated cells according to Invention 1014, wherein the cells are selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, bone marrow cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells. [Invention 1016] The cells further contain a protease capable of cleaving the protease cleavage site, and optionally the protease is an endogenous protease and / or optionally the endogenous protease is a type 1 transmembrane protease, a type 2 transmembrane protease, a GPI-anchored protease, an ADAM8 protease, an ADAM9 protease, an ADAM10 protease, an ADAM12 protease, an ADAM15 protease, an ADAM17 protease, an ADAM19 protease, an ADAM20 protease. Isolated cells according to Invention 1014 or Invention 1015, selected from the group consisting of rotease, 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, matryptase protease, matryptase 2 protease, and MMP9 protease. [Invention 1017] Isolated cells according to any of items 1014 to 1016 of the present invention, wherein the cells further contain an antigen-recognizing receptor, and optionally the antigen-recognizing receptor is a CAR. [Invention 1018] A composition comprising a membrane-cleavable chimeric protein of any of Invention 1001 to 1011, an engineered nucleic acid of Invention 1012, an expression vector of Invention 1013, or an isolated cell of any of Invention 1014 to 1017, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. [Invention 1019] Methods of treating those who need it, including the following: Administer a therapeutically effective dose of any isolated cells from any of Invention 1014 to 1017 or any of the compositions from Invention 1018. [Invention 1020] Methods for inducing the release of membrane-anchored effector molecules, including the following: (a) To provide any of the cells described in invention 1014 to 1017, and (b) Culturing the cells under conditions suitable for the expression of the membrane-bound protease and the membrane-cleavable chimeric protein, Upon expression, the membrane-cleavable chimeric protein is anchored to the cell membrane of the cell, and Upon expression, the membrane-bound protease cleaves the homologous membrane-bound protease cleavage site of the membrane-cleavable chimeric protein, thereby releasing the secreted effector molecule from the cell membrane. thing. [Brief explanation of the drawing]

[0048] [Figure 1A] Figure 1A illustrates a schematic diagram of a membrane-cleaving system described herein, in which the desired payload is expressed as a chimeric protein in which a protease cleavage site is inserted between the payload and a membrane anchoring domain. The left panel illustrates a schematic diagram of a membrane-cleaving system using a transmembrane structure (e.g., the chimeric protein contains a transmembrane domain). The right panel illustrates a schematic diagram of a membrane-cleaving system using a membrane-associating structure (e.g., the chimeric protein contains a post-translational modification tag that enables association with the cell membrane). [Figure 1B]Figure 1B illustrates a typical membrane-cleavable system for regulated IL-15 secretion, in which IL-15 is expressed as a chimeric protein having a TACE-mediated cleavage site inserted between the IL-15 payload and the membrane anchoring 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 2] Figure 2 provides plots of membrane-bound expression and IL-15 secretion determined by ELISA (pg / ml, measured on the x-axis) for each membrane-cleavable IL-15 construct, assayed by flow cytometry (geometric mean fluorescence intensity, "gMFI", calculated as the y-axis). [Figure 3A] Figures 3A-3C illustrate the membrane-bound expression and secretion of IL-12 membrane-cleaving systems. Figure 3A shows the membrane-bound expression of each membrane-cleaving IL-12 system in NK cells, as measured by flow cytometry. [Figure 3B] Figures 3A-3C show the membrane-bound expression and secretion of IL-12 membrane-cleaving systems. Figure 3B shows the membrane-bound expression of each membrane-cleaving IL-12 system in T cells, as measured by flow cytometry. [Figure 3C] Figures 3A-3C illustrate the membrane-bound expression and secretion of IL-12 membrane-cleavable systems. Figure 3C shows IL-12 secretion for each membrane-cleavable IL-12 system expressed in NK cells and T cells. [Figure 4A] Figures 4A-4B show the expression of membrane-bound NK cells and the secretion of various IL-15 membrane-cleaving systems. Figure 4A shows the membrane-bound expression of each membrane-cleaving IL-15 system in NK cells, as measured by flow cytometry. [Figure 4B]Figures 4A-4B show the expression of membrane-bound NK cells and the secretion of various IL-15 membrane-cleaving systems. Figure 4B shows the IL-15 secretion for each membrane-cleaving IL-15 system expressed in NK cells. [Modes for carrying out the invention]

[0049] Detailed explanation Chimeric proteins (or engineered nucleic acids encoding chimeric proteins) are provided herein, having the formula SC-MT or MT-CS, oriented N-terminus to C-terminus, and expressed as a single polypeptide. S refers to a secretory effector molecule. C refers to a protease cleavage site. MT refers to a cell membrane anchoring domain. Membrane-cleavable chimeric proteins are engineered to enable the secretion of effector molecules to be regulated in a protease-dependent manner. Specifically, membrane-cleavable chimeric proteins are engineered so that the secretion of effector molecules can be regulated as part of a “membrane-cleavable” system, where the incorporation of a protease cleavage site ("C") and a cell membrane anchoring domain ("MT") enables the regulated secretion of effector molecules in a protease-dependent manner. While we do not wish to be constrained by theory, secretion is generally regulated through the following cellular processes by the components of the membrane-cleavable system present in membrane-cleavable chimeric proteins: -MT: The cell membrane anchoring domain includes a transmembrane domain (or transmembrane-intracellular domain) that directs the cellular transport of the chimeric protein so as to be inserted into or associated with the cell membrane ("anchored"). -C: Following the expression and localization of the chimeric protein into the cell membrane, the protease cleavage site directs the cleavage of the chimeric protein, causing the effector molecule to be released into the extracellular space ("secreted"). Generally, the protease cleavage site is protease-specific and includes sites that have been engineered to be protease-specific. The protease cleavage site may be selected or engineered to achieve optimal protein expression, cell type-specific cleavage, cell state-specific cleavage, and / or cleavage and release of the payload in the desired dynamics (e.g., ratio of membrane-bound chimeric protein to secreted chimeric protein levels).

[0050] In some embodiments, a membrane-cleavable chimeric protein (or an engineered nucleic acid encoding a membrane-cleavable chimeric protein) is provided herein, having a protein of interest (e.g., any of the effector molecules described herein), a protease cleavage site, and a cell membrane anchoring domain.

[0051] An "effector molecule" refers to a molecule that binds to another molecule and modulates the biological activity of that molecule (e.g., nucleic acids, such as DNA or RNA, or proteins (polypeptides) or peptides). For example, an effector molecule may act as a ligand to increase or decrease enzyme activity, gene expression, or cell signaling. Thus, in some embodiments, an effector molecule modulates (activates or inhibits) different immunomodulatory mechanisms. By directly binding to and modulating another molecule, an effector molecule may also indirectly modulate a second, downstream molecule.

[0052] 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 formula SC-MT or MT-CS for the membrane-cleavable chimeric proteins described herein). Non-limiting examples of effector molecules include cytokines, chemokines, enzymes that modulate metabolite levels, growth factors, co-activating molecules, tumor microenvironment modifiers, ligands, peptides, enzymes, antibodies, cytokines, homing molecules, and / or antibodies or decoy molecules that modulate integrins.

[0053] The term “modulate” encompasses the maintenance of biological activity, the inhibition (partial or complete) of biological activity, and the stimulation / activation (partial or complete) of biological activity. The term also encompasses the reduction or increase (e.g., enhancement) of 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 other effector molecule (e.g., stimulating antigen presentation and / or processing).

[0054] Regulation by effector molecules can be direct or indirect. Direct regulation occurs when an effector molecule binds to another molecule and modulates its activity. Indirect regulation occurs when an effector molecule binds to another molecule and modulates its activity, which in turn modulates the activity of yet another molecule (not to which an effector molecule is bound).

[0055] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in an increase of at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%) in immunostimulatory and / or antitumor immune responses (e.g., systemically or in the tumor microenvironment). For example, modulation of tumor-mediated immunosuppressive mechanisms may result in an increase of 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 immunostimulatory and / or antitumor immune responses. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in an increase of 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% in the immunostimulatory and / or antitumor immune response. It should be understood that the “increase” in immunostimulatory and / or antitumor immune responses is related to the immunostimulatory and / or antitumor immune responses that would otherwise occur, for example, systemically or within the tumor microenvironment, in the absence of effector molecules.

[0056] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in an increase of at least twofold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100fold) in immunostimulatory and / or antitumor immune responses (e.g., systemically or in the tumor microenvironment). For example, modulation of tumor-mediated immunosuppressive mechanisms may result in an increase of at least threefold, at least fivefold, at least tenfold, at least twentyfold, at least fiftyfold, or at least 100fold in immunostimulatory and / or antitumor immune responses. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in an increase of 2-10fold, 2-20fold, 2-30fold, 2-40fold, 2-50fold, 2-60fold, 2-70fold, 2-80fold, 2-90fold, or 2-100fold in immunostimulatory and / or antitumor immune responses.

[0057] Non-limiting examples of immunostimulatory and / or antitumor immune mechanisms include T cell signaling, activity and / or recruitment, antigen presentation and / or processing, natural killer cell-mediated cytotoxic signaling, activity and / or recruitment, dendritic cell differentiation and / or maturation, immune cell recruitment, pro-inflammatory macrophage signaling, activity and / or recruitment, stromal degradation, production of immunostimulatory metabolites, interferon gene stimulator (STING) signaling (which increases the secretion of IFN and Th1 polarization and promotes an antitumor immune response), and / or type I interferon signaling. Effector molecules may stimulate at least one (or more) of the aforementioned immunostimulatory mechanisms, thus potentially leading to an increase in the immunostimulatory response. Changes in the aforementioned immunostimulatory and / or antitumor immune mechanisms can be evaluated, 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 cell secretion assays (e.g., of cytokines).

[0058] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in a reduction of at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%) in the immunosuppressive response (e.g., systemically or in the tumor microenvironment). For example, modulation of tumor-mediated immunosuppressive mechanisms may result in a reduction of 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 the immunosuppressive response. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in a reduction of 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% in the immunosuppressive response. It should be understood that the “reduction” in the immunosuppressive response relates to the immunosuppressive response that would otherwise occur, for example, systemically or in the tumor microenvironment, in the absence of effector molecules.

[0059] In some embodiments, modulation of tumor-mediated immunosuppression by at least one effector molecule results in a reduction of at least twofold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100fold) in the immunosuppressive response (e.g., systemically or in the tumor microenvironment). For example, modulation of tumor-mediated immunosuppression may result in a reduction of at least threefold, at least fivefold, at least tenfold, at least twentyfold, at least fiftyfold, or at least 100fold in the immunosuppressive response. In some embodiments, modulation of tumor-mediated immunosuppression may result in a reduction of 2-10fold, 2-20fold, 2-30fold, 2-40fold, 2-50fold, 2-60fold, 2-70fold, 2-80fold, 2-90fold, or 2-100fold in the immunosuppressive response.

[0060] Non-limiting examples of immunosuppressive mechanisms include negative co-stimulatory 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, bone marrow-derived suppressor cell signaling, activation, 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 reduction in the immunosuppressive response. Changes in the aforementioned immunosuppressive mechanisms may include, for example, an increase in T cell proliferation and / or an increase in IFNγ production (negative co-stimulatory signaling, T reg Cellular 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 and enzyme assays via ELISA, LUMINEX®, qPCR, e.g., IDO tryptophan catabolism (immunosuppressive factor / metabolite production); and phosphorylation of PI3K, Akt, and p38 (VEGF signaling) can be evaluated.

[0061] In some embodiments, effector molecules function additively: the effect of two effector molecules may be equal to, for example, the sum of the effects of two effector molecules functioning separately. In other embodiments, effector molecules function synergistically: the effect of two effector molecules may be greater than, for example, the combined effect of two effector molecules.

[0062] Effector molecules that modulate tumor-mediated immunosuppressive mechanisms and / or modify the tumor microenvironment may include, for example, secretory factors (e.g., cytokines, chemokines, antibodies, and / or decoy receptors that modulate extracellular mechanisms involved in the immune system), inhibitors (e.g., antibodies, antibody fragments, ligands, TRAP, and / or small blockade peptides), intracellular factors that regulate cellular state (e.g., microRNAs and / or transcription factors that modulate cellular state to enhance pro-inflammatory activity), factors packaged in exosomes (e.g., microRNAs, cytosolic factors, and / or extracellular factors), surface-presented factors (e.g., checkpoint inhibitors, TRAIL), and and / or metabolic genes (e.g., enzymes that produce / regulate or degrade metabolites or amino acids).

[0063] In some embodiments, at least one of the effector molecules stimulates an immunostimulatory mechanism in the tumor microenvironment and / or inhibits an immunosuppressive mechanism in the tumor microenvironment.

[0064] In some embodiments, at least one effector molecule (a) stimulates T cell signaling, activity, and / or recruitment; (b) stimulates antigen presentation and / or processing; (c) stimulates natural killer cell-mediated cytotoxic signaling, activity, and / or recruitment; (d) stimulates dendritic cell differentiation and / or maturation; (e) stimulates immune cell recruitment; (f) stimulates pro-inflammatory macrophage signaling, activity, and / or recruitment, or inhibits anti-inflammatory macrophage signaling, activity, and / or recruitment; (g) stimulates stromal degradation; (h) stimulates immunostimulatory metabolite production; (i) stimulates type I interferon signaling; (j) inhibits negative co-stimulatory signaling; (k) inhibits pro-apoptotic signaling of anti-tumor immune cells; and (l) regulates T(T) reg(m) inhibit cell signaling, activity, and / or recruitment; (n) inhibit tumor checkpoint molecules; (o) stimulate interferon gene stimulator (STING) signaling; (p) inhibit bone marrow-derived suppressor cell signaling, activity, and / or recruitment; (q) degrade immunosuppressive factors / metabolites; (r) inhibit vascular endothelial growth factor signaling; and / or directly kill tumor cells.

[0065] In some embodiments, the effector molecule may be selected from the following non-limiting classes of molecules: cytokines, antibodies, chemokines, nucleotides, peptides, and enzymes. Non-limiting examples of the aforementioned classes of effector molecules are listed in Table 1, and specific sequences encoding exemplary effector molecules are listed in Table 2. The effector molecule may be human, as listed in Table 1 or Table 2, or a human equivalent of a mouse effector molecule listed in Table 1 or Table 2. The effector molecule may be of human origin, but may be, for example, an endogenous human effector molecule or an effector molecule modified and / or optimized for function, e.g., optimized for improved expression, modified for improved stability, or modified in its signal sequence (see below). Various programs and algorithms for optimizing function are known to those skilled in the art and can be selected based on desired improvements, e.g., codon optimization for a specific species (e.g., human, mouse, bacteria, etc.).

[0066] In some embodiments, the effector molecule comprises interleukin-12 (IL-12), for example, p35 and p40 as dimers commonly referred to in the art as IL-12p70. In some embodiments, the first effector molecule comprises an IL-12p70 fusion protein. In some embodiments, the IL-12p70 fusion protein is a human IL-12p70 fusion protein. In some embodiments, the human IL-12p70 fusion protein comprises the sequence shown in SEQ ID NO: 203.

[0067] In some embodiments, the effector molecule comprises interlekin 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 containing IL-15 and the extracellular portion of IL-15 receptor α (IL-15Rα), such as the sushi domain shown in SEQ ID NO: 201. An exemplary IL-15 / IL-15Rα sushi domain fusion is provided as SEQ ID NO: 202.

[0068] (Table 1) Exemplary effector molecules TIFF0007865603000001.tif213159TIFF0007865603000002.tif152159

[0069] (Table 2) Exemplary effector molecular arrangements TIFF0007865603000003.tif42160TIFF0007865603000004.tif245160TIFF0007865603000005.tif245160TIFF0007865603000006.ti f245160TIFF0007865603000007.tif245160TIFF0007865603000008.tif246160TIFF0007865603000009.tif242160TIFF00078656030 00010.tif247160TIFF0007865603000011.tif243160TIFF0007865603000012.tif245160TIFF0007865603000013.tif246160TIFF000 7865603000014.tif245160TIFF0007865603000015.tif246160TIFF0007865603000016.tif245160TIFF0007865603000017.tif223160

[0070] Secretory signals and signal anchors One or more effector molecules of chimeric proteins provided herein may 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 a newly synthesized protein destined for secretion or membrane localization (also referred to as membrane insertion) to a suitable protein processing pathway. For chimeric proteins having formula MT-CS, the membrane anchoring domain generally has a signal anchor sequence (e.g., a signal anchor sequence of a type II transmembrane protein) that directs a newly synthesized protein destined for membrane localization to a suitable protein processing pathway. For chimeric proteins having formula SC-MT, a membrane anchoring domain having a reverse signal anchor sequence (e.g., a signal anchor sequence of a certain type III transmembrane protein) can be used, generally without the involvement of a separate secretory signal peptide, thereby directing a newly synthesized protein destined for membrane localization to a suitable protein processing pathway.

[0071] Generally, for all membrane-cleavable chimeric proteins described herein, one or more effector molecules are secretory effector molecules (referred to as "S" in formulas SC-MT or MT-CS). In embodiments involving two or more chimeric proteins, each chimeric protein may contain a secretory signal. In embodiments involving two or more chimeric proteins, each chimeric protein may contain a secretory signal, but each effector molecule enables secretion from the manipulated cell after cleavage of the protease cleavage site.

[0072] Secretory signaling peptides operably associated with effector molecules may be intrinsic secretory signaling peptides (e.g., generally, secretory signaling peptides endogenously associated with a given effector molecule). Secretory signaling peptides operably associated with effector molecules may be non-intrinsic secretory signaling peptides or intrinsic secretory signaling peptides. Non-intrinsic secretory signaling peptides can promote improved expression and function, such as maintained secretion, in specific environments, such as the tumor microenvironment. Non-exclusive examples of non-intrinsic secretory signaling peptides are shown in Table 3.

[0073] (Table 3) Exemplary signaling peptides TIFF0007865603000018.tif220158TIFF0007865603000019.tif225158TIFF0007865603000020.tif42158

[0074] Protease cleavage site In certain embodiments, the chimeric proteins provided herein (e.g., generally for all membrane-cleavable chimeric proteins described herein) include protease cleavage sites (e.g., referred to as "C" in formulas SC-MT or MT-CS for the membrane-cleavable chimeric proteins described herein). Generally, the protease cleavage sites can be any amino acid sequence motif that can be cleaved by a protease. Examples of protease cleavage sites are not limited to, but include type 1 transmembrane protease cleavage sites, type II transmembrane protease cleavage sites, GPI-anchored protease cleavage sites, ADAM8 protease cleavage sites, ADAM9 protease cleavage sites, ADAM10 protease cleavage sites, ADAM12 protease cleavage sites, ADAM15 protease cleavage sites, ADAM17 protease cleavage sites, ADAM19 protease cleavage sites, ADAM20 protease cleavage sites, ADAM21 protease cleavage sites, ADAM28 protease cleavage sites, ADAM30 protease cleavage sites, and ADAM33 protease cleavage sites. Includes protease cleavage sites, BACE1 protease cleavage sites, BACE2 protease cleavage sites, SIP protease cleavage sites, MT1-MMP protease cleavage sites, MT3-MMP protease cleavage sites, MT5-MMP protease cleavage sites, furin protease cleavage sites, PCSK7 protease cleavage sites, matryptase protease cleavage sites, matryptase-2 protease cleavage sites, MMP9 protease cleavage sites, or NS3 protease cleavage sites.

[0075] One example of a protease cleavage site is the hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease cleavage site, which includes, but is not limited to, NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, or NS5A / NS5B cleavage sites. 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 text is incorporated herein by reference. For example, sequences of the HCV NS4A / 4B protease cleavage site; the HCV NS5A / 5B protease cleavage site; the C-terminal degron with the NS4A / 4B protease cleavage site; and the N-terminal degron with the HCV NS5A / 5B protease cleavage site are provided. Representative NS3 sequences are listed in the National Center for Biotechnology Information (NCBI) database. For example, 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 these sequences (as entered by the filing date of this application) are incorporated herein by reference.

[0076] Another example of a protease cleavage site is the ADAM17-specific protease (also referred to as tumor necrosis factor α-converting enzyme [TACE]) cleavage site. The ADAM17-specific protease cleavage site may be an endogenous sequence of a substrate spontaneously cleaved by ADAM17. The ADAM17-specific protease cleavage site may be an engineered sequence capable of being cleaved by ADAM17. Engineered ADAM17-specific protease cleavage sites may be engineered for specific desired properties, including, but are not limited to, optimal expression of the chimeric protein, specificity for ADAM17, cleavage rate by ADAM17, ratio of secreted and membrane-bound chimeric protein levels, and cleavage in different cellular states. Protease cleavage sites can be selected for specific cleavage by ADAM17. For example, a specific protease cleavage site capable of being cleaved by ADAM17 may also be capable of cleavage by additional ADAM family proteases, such as ADAM10. Therefore, ADAM17-specific protease cleavage sites can be selected and / or manipulated so that cleavage by other proteases, such as ADAM10, is reduced or eliminated. Protease cleavage sites can be selected in terms of the cleavage rate by ADAM17. For example, it may be desirable to select a protease cleavage site that exhibits a specific cleavage rate by ADAM17, such as reduced cleavage kinetics with respect to the endogenous sequence of the substrate that is naturally cleaved by ADAM17. In such cases, a specific 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. Therefore, ADAM17-specific protease cleavage sites can be selected and / or manipulated so that the sequence exhibits a desired cleavage rate by ADAM17. Protease cleavage sites can be selected in terms of both specific cleavage by ADAM17 and the cleavage rate by ADAM17. Exemplary ADAM17-specific protease cleavage sites, including those exhibiting specific specificity and cleavage rate dynamics, 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 their expression and protease cleavage sites, are described in Shamla, 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 incorporated herein by reference for the purposes of this document.

[0077] (Table 4A) Various ADAM17 protease cleavage site arrangements TIFF0007865603000021.tif71142

[0078] In some embodiments, the protease cleavage site includes a first region having the amino acid sequence of PRAE (SEQ ID NO: 176). In some embodiments, the protease cleavage site includes a second region having the amino acid sequence of KGG (SEQ ID NO: 177). In some embodiments, the first region is located at the N-terminus of the second region. In some embodiments, the protease cleavage site includes the amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 177). 219) includes, where X1 is A, Y, P, S, or F, and where X2 is V, L, S, I, Y, T, or A. In some embodiments, the protease cleavage site includes the amino acid sequence PRAEX1X2KGG (SEQ ID NO: 178), where X1 is A, Y, P, S, or F, and where X2 is V, L, S, I, Y, or T. In some embodiments, the protease cleavage site includes the amino acid sequence PRAEAVKGG (SEQ ID NO: 179). In some embodiments, the protease cleavage site includes the amino acid sequence PRAEALKGG (SEQ ID NO: 180). In some embodiments, the protease cleavage site includes the amino acid sequence PRAEYSKGG (SEQ ID NO: 181). In some embodiments, the protease cleavage site includes the amino acid sequence PRAEPIKGG (SEQ ID NO: 182). In some embodiments, the protease cleavage site includes the amino acid sequence PRAEAYKGG (SEQ ID NO: 183). In some embodiments, the protease cleavage site includes the amino acid sequence PRAESSKGG (SEQ ID NO: 184). In some embodiments, the protease cleavage site includes the amino acid sequence PRAEFTKGG (SEQ ID NO: 185). In some embodiments, the protease cleavage site includes the amino acid sequence PRAEAAKGG (SEQ ID NO: 186).

[0079] In some embodiments, the protease cleavage site includes the amino acid sequence of DEPHYSQRR (SEQ ID NO: 187). In some embodiments, the protease cleavage site includes the amino acid sequence of PPLGPIFNPG (SEQ ID NO: 188). In some embodiments, the protease cleavage site includes the amino acid sequence of PLAQAYRSS (SEQ ID NO: 189). In some embodiments, the protease cleavage site includes the amino acid sequence of TPIDSSFNPD (SEQ ID NO: 190). In some embodiments, the protease cleavage site includes the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191). The protease cleavage sites of SEQ ID NOs: 187, 189, and 191 can be cleaved by ADAM17.

[0080] In some embodiments, the protease cleavage site comprises the amino acid sequence ITQGLAVSTISSFF (SEQ ID NO: 198), which is native to CD16 and cleavable by ADAM17.

[0081] The protease cleavage site may be at the C-terminus of the secreted effector molecule. The protease cleavage site may be at the N-terminus of the secreted effector molecule. Generally, for all membrane-cleavable chimeric proteins described herein, the protease cleavage site is one of the following: (1) the C-terminus of the secreted effector molecule and the N-terminus of the cell membrane anchoring domain (in other words, the protease cleavage site is between the secreted effector molecule and the cell membrane anchoring domain); or (2) the N-terminus of the secreted effector molecule and the C-terminus of the cell membrane anchoring domain (also between the secreted effector molecule and the cell membrane anchoring domain with reversed domain orientation). The protease cleavage site can be linked to the secreted effector molecule by a polypeptide linker, i.e., a polypeptide sequence that is 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 that is not generally considered to be part of the cell membrane anchoring domain or the protease cleavage site. 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 mobile linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers are, but are not limited to, GSG linkers (e.g., [GS]4GG [SEQ ID NO: 1]). 220 ]), A(EAAAK)3A(Sequence ID) 221 ), and Whitlow linkers (e.g., "KEGS" linker, for example, amino acid sequence KESGSVSSEQLAQFRSLD(Sequence ID) 222 ), eGK linker, for example amino acid sequence EGKSSGSGSESKST (sequence number) 223This includes, and linkers as described in more detail in U.S. Patent No. 5,990,275, which is incorporated herein by reference. Additional, exemplary polypeptide linkers include SEQ ID NOs. 194, 195, 196, and 197. Other polypeptide linkers may be selected based on desired properties (e.g., length, mobility, amino acid composition, etc.) and are known to those skilled in the art.

[0082] In membrane-cleaving systems, following the expression and localization of chimeric proteins within the cell membrane, the cleavage of the chimeric proteins is directed by protease cleavage sites, causing effector molecules to be released ("secreted") into the extracellular space of the cell.

[0083] Generally, a protease that cleaves a protease cleavage site is a protease specific to that particular protease cleavage site. For example, in the case of disintegrin and metalloproteinase ("ADAM") family proteases, a protease that cleaves a specific ADAM protease cleavage site is generally limited to an ADAM protease that specifically recognizes a particular ADAM protease cleavage site motif. Protease cleavage sites can be selected and / or manipulated to reduce or eliminate cleavage by undesirable proteases. Proteases can be membrane-bound or membrane-associated. Proteases can be secreted, for example, in specific cellular environments, such as the tumor microenvironment ("TME").

[0084] The proteases that cleave the protease cleavage sites of chimeric proteins may be expressed in the same cells that express the chimeric protein. The proteases that cleave the protease cleavage sites of chimeric proteins may be endogenous to the cells expressing the chimeric protein. In other words, cells engineered to express chimeric proteins can endogenously express proteases specific to the protease cleavage sites present in the chimeric protein. Endogenous protease expression 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). Protease cleavage sites can be selected based on known proteases endogenously expressed by the desired cell population. In such cases, the cleavage of the protease cleavage site (and thus the release / secretion of the payload) can generally be restricted to only the target cells due to a cell-restricting protease that needs to come into contact with the protease cleavage site of the chimeric protein expressed in the same cell. For example, although we do not wish to be constrained by theory, ADAM17 is thought to have restricted endogenous expression in NK cells and T cells. Thus, the selection of an ADAM17-specific protease cleavage site can restrict the cleavage of the protease cleavage site to NK cells and T cells co-expressing the chimeric protein. In another example, the protease cleavage site can be selected for a specific tumor-associated protease that is known to be expressed in a particular tumor population of interest (e.g., in specific tumor cells engineered to express the chimeric protein).Using protease and / or expression databases, suitable protease cleavage sites are selected, and each of these is incorporated by reference for all purposes, such as selecting protease cleavage sites to be cleaved by tumor-associated proteases through consultation with, for example, Oncomine (www.oncomine.org), the European Institute for Bioinformatics (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).

[0085] The proteases that cleave the protease cleavage sites of chimeric proteins may be heterologous to the cells expressing the chimeric proteins. For example, cells engineered to express chimeric proteins may be engineered to express proteases that are specific to the protease cleavage sites present in the chimeric proteins and are not generally expressed by the cells. Cells engineered to express both chimeric proteins and proteases may be engineered to express each from separately engineered nucleic acids or from multi-cistron systems (multi-cistron and multi-promoter systems are described in more detail in the section of this specification titled "Multi-cistron and Multi-promoter Systems"). Heterologous proteases and their corresponding protease cleavage sites may be selected with reference to endogenous proteases as described above.

[0086] The proteases that cleave the protease cleavage sites of chimeric proteins may be expressed on different cells rather than on the cells expressing the chimeric protein. For example, proteases may generally be expressed in specific cellular environments, such as the tumor microenvironment. In such cases, cleavage of the protease cleavage site may generally be restricted to the target cellular environment (e.g., the tumor microenvironment) due to an environment-restricting protease that needs to come into contact with the protease cleavage site. In embodiments having membrane-cleavable chimeric proteins, the secretion of effector molecules may generally be restricted to the target cellular environment (e.g., the tumor microenvironment) due to an environment-restricting protease that needs to come into contact with the protease cleavage site. The proteases that cleave the protease cleavage sites of chimeric proteins may be endogenous to different cells. The proteases that cleave the protease cleavage sites of chimeric proteins may be heterogeneous to different cells. For example, different cells can be manipulated to express proteases that are not generally expressed by different cells.

[0087] Proteases include, but are not limited to, type 1 transmembrane proteases, type 2 transmembrane proteases, GPI-anchored proteases, ADAM8 proteases, ADAM9 proteases, ADAM10 proteases, ADAM12 proteases, ADAM15 proteases, ADAM17 proteases, ADAM19 proteases, ADAM20 proteases, ADAM21 proteases, ADAM28 proteases, ADAM30 proteases, ADAM33 proteases, BACE1 proteases, BACE2 proteases, SIP proteases, MT1-MMP proteases, MT3-MMP proteases, MT5-MMP proteases, furin proteases, PCSK7 proteases, matryptase proteases, matryptase 2 proteases, and MMP9 proteases. The protease may be an NS3 protease. The protease may be an ADAM17 protease.

[0088] The protease may be a tumor-associated protease, such as cathepsin, cysteine ​​protease, aspartyl protease, serine protease, or 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, those listed in Table 4B below. Exemplary homologous protease cleavage sites for specific proteases are also listed in Table 4B.

[0089] (Table 4B) Exemplary proteases with homologous cleavage sites and inhibitors TIFF0007865603000022.tif223159TIFF0007865603000023.tif229159TIFF0007865603000024.t if228159TIFF0007865603000025.tif229159TIFF0007865603000026.tif229159TIFF00078656030 00027.tif229159TIFF0007865603000028.tif228159TIFF0007865603000029.tif229159TIFF000 7865603000030.tif229159TIFF0007865603000031.tif230159TIFF0007865603000032.tif112159

[0090] The protease may be one 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) (MER037291), hCG1733572 (Homo sapiens) type presumptive peptidase (MER107386), napsin B pseudogene (MER004982), CYMP gp (Homo sapiens) (MER002929), subfamily A1A unassigned peptidase (MER181559), mouse mammary gland tumor virus retropepsin (MER048030), rabbit endogenous retrovirus endopeptidase (MER043650), S71-related human endogenous retropepsin (MER001812), RTVL-H type presumptive peptidase (MER047117), RTVL-H type presumptive peptidase (MER047133), RTVL-H type presumptive peptidase (MER047160), RTVL-H type presumptive peptidase (MER04 7206), RTVL-H type presumptive peptidase (MER047253), RTVL-H type presumptive peptidase (MER047260), RTVL-H type presumptive peptidase (MER047291), RTVL-H type presumptive peptidase (MER047418), RTVL-H type presumptive peptidase (MER047440), RTVL-H type presumptive peptidase (MER047479), RTVL-H type presumptive peptidase (MER047559), RTVL-H type presumptive peptidase (MER047583),RTVL-H type presumptive peptidase (MER015446), human endogenous retrovirus retropepsin homolog 1 (MER015479), human endogenous retrovirus retropepsin homolog 2 (MER015481), endogenous retrovirus retropepsin pseudogene 1 (Homo sapiens chromosome 14) (MER029977), endogenous retrovirus retropepsin pseudogene 2 (Homo sapiens chromosome 8) (MER029665), endogenous retrovirus retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER002660), Endogenous retrovirus retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER030286), Endogenous retrovirus retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER047144), Endogenous retrovirus retropepsin pseudogene 5 (Homo sapiens chromosome 12) (MER029664), Endogenous retrovirus retropepsin pseudogene 6 (Homo sapiens chromosome 7) (MER002094), Endogenous retrovirus retropepsin pseudogene 7 (Homo sapiens chromosome 6) (MER029776), Endogenous Endogenous retrovirus retropepsin pseudogene 8 (Homo sapiens chromosome Y) (MER030291), endogenous retrovirus retropepsin pseudogene 9 (Homo sapiens chromosome 19) (MER029680), endogenous retrovirus retropepsin pseudogene 10 (Homo sapiens chromosome 12) (MER002848), endogenous retrovirus retropepsin pseudogene 11 (Homo sapiens chromosome 17) (MER004378), endogenous retrovirus retropepsin pseudogene 12 (Homo sapiens chromosome 11) (MER003344), endogenous Retrovirus retropepsin pseudogene 13 (Homo sapiens chromosome 2 and similar) (MER029779), Endogenous retrovirus retropepsin pseudogene 14 (Homo sapiens chromosome 2) (MER029778), Endogenous retrovirus retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047158), Endogenous retrovirus retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047332), Endogenous retrovirus retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER003182),Endogenous retrovirus retropepsin pseudogene 16 (MER047165), Endogenous retrovirus retropepsin pseudogene 16 (MER047178), Endogenous retrovirus retropepsin pseudogene 16 (MER047200), Endogenous retrovirus retropepsin pseudogene 16 (MER047315), Endogenous retrovirus retropepsin pseudogene 16 (MER047405), Endogenous retrovirus retropepsin pseudogene 16 (MER030292), Endogenous retrovirus retropepsin pseudogene 17 (Homo sapiens staining) Body 8) (MER005305), Endogenous retrovirus retropepsin pseudogene 18 (Homo sapiens chromosome 4) (MER030288), Endogenous retrovirus retropepsin pseudogene 19 (Homo sapiens chromosome 16) (MER001740), Endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER047222), Endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER047454), Endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER047477), Endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER004403), Endogenous retrovirus 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 homolog G (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 homolog (MER047101), subfamily A2A nonpeptidase homolog (MER047102), subfamily A2A nonpeptidase homolog (MER047107), subfamily A2A nonpeptidase homolog (MER047108), subfamily A2A nonpeptidase homolog (MER047109), subfamily A2A nonpeptidase homolog (MER047110), subfamily A2A nonpeptidase homolog (MER047111), subfamily A2A nonpeptidase homolog (MER047111), subfamily A2A nonpeptidase homolog Molog (MER047114), 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), Sa Subfamily A2A nonpeptidase homolog (MER047135), subfamily A2A nonpeptidase homolog (MER047137), subfamily A2A nonpeptidase homolog (MER047140), subfamily A2A nonpeptidase homolog (MER047141), subfamily A2A nonpeptidase homolog (MER047142), subfamily A2A nonpeptidase homolog (MER047148), subfamily A2A nonpeptidase homolog (MER047149), subfamily A2A nonpeptidase Homolog (MER047151), subfamily A2A 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 Ze homolog (MER047190), subfamily A2A nonpeptidase homolog (MER047191), 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 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 Ze homolog (MER047213), subfamily A2A nonpeptidase homolog (MER047215), subfamily A2A 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 Ze 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), 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 Ze homolog (MER047279), subfamily A2A nonpeptidase homolog (MER047280), subfamily A2A nonpeptidase homolog (MER047281), subfamily A2A nonpeptidase homolog (MER047282), subfamily A2A nonpeptidase homolog (MER047284), subfamily A2A nonpeptidase homolog (MER047285), 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 Ze homolog (MER047306), subfamily A2A nonpeptidase homolog (MER047307), subfamily A2A nonpeptidase homolog (MER047310), 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 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 Ze homolog (MER047369), subfamily A2A nonpeptidase homolog (MER047370), subfamily A2A nonpeptidase homolog (MER047371), subfamily A2A 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 Ze homolog (MER047401), subfamily A2A nonpeptidase homolog (MER047403), subfamily A2A nonpeptidase homolog (MER047406), subfamily A2A nonpeptidase homolog (MER047407), 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 homolog (MER047423), subfamily A2A nonpeptidase homolog (MER047424), subfamily A2A nonpeptidase homolog (MER047428), subfamily A2A nonpeptidase homolog (MER047429), subfamily A2A nonpeptidase Ze homolog (MER047431), subfamily A2A nonpeptidase homolog (MER047434), subfamily A2A nonpeptidase homolog (MER047439), subfamily A2A nonpeptidase homolog (MER047442), subfamily A2A 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 Ze homolog (MER047470), subfamily A2A nonpeptidase homolog (MER047476), subfamily A2A nonpeptidase homolog (MER047478), subfamily A2A nonpeptidase homolog (MER047483), subfamily A2A nonpeptidase homolog (MER047488), subfamily A2A nonpeptidase homolog (MER047489), subfamily A2A nonpeptidase homolog (MER047490), subfamily A2A nonpeptidase homolog (MER047493), Subfamily A2A nonpeptidase homolog (MER047494), subfamily A2A nonpeptidase homolog (MER047495), subfamily A2A nonpeptidase homolog (MER047496), subfamily A2A nonpeptidase homolog (MER047497), subfamily A2A nonpeptidase homolog (MER047499), subfamily A2A nonpeptidase homolog (MER047502), subfamily A2A nonpeptidase homolog (MER047504), subfamily A2A nonpeptidase Ze homolog (MER047511), subfamily A2A nonpeptidase homolog (MER047513), subfamily A2A nonpeptidase homolog (MER047514), subfamily A2A nonpeptidase homolog (MER047515), subfamily A2A nonpeptidase homolog (MER047516), subfamily A2A 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 Ze homolog (MER047649), subfamily A2A nonpeptidase homolog (MER047662), subfamily A2A nonpeptidase homolog (MER048004), 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), cutaneous aspartate protease (MER057097), presenilin 1 (MER005221), Presenilin 2 (MER005223), Impass 1 peptidase (MER019701), Impass 1 peptidase (MER184722), Impass 4 peptidase (MER019715), Impass 2 peptidase (MER019708), Impass 5 peptidase (MER019712), Impass 3 peptidase (MER019711), Possible family A22 pseudogene (Homo sapiens chromosome 18) (MER029974), Possible family A22 pseudogene (Homo sapiens chromosome 11) (MER023159), Cathepsin V (MER004437), Cathepsin X (MER004508), Cathepsin F (MER004980),Cathepsin L (MER000622), Cathepsin S (MER000633), Cathepsin O (MER001690), Cathepsin K (MER000644), Cathepsin W (MER003756), Cathepsin H (MER000629), Cathepsin B (MER000686), Dipeptidyl peptidase I (MER001937), Bleomycin hydrolase (animal) (MER002481), Tubulointerstitial nephritis antigen (MER016137), Tubulointerstitial nephritis antigen-related protein (MER021799), Cathepsin L-like pseudogene 1 (Homo sapiens) (MER002789), Cathepsin B-like pseudogene (chromosome 4, Homo sapiens) (MER029469), Cathepsin B-like pseudogene (chromosome 1, Homo sapiens) (MER029457), CTSLL2 gp (Homo sapiens) (MER005210), CTSLL3 gp (Homo sapiens) (MER005209), Calpain-1 (MER000770), Calpain-2 (MER000964), Calpain-3 (MER001446), Calpain-9 (MER004042), Calpain-8 (MER021474), Calpain-15 (MER004745), Calpain-5 (MER002939), Calpain-11 (MER005844), Calpain In-12 (MER029889), Calpain-10 (MER013510), Calpain-13 (MER020139), Calpain-14 (MER029744), Mername-AA253 peptidase (MER005537), Carpamodulin (MER000718), Virtual 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 Ze8 (MER001884), Ubiquitin-specific peptidase 13 (MER002627), Ubiquitin-specific peptidase 2 (MER004834), Ubiquitin-specific peptidase 11 (MER002693), Ubiquitin-specific peptidase 14 (MER002667), Ubiquitin-specific peptidase 7 (MER002896), Ubiquitin-specific peptidase 9X (MER005877),Ubiquitin-specific peptidase 10 (MER004439), Ubiquitin-specific peptidase 1 (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 (MER 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 -ze 25 (MER011115), ubiquitin-specific peptidase 36 (MER014033), 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 (MER00 14633), Ubiquitin-specific peptidase 28 (MER014634), Ubiquitin-specific peptidase 47 (MER014636), Ubiquitin-specific peptidase 38 (MER014637), Ubiquitin-specific peptidase 44 (MER014638), Ubiquitin-specific peptidase 50 (MER030315), Ubiquitin-specific peptidase 35 (MER014646), Ubiquitin-specific peptidase 30 (MER014649), Mername-AA091 Peptidase (MER014743), ubiquitin-specific peptidase 45 (MER030314), ubiquitin-specific peptidase 51 (MER014769), ubiquitin-specific peptidase 34 (MER014780), ubiquitin-specific peptidase 48 (MER064620),Ubiquitin-specific peptidase 40 (MER015483), Ubiquitin-specific peptidase 41 (MER045268), Ubiquitin-specific peptidase 31 (MER015493), Mername-AA129 peptidase (MER016485), Ubiquitin-specific peptidase 49 (MER016486), Mername-AA187 peptidase (MER052579), USP17 Peptidase (MER030192), Ubiquitin-specific peptidase 54 (MER028714), Ubiquitin-specific peptidase 53 (MER027329), Ubiquitin-specific endopeptidase 39 [misread] (MER064621), Mername-AA090 nonpeptidase homolog (MER014739), Ubiquitin-specific peptidase 43 [misread] (MER030140), Ubiquitin-specific peptidase 52 [misread] (MER030317), NEK2 pseudogene (MER014736), C19 pseudogene (Homo sapiens: 5th Chromosome (MER029972), Mername-AA088 peptidase (MER014750), Autofadin-2 (MER013564), Autofadin-1 (MER013561), Autofadin-3 (MER014316), Autofadin-4 (MER064622), Cezanne deubiquitinated peptidase (MER029042), Cezanne-2 peptidase (MER029044), Tumor necrosis factor alpha-inducible protein 3 (MER029050), Trabid peptidase (MER029052), VCIP135 deubiquitinated peptidase (MER152304), Otubain-1 (MER029056), Otubain-2 (MER029061), CylD Protein (MER030104), UfSP1 peptidase (MER042724), UfSP2 peptidase (MER060306), DUBA deubiquitinase (MER086098), KIAA0459 (Homo sapiens)-like protein (MER122467), Otud1 protein (MER125457), glycosyltransferase containing 28 domains 1, isoform CRA_c (Homo sapiens)-like (MER123606), hin1L gp (Homo sapiens) (MER139816),Ataxin-3 (MER099998), ATXN3L presumptive peptidase (MER115261), Josephine domain-containing 1 (Homo sapiens) (MER125334), Josephine domain-containing 2 (Homo sapiens) (MER124068), YOD1 peptidase (MER116559), Regmine (plant alpha type) (MER044591), Regmine (MER001800), Glycosylphosphatidylinositol:protein transamidase (MER002479), Regmine 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 (MER00284) 9) Caspase-9 (MER002707), Caspase-10 (MER002579), Caspase-14 (MER012083), Paracaspase (MER019325), Mername-AA143 peptidase (MER021304), Mername-AA186 peptidase (MER020516), Presumed caspase (Homo sapiens) (MER021463), FLIP Protein (MER003026), Mername-AA142 protein (MER021316), caspase-12 pseudogene (Homo sapiens) (MER019698), Mername-AA093 caspase pseudogene (MER014766), subfamily C14A nonpeptidase homolog (MER185329), subfamily C14A nonpeptidase homolog (MER179956), separin (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), prenyl peptidase 1 (MER004246), aminopeptidase N (MER000997), aminopeptidase A (MER001012), leukotriene A4 hydrolase (MER001013), pyroglutamyl peptidase II (MER012221), cytozolaranyl aminopeptidase (MER002746), cystinyl aminopeptidase (MER002060), aminopeptidase B (MER001494), aminopeptidase PILS (MER005331), arginyl aminopeptidase-like 1 (MER012271), leukocyte-derived arginine aminopeptidase (MER002968), aminopeptidase Q (MER052595), aminopeptidase O (MER019730), Tata-binding protein-related factor (MER026493), angiotensin-converting enzyme peptidase unit 1 (MER004967), angiotensin-converting enzyme peptidase unit 2 (MER001019), Angiotensin-Converting Enzyme-2 (MER011061), Mername-AA153 Protein (MER020514), Chimet Oligo Peptidase (MER001737), Neurolysin (MER010991), Mitochondrial Intermediate Peptidase (MER003665), Mername-AA154 Protein (MER021317), Reishi Manorisin-2 (MER014492), Reishi Manorisin-3 (MER180031), Matrix Metallopeptidase-1 (MER001063), Matrix Metallopeptidase-8 (MER001084),Matrix metallopeptidase-2 (MER001080), Matrix metallopeptidase-9 (MER001085), Matrix metallopeptidase-3 (MER001068), Matrix metallopeptidase-10 (Homo sapiens type) (MER001072), Matrix metallopeptidase-11 (MER001075), Matrix metallopeptidase-7 (MER001092), Matrix metallopeptidase-12 (MER001089), Matrix metallopeptidase-13 (MER001411), Membrane-type matrix metallopeptidase-1 (MER 001077), Membrane-type matrix metallopeptidase-2 (MER002383), Membrane-type matrix metallopeptidase-3 (MER002384), Membrane-type matrix metallopeptidase-4 (MER002595), Matrix metallopeptidase-20 (MER003021), Matrix metallopeptidase-19 (MER002076), Matrix metallopeptidase-23B (ME R004766), 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 (MER 013587), Matrix metallopeptidase-23A (MER037217), Macrophage elastase homolog (chromosome 8, Homo sapiens) (MER030035), Mername-AA156 protein (MER021309), Matrix metallopeptidase-like 1 (MER045280), Subfamily M10A non-peptidase homolog (MER175912), Subfamily M10A non-peptidase homolog (MER187997), subfamily M10A non-peptidase homolog (MER187998), subfamily M10A non-peptidase homolog (MER180000), meprine alpha subunit (MER001111), meprine beta subunit (MER005213), procollagen C-peptidase (MER001113), mammalian toroid-like 1 protein (MER005124), mammalian toroid-like2 protein (MER005866), ADAMTS9 peptidase (MER012092), ADAMTS14 peptidase (MER016700), ADAMTS15 peptidase (MER017029), ADAMTS16 peptidase (MER015689), ADAMTS17 peptidase (MER016302), ADAMTS18 peptidase (MER016090), ADAMTS19 peptidase (MER015663), ADAM8 peptidase ( MER003902), ADAM9 peptidase (MER001140), ADAM10 peptidase (MER002382), ADAM12 peptidase (MER005107), ADAM19 peptidase (MER012241), ADAM15 peptidase (MER002386), ADAM17 peptidase (MER003094), ADAM20 peptidase (MER004725), ADAM MEC1 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 ADAM21 peptidase (MER006268), ADAMTS10 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 IN-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), AD AM3B protein (Homo sapiens type) (MER005199), ADAM11 protein (MER001146), ADAM22 protein (MER005102), ADAM23 protein (MER005103), ADAM29 protein (MER006267), a 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 peptidase (MER005197), Neprilysin-2 (MER013406), Kell blood group protein (MER001054), PHEX peptidase (MER002062), i-AAA peptidase (MER001246), i-AAA peptidase (MER005755), Parapregin (MER004454), Afg3-like protein 2 (MER005496), Afg3Protein 1A (MER014306), Paparicin-1 (MER002217), Paparicin-2 (MER014521), Farnesyl-converting enzyme 1 (MER002646), Metalloproteinase-related protein-1 (MER030873), Aminopeptidase AMZ2 (MER011907), Aminopeptidase AMZ1 (MER058242), Carboxypeptidase Peptidase A1 (MER001190), Carboxypeptidase A2 (MER001608), Carboxypeptidase B (MER001194), Carboxypeptidase N (MER001198), Carboxypeptidase E (MER001199), Carboxypeptidase M (MER001205), Carboxypeptidase U (MER001193), Carboxypeptidase Cytosol Carboxypeptidase-like Protein 5 (MER033174), Cytosol Carboxypeptidase 3 (MER033176), Cytosol Carboxypeptidase 6 (MER033178), Cytosol Carboxypeptidase 1 (MER033179), Cytosol 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), Insulin (MER001214), MitochondriaProcessing peptidase beta subunit (MER004497), Nardilysin (MER003883), Eupitrilidine (MER004877), Mitochondrial processing peptidase non-peptidase alpha subunit (MER001413), Ubiquinol-cytochrome c reductase core protein I (MER003543), Ubiquinol-cytochrome c reductase core protein II (MER003544), Ubiquinol-cytochrome c reductase core protein domain 2 (MER043998), Insulin unit 2 (MER046821), Nardilysin unit 2 (MER046874), Insulin unit 3 (MER078753), Mitochondrial processing peptidase subunit alpha Unit 2 (MER124489), Nardi Ryzin Unit 3 (MER142856), LOC133083 gp (Homo sapiens) (MER021876), Subfamily M16B non-peptidase homolog (MER188757), Leucylaminopeptidase (animal) (MER003100), Mername-AA040 peptidase (MER003919), Leucylaminopeptidase-1 (nematode type) (MER013416), Methionylaminopeptidase 1 (MER001342), Methionylaminopeptidase 2 (MER001728), Aminopeptidase P2 (MER004498), Xaa-Pro Dipeptidase (eukaryote) (MER001248), Aminopeptidase P1 (MER004321), Mitochondrial intermediate cleavage peptidase 55kDa (MER013463), Mitochondrial methionylaminopeptidase (MER014055), Mername-AA020 peptidase homolog (MER010972), Growth-related protein 1 (MER005497), Chromatin-specific transcription elongation factor 140kDa subunit (MER026495), proliferation-related protein 1-like (Homo sapiens chromosome X) (MER029983), Mername-AA226 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 (MER005152), Glutaminylcyclis (MER015095), glutamate carboxypeptidase II (Homo sapiens) type non-peptidase homolog (MER026971), nicarin (MER044627), membrane dipeptidase (MER001260), membrane-bound dipeptidase-2 (MER013499), membrane-bound dipeptidase-3 (MER013496), dihydroorotase (MER005767), dihydropyrimidinase (MER033266), dihydropyrimidinase-related protein-1 (MER030143), dihydropyrimidinase-related protein-2 (MER030155), dihydropyrimidinase-related protein-3 (MER030151), dihydropyrimidinase-related protein-4 (MER030149), dihydropyrimidinase-related Hypothetical proteins such as Protein-5 (MER030136), 5730457F11RIK (MER033184), 1300019j08rik protein (MER033186), guanine aminohydrolase (MER037714), Kae1 putative peptidase (MER001577), OSGEPL1-like protein (MER013498), S2P peptidase (MER004458), subfamily M23B nonpeptidase homolog (MER199845), subfamily M23B nonpeptidase homolog (MER199846), subfamily M23B nonpeptidase homolog (MER199847), subfamily M23B nonpeptidase homolog (MER137320), subfamily M23B nonpeptidase homolog (MER 201557), subfamily M23B nonpeptidase homolog (MER199417), subfamily M23B nonpeptidase homolog (MER199418), subfamily M23B nonpeptidase homolog (MER199419), subfamily M23B nonpeptidase homolog (MER199420), subfamily M23B nonpeptidase homolog (MER175932), subfamily M23B nonpeptidase homolog (MER199665), Poh1 peptidase (MER020382), Jab1 / MPN domain metalloenzyme (MER022057), Mername-AA165 peptidase (MER021865),Brcc36 isopeptidase (MER021890), histone H2A deubiquitinating enzyme MYSM1 (MER021887), AMSH deubiquitinating peptidase (MER030146), putative peptidase (Homo sapiens chromosome 2) (MER029970), Mername-AA168 protein (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), testosin (MER005212), tryptase beta (MER000136), kallikrein-related peptidase 5 (MER005544), choline (MER005881), kallikrein-related peptidase 12 (MER006038), DESC1 peptidase (MER006298), tryptase gamma 1 (MER011036), kallikrein-related peptidase 14 (MER011038), hyaluronic acid-binding peptidase (MER003612), transmembrane peptidase, serine 4 (MER011104), enteric serine peptidase (rodents) (MER016130), adrenal secretory serine peptidase (MER003734), tryptase delta 1 (Homo sapiens) (MER005948), Matryptase-3 (MER029902), Malapsin (MER006119), Tryptase-6 (MER006118), Ovochymase-1 Domain 1 (MER099182), Transmembrane Peptidase, Serine 3 (MER005926), Kallikrein-related Peptidase 15 (MER000064), Mernam e-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-related 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 Syn 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-related serine peptidase 1 (MER000244), Complement factor I (MER000228), Pancreatic endopeptidase E form B (MER000150), Pancreatic elastase IIB (MER000147), Coagulation factor XIIa (MER000187), Plasma kallikrein (MER000203), Coagulation factor Xia (MER000210), Coagulation factor IXa (MER000216), Coagulation factor Vila (MER000215), Coagulation factor Xa (MER000212), Thrombin (MER000188), Protein C (active (MER000222), acrosin (MER000078), hepsin (MER000156), hepatocyte growth factor activator (MER000186), mannan-binding lectin-related serine peptidase 2 (MER002758), u-plasminogen activator (MER000195), t-plasminogen activator (MER000192), plasmin (MER000175),Kallikrein-related peptidase 6 (MER002580), neurotrypsin (MER004171), kallikrein-related peptidase 8 (MER005400), kallikrein-related peptidase 10 (MER003645), epiceriacin (MER003736), kallikrein-related peptidase 4 (MER005266), procemin (MER004214), chymopathin (MER001503), kallikrein-related peptidase 11 (MER004861), kallikrein HtrA1 peptidase (Homo sapiens type) (MER002577), HtrA2 peptidase (MER208413), HtrA2 peptidase (MER004093), HtrA3 peptidase (MER014795), HtrA4 peptidase (MER016351), Tysnd1 peptidase (MER050461), TMPRSS12 peptidase (MER01708 5) HAT-like presumed peptidase 2 (MER021884), trypsin C (MER021898), kallikrein-related peptidase 7 (MER002001), matryptase (MER003735), kallikrein-related peptidase 13 (MER005269), kallikrein-related peptidase 9 (MER005270), matryptase-2 (MER005278), umbilical vein peptidase (MER005421), LCLP peptidase (MER001900), spinesin N (MER014385), Malapsin-2 (MER021929), Complement Factor D-like presumptive peptidase (MER056164), Ovochymase-2 (MER022410), HAT-like 4-peptidase (MER044589), Ovochymase 1-domain 1 (MER022412), Epidermal-specific SP-like presumptive peptidase (MER029900), Testicular serine peptidase 5 (MER029901), Mername-AA258 peptidase (MER000285), Polycellase-IA Unit 1 (MER030879), Polycellase-IA Unit 2 (MER030880), Testicular Serine Peptidase 2 (Human Type) (MER033187), Virtual Acrosin-like Peptidase (Homo Sapiens) (MER033253), HAT-like 5-peptidase (MER028215),Polycellase 3 Unit 1 (MER061763), Polycellase 3 Unit 2 (MER061748), Tryptophan / Serine Protease (MER056263), Peptidase similar to Polycellase 2 Unit 1 (MER061777), Mername-AA123 Peptidase (MER021930), HAT-like Peptidase 2 (MER099184), hCG2041452-like Protein (MER099172), hCG22067 (Homo sapiens) (MER099169), Brain Rescue Factor-1 (Homo sapiens) (MER098873), hCG2041108 (Homo sapiens) (MER099173), Polycellase-2 Unit 2 (MER061760), Polycellase-2 Unit 3 (MER065694), Mername-AA201 (peptidase homolog) MER099175, secretory trypsin-like serine peptidase homolog (MER030000), polycellase 1A Unit 3 (MER029880), Azulocidine (MER000119), Haptoglobin-1 (MER000233), Haptoglobin-related protein (MER000235), Macrophage-stimulating protein (MER001546), Hepatocyte growth factor (MER000185), Protein Z (MER000227), TESP1 protein (MER047214), LOC136242 protein (MER016132), Plasma kallikrein-like protein 4 (MER016346), PRSS35 protein (MER016350), DKFZp586H2123-like protein (MER066474), Apolipoprotein (MER0001 83) 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), amide phosphoribosyltransferase precursor (MER003314), glutamine-fructose-6-phosphate transaminase 1 (MER003322), glutamine:fructose-6-phosphate amidetransferase (MER012158),Mername-AA144 protein (MER021319), asparagine synthetase (MER033254), family C44 nonpeptidase homolog (MER159286), Family C44 unassigned peptidase (MER185625), Family C44 unassigned peptidase (MER185626), Cesernin 1 (MER045376), Cesernin 2 (MER064573), Cesernin 3 (MER064582), Acid ceramidase precursor (MER100794), N-acylethanolamine 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 Proteasome 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 (MER001710), 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), Glycosyl asparaginase precursor (MER003299), Isoaspartyl dipeptidase (threonine type) (MER031622), Tassase-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-glutamyl Transpeptidase homolog (chromosome 2, Homo sapiens) (MER037241), polycystin-1 (MER126824), KIAA1879 protein (MER159329), polycystic kidney disease type 1-3 (MER172554), gamma-glutamyl hydrolase (MER002963), guanine 5″ monophosphate synthetase (MER043387), carbamoyl phosphate synthase (Homo sapiens type) (MER078640), dihydroorotase (N-terminal unit) (Homo sapiens type) (MER060647), DJ-1 putative peptidase (MER003390), Mername-AA100 putative peptidase This includes -ase (MER014802), Mername-AA101 nonpeptidase homolog (MER014803), KIAA0361 protein (Homo sapiens type) (MER042827), F1134283 protein (Homo sapiens) (MER044553), nonpeptidase homolog chromosome 21 open reading frame 33 (Homo sapiens) (MER160094), family C56 nonpeptidase homolog (MER177016), family C56 nonpeptidase homolog (MER176613), family C56 nonpeptidase homolog (MER176918), and mucin-like hormone receptor-like 2. EGF-like module (MER037230), CD97 antigen (human type) (MER037286), mucin-like hormone receptor 3 (MER037288) included EGF-like module,EGF-like module containing mucin-like hormone receptor-like 1 (MER037278), EGF-like module containing mucin-like hormone receptor-like 4 (MER037294), cadherin EGF LAG Sevenpass G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (mouse) type protein (MER123205), GPR56 (Homo sapiens) type protein (MER122057), latrophyllin 2 (MER122199), latrophyllin-1 (MER126380), latrophyllin 3 (MER124612), protocadherin flamingo 2 (MER124239), ETL protein (MER126267), G protein-coupled receptor 112 (MER126114), 7-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 self-cleaving mucin (MER074260), dystroglycan (MER054741), proprotein converter 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 (eukaryotes) (MER000401), acylaminoacyl peptidase (MER000408), fibroblast-activating protein alpha subunit (MER000399), PREPL Protein A (MER004227), dipeptidyl-peptidase 8 (MER013484), dipeptidyl-peptidase 9 (MER004923), FLJ1 presumptive peptidase (MER017240), Mername-AA194 presumptive peptidase (MER017353), Mername-AA195 presumptive peptidase (MER017367), Mername-AA196 presumptive peptidase (MER017368), Mername-AA197 presumptive peptidase (MER017371), C14orf29 protein Protein (MER033244), virtual protein (MER033245), virtual esterase / lipase / thioesterase (MER047309), protein bat5 (MER037840), virtual protein flj40219 (MER033212), virtual protein flj37464 (MER033240), virtual 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), neurolysin 3 (MER033232), neuro Lysine 4, X-linked (MER033235), Neurolysine 4, Y-linked (MER033236), Esterase D (MER043126), Arylacetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), Hormone-sensitive lipase (MER033274), Neurolysine 1 (MER033280), Neurolysine 2 (MER033283), Family S9 non-peptidase homolog (MER212939), Family S9 non-peptidase homolog (MER211490), Subfamily S 9C 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), Yolk-forming carboxypeptidase-like protein (MER005492), RISC peptidase (MER010960), Family S15 unassigned peptidase (ME R199442), Family S15 unassigned peptidase (MER200437), Family S15 unassigned peptidase (MER212825), Lysosomal Pro-Xaa carboxypeptidase (MER000446), Dipeptidyl peptidase II (MER004952), Thymus-specific serine peptidase (MER005538), Epoxyd hydrolase-like putative peptidase (MER031614), Loc328574-like protein (MER033246), Abhydrolase domain-containing protein 4 (MER031616),Epoxydohydrolase (MER000432), mesoderm-specific transcription protein (MER199890), mesoderm-specific transcription protein (MER017123), cytosolic epoxydohydrolase (MER029997), Cytosolic epoxide hydrolase (MER213866), virtual protein FLJ22408 (MER031608), CGI-58 putative peptidase (MER030163), important region protein 21 epoxide hydrolase of Williams-Beuren syndrome (MER031610), epoxide hydrolase (MER031612), virtual protein 922408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), virtual protein (MER033249), valacyclovir hydrolase (MER033259) ), Ccg1 interaction factor b (MER210738), glycosyl asparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622), tapase-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). Polycystic kidney disease-like syndrome (MER172554). Gamma-glutamyl hydrolase (MER002963). Guanine 5″ monophosphate synthetase (MER043387). Carbamoyl phosphate 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 Mu33 (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 2 (MER037230). CD97 antigen (human type) (MER037286). EGF-like module containing mucin-like hormone receptor 3 (MER037288). EGF-like module containing mucin-like hormone receptor 1 (MER037278). EGF-like module containing mucin-like hormone receptor 4 (MER037294). Cadherin EGF LAG 7-transmembrane G receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (mouse) type protein (MER123205), GPR56 (Homo sapiens) type protein (MER122057), latrophyllin 2 (MER122199), latrophyllin-1 (MER126380), latrophyllin 3 (MER124612),Protocadherin Flamingo 2 (MER124239). ETL protein (MER126267). G protein-coupled receptor 112 (MER126114). 7-transmembrane helix receptor (MER125448). Gpr114 protein (MER159320). GPR126 vascular-induced G protein-coupled receptor (MER140015). GPR125 (Homo sapiens) type protein (MER159279). GPR116 (Homo sapiens) type G protein-coupled receptor (MER159280). GPR128 (Homo sapiens) type G protein-coupled receptor (MER162015). GPR133 (Homo sapiens) type protein (MER159334). GPR110 G protein-coupled receptor (MER159277), GPR97 protein (MER159322), KPG_006 protein (MER161773), KPG_008 protein (MER161835), KPG_009 protein (MER159335), 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 converter 9 (MER022416), site 1 peptidase (MER00 1948), 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 (eukaryotes) (MER000401), acylaminoacyl peptidase (MER000408), fibroblast-activating protein alpha subunit (MER000399), PREPL Protein A (MER004227), dipeptidyl peptidase 8 (MER013484), dipeptidyl peptidase 9 (MER004923), FLJ1 presumptive peptidase (MER017240), Mername-AA194 presumptive peptidase (MER017353), Mername-AA195 presumptive peptidase (MER017367), Mername-AA196 presumptive peptidase (MER017368), Mername-AA197 presumptive peptidase -ase (MER017371), C14orf29 protein (MER033244), virtual protein (MER033245), virtual esterase / lipase / thioesterase (MER047309), protein bat 5 (MER037840), hypothetical protein flj40219 (MER033212), hypothetical protein flj37464 (MER033240), hypothetical protein flj33678 (MER033241), dipeptidyl peptidase homolog DPP6 (MER000403), dipeptidyl peptidase homolog DPP10 (MER005988), a protein similar to mouse chromosome 20 open reading frame 135 (MER037845), kynurenine formamidase (MER046020), thyroglobulin precursor (MER011604), acetylcholinesterase (MER033188), cholinesterase (MER033198), carboxylesterase D1 (MER033213), liver carboxylesterase (MER033220), carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033226), bile salt-dependent lipase (MER033227), carboxylesterase-related protein (MER033231), neurolysin 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 (MER211 490), 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), yolk-forming carboxypeptidase-like protein (MER00549 2) 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), Epoxydohydrolase Presumptive 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 virtual protein FLJ22408 (MER031608), CGI-58 presumptive peptidase (MER030163), Williams-Buren syndrome critical region protein 21 epoxide hydrolase (MER031610), epoxide hydrolase (MER031612),Hypothetical proteins: flj22408 (epoxydohydrolase) (MER031617), monoglyceride lipase (MER033247), virtual protein (MER033249), valacyclovir hydrolase (MER033259), Ccg1 interacting factor b (MER210738).

[0091] Protease enzyme activity can be regulated. For example, certain proteases can be inactivated by the presence or absence of certain drugs (e.g., proteases that bind to certain small molecule inhibitors). Such proteases may 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, silprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and boxylaprevir. In another embodiment, protease activity can be regulated by modulating the expression of the protease itself, for example, by manipulating cells to express the protease using an inducible promoter system (e.g., a Tet On / Off system) or a cell-specific promoter (promoters that can be used to express heterologous proteases are described in more detail in a section titled “Promoters” herein). The protease may also include degron, for example, any of the degrons described herein, and can be regulated using any of the degron systems described herein.

[0092] Protease enzyme activity can also be regulated through the selection of specific protease cleavage sites. For example, protease cleavage sites may be selected and / or manipulated so that the sequence exhibits a desired cleavage rate by the desired protease, such as a reduced cleavage rate relative to the endogenous sequence of a substrate spontaneously cleaved by the desired protease. Another example is selecting and / or manipulating protease cleavage sites so that the sequence exhibits a desired cleavage rate in a cell state-specific manner. For example, various cell states (e.g., after cell signaling, e.g., immune cell activation) can affect the expression and / or localization of specific proteases. As an illustrative example, ADAM17 protein levels and localization are known to be affected by signaling, for example, through the protein kinase C (PKC) signaling pathway (e.g., activation by the PKC activator phorbol-12-myristate-13-acetat [PMA]). Therefore, protease cleavage sites can be selected and / or manipulated so that the cleavage of the protease cleavage site and the subsequent release of effector molecules are increased or decreased, as desired, depending on the protease properties (e.g., expression and / or localization) of a particular cellular state. As another example, protease cleavage sites (particularly in combination with a specific membrane anchoring domain) can be selected and / or manipulated for optimal protein expression of the chimeric protein.

[0093] Cell membrane anchoring domain The membrane-cleavable chimeric proteins provided herein include a cell membrane anchoring domain (referred to as "MT" in formulas SC-MT or MT-CS). Generally, the cell membrane anchoring domain may be any amino acid sequence motif that is localized to the cell membrane of a cell expressing the chimeric protein (e.g., inserted into it) or otherwise capable of orienting the chimeric protein to associate with it. The cell membrane anchoring domain may be a transmembrane-intracellular domain. The cell membrane anchoring domain may be a transmembrane domain. The cell membrane anchoring domain may be an endogenous protein domain (e.g., a transmembrane domain). The cell membrane anchoring domain may be derived from a type I, type II, or type III transmembrane protein. The cell membrane anchoring domain may include a post-translational modification tag, or a post-translationally modifiable motif for modifying the chimeric protein to include a post-translational modification tag, where the post-translational modification tag enables association with the cell membrane. Examples of post-translational modification tags include, but are not limited to, lipid anchoring domains (e.g., GPI lipid anchors, myristoylation tags, or palmitoylation tags). Examples of cell membrane anchoring 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. Cell membrane anchoring domains may include cell surface receptors or their cell membrane binding portions.

[0094] In some embodiments, the cell membrane anchoring domain includes a transmembrane domain derived from the B-71 polypeptide. In some embodiments, the transmembrane domain includes the sequence LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 204).

[0095] In some embodiments, the cell membrane anchoring domain includes 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 includes the sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 205). In some embodiments, the transmembrane domain includes the sequence IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 206). In some embodiments, the transmembrane domain includes the sequence IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 207). In some embodiments, the cell membrane anchoring domain includes a hinge and a transmembrane domain derived from CD8. In some embodiments, the CD8 hinge includes the sequence TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 208). In some embodiments, the CD8 hinge includes the sequence AAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 209).

[0096] Generally, for all membrane-cleavable chimeric proteins described herein, the cell membrane anchoring domain is either (1) the C-terminus of the protease cleavage site and the N-terminus of any intracellular domain, if present (in other words, the cell membrane anchoring domain is between the protease cleavage site and, if present, the intracellular domain), or (2) the N-terminus of the protease cleavage site and the C-terminus of any intracellular domain, if present (also between the protease cleavage site and, if present, the intracellular domain with reversed domain orientation). In embodiments characterized by a degron associated with a chimeric protein, the degron domain is specifically a terminal cytoplasm-directed domain with respect to cell membrane anchoring (in other words, the cell membrane anchoring domain is between the protease cleavage site and the degron). The cell membrane anchoring domain can be linked to the protease cleavage site by a polypeptide linker, i.e., a polypeptide sequence that is not generally considered to be part of the cell membrane anchoring domain or the protease cleavage site. The cell membrane anchoring domain, if present, can be linked 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. The cell membrane anchoring domain, if present, can be linked 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 linking the first polypeptide sequence and the second polypeptide sequence. The polypeptide linker can be a mobile linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers are, but are not limited to, GSG linkers (e.g., [GS]4GG [SEQ ID NO: 1]). 220 ]), A(EAAAK)3A(Sequence ID) 221 ), and Whitlow linkers (e.g., "KEGS" linker, for example, amino acid sequence KESGSVSSEQLAQFRSLD(Sequence ID) 222 ), eGK linker, for example amino acid sequence EGKSSGSGSESKST (sequence number) 223This includes the linkers described in more detail in U.S. Patent No. 5,990,275, which is incorporated herein by reference. Additional polypeptide linkers include SEQ ID NOs. 194, 195, 196, and 197. Other polypeptide linkers may be selected based on desired properties (e.g., length, mobility, amino acid composition, etc.) and are known to those skilled in the art.

[0097] Generally, the cell membrane anchoring domain is oriented so that the secreted effector molecule and protease cleavage site are exposed extracellularly after insertion into or association with the cell membrane, and the protease cleavage site is cleaved by its respective protease, allowing the effector molecule to be released into the extracellular space ("secreted").

[0098] Deglon system and domain In some embodiments, any of the proteins described herein may include a degron domain, but is not limited to, a protease, a transcription factor, a promoter or component of a promoter system (e.g., ACP), and / or any membrane-cleavable chimeric protein described herein. Generally, a degron domain can be any amino acid sequence motif capable of directing regulated degradation, such as regulated degradation via a ubiquitin-mediated pathway. In the presence of an immunomodulatory drug (IMiD), the degron domain directs the degradation of the ubiquitin-mediated degron fusion protein.

[0099] The degron domain may be a cerebron (CRBN) polypeptide substrate domain capable of binding to 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, as well as a fragment thereof capable of drug-inducible binding to CRBN. The CRBN polypeptide substrate domain may be a chimeric fusion product of a natural CRBN polypeptide sequence, such as an IKZF3 / ZFP91 / IKZF3 chimeric fusion product having the amino acid sequence FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 175). The Degron domain, and in particular the CRBN degron system, are described in more detail in International Patent Publication No. WO2019 / 089592Al, which is incorporated herein by reference for all purposes.Other examples of degron domains include, but are not limited to, HCVNS4 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 repeat of SP2 and NB (SP2-NB-SP2 of influenza A or influenza B, e.g., SEQ ID NO: 164), RPB (four copies of residues 1688-1702 of yeast RPB; SEQ ID NO: 165), SPmix (tandem repeat of SP1 and SP2 (SP2-SP1-SP2-SP1-SP2 of influenza A virus M2 protein; SEQ ID NO: 166), NS2 (three copies of residues 79-93 of influenza A virus NS protein; SEQ ID NO: 167)). ODC (ornithine decarboxylase residues 106-142; SEQ ID NO: 168), Nek2A, mouse ODC (residues 422-461, SEQ ID NO: 169), mouse ODC_DA (residues 422-461 of mODC including D433A and D434A point mutations), APC / C degron, COP1E3 ligase-binding degron motif, CRL4-Cdt2-binding PIP degron, actinphylline-binding degron, KEAP1-binding degron, KLHL2 and KLHL3-binding degron, MDM2-binding motif, N-degron, hydroxyproline modification in hypoxia signaling, plant hormone-dependent SCF-LRR-binding degron, SCF ubiquitin ligase-binding phosphodegron, plant hormone-dependent SCF-LRR-binding degron, DSGxxS phosphate-dependent degron, Siah-binding motif, SPOP Includes an SBC docking motif or a PCNA-coupled PIP box.

[0100] Modulated degradation can be drug-inducible. Drugs capable of mediating / modulating degradation can be small molecule compounds. Drugs capable of mediating / modulating degradation may 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 or other proteins of interest as described herein). For degron domains having a CRBN polypeptide substrate domain, examples of imide-containing IMiDs include, but are not limited to, thalidomide, lenalidomide, or pomalidomide. IMiDs may be FDA-approved drugs.

[0101] Chimeric proteins described herein may include a degron domain (e.g., referred to as "D" in formulas SC-MT-D or D-MT-CS for membrane-cleavable chimeric proteins described herein). In the absence of an IMiD, degron / ubiquitin-mediated degradation of the chimeric protein does not occur. Following the expression and localization of the chimeric protein into the cell membrane, the protease cleavage site directs the cleavage of the chimeric protein, causing the effector molecule to be released ("secreted") into the extracellular space. In the presence of an immunomodulatory drug (IMiD), the degron domain directs the ubiquitin-mediated degradation of the chimeric protein, causing the secretion of the effector molecule to be reduced or eliminated. Generally, for membrane-cleavable chimeric proteins fused to a degron domain, the degron domain is specifically a terminal cytoplasm-directing domain relative to the cell membrane anchoring domain, e.g., the C-terminal domain in formula SC-MT-D or the N-terminal domain in formula D-MT-CS. The degron domain can be linked by a polypeptide linker to a polypeptide sequence that is not generally considered to be part of the cell membrane anchoring domain or the degron domain, i.e., a polypeptide sequence that is not generally considered to be part of the cell membrane anchoring domain or the degron domain. The polypeptide linker can be any amino acid sequence that links the first polypeptide sequence and the second polypeptide sequence. The polypeptide linker can be a mobile linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers are, but are not limited to, GSG linkers (e.g., [GS]4GG [SEQ ID NO: 1]). 220 ]), A(EAAAK)3A(Sequence ID) 221 ), and Whitlow linkers (e.g., "KEGS" linker, for example, amino acid sequence KESGSVSSEQLAQFRSLD(Sequence ID) 222 ), eGK linker, for example amino acid sequence EGKSSGSGSESKST (sequence number) 223This includes degrons, and linkers as described in more detail in U.S. Patent No. 5,990,275, which is incorporated herein by reference. Additional polypeptide linkers include SEQ ID NOs. 194, 195, 196, and 197. Other polypeptide linkers may be selected based on desired properties (e.g., length, mobility, amino acid composition, etc.) and are known to those skilled in the art. Generally, degrons are oriented in relation to the cell membrane anchoring domain, and after localization to the cell membrane, the degrons are exposed to the cytosol, allowing the degron domain to mediate degradation (e.g., exposure to the cytosol and cytosol), and to mediate ubiquitin-mediated degradation.

[0102] For degron fusion proteins, the degron domain can be the N-terminus or C-terminus of the target protein, for example, an effector molecule. The degron domain can be linked to the target protein by a polypeptide linker, i.e., a polypeptide sequence that is not generally considered to be part of the target protein or the degron domain. The polypeptide linker can be any amino acid sequence linking the first polypeptide sequence and the second polypeptide sequence. The polypeptide linker can be a mobile linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers are, but are not limited to, GSG linkers (e.g., [GS]4GG [SEQ ID NO: 1]). 220 ]), A(EAAAK)3A(Sequence ID) 221 ), and Whitlow linkers (e.g., "KEGS" linker, for example, amino acid sequence KESGSVSSEQLAQFRSLD(Sequence ID) 222 ), eGK linker, for example amino acid sequence EGKSSGSGSESKST (sequence number) 223This includes, and linkers as described in more detail in U.S. Patent No. 5,990,275, which is incorporated herein by reference. Additional polypeptide linkers include SEQ ID NOs. 194, 195, 196, and 197. Other polypeptide linkers may be selected based on desired properties (e.g., length, mobility, amino acid composition, etc.) and are known to those skilled in the art. Polypeptide linkers may be cleavable, for example, any of the protease cleavage sites described herein.

[0103] Homing molecules The "tumor microenvironment" is the cellular environment in which a tumor exists, including surrounding blood vessels, immune cells, fibroblasts, myeloid-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM) (see, e.g., Pattabiraman, DR & Weinberg, RANature Reviews Drug Discovery 13, 497-512 (2014); Balkwill, F et al. J Cell Sci 125, 5591-5596, 2012; and Li, H. et al. J Cell Biochem 101(4), 805-15, 2007).

[0104] 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, which includes a secreted effector molecule, the secreted effector molecule may be a homing molecule. "Homing" refers to the active navigation (migration) of a cell to a target site (e.g., a cell, a tissue (e.g., a tumor), or an organ). "Homing molecule" refers to a molecule that directs a cell toward a target site. In some embodiments, the homing molecule functions to recognize and / or initiate an interaction of the engineered cell toward the target site. Non-limiting examples of homing molecules include CXCR1, CCR9, CXCR2, CXCR3, CXCR4, CCR2, CCR4, FPR2, VEGFR, IL6R, CXCR1, CSCR7, and PDGFR.

[0105] 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 within cells. Chemokines can be classified into four main 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 chemokine receptors that allow the engineered cells 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 may be encoded by the manipulated nucleic acids of the present disclosure include: CXC chemokine receptors (e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7), CC chemokine receptors (CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, and CCR11), CX3C chemokine receptors (e.g., CX3CR1 that binds to CX3CL1), and XC 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 manipulated nucleic acids are configured to produce CXCL8, CXCL9, and / or CXCL10 (which promotes T cell recruitment), CCL3 and / or CXCL5, CCL21 (which promotes Th1 recruitment and polarization).

[0106] In some embodiments, the manipulated nucleic acids are configured to produce G protein-coupled receptors (GPCRs) that detect N-formylated oligopeptides (including, but not limited to, FPR2 and FPRL1).

[0107] In some embodiments, the manipulated nucleic acid is configured to produce receptors that detect interleukins (including, but not limited to, IL6R).

[0108] In some embodiments, the engineered nucleic acid is configured to produce receptors (including, but not limited to, FGFR, PDGFR, and EGFR, as well as receptors of the VEGF family, including, but not limited to, VEGF-C and VEGF-D) that detect growth factors secreted from other cells, tissues, or tumors.

[0109] 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). The α subunit of an integrin may, but are not limited to, ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, IGTA7, ITGA8, ITGA9, IGTA10, IGTA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGA2B, ITGAX. The β subunit of an integrin may, but are not limited to, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, and ITGB8. Manipulated nucleic acids may be configured to produce any combination of integrin α and β subunits.

[0110] In some embodiments, the homing molecule is a matrix metalloproteinase (MMP). MMPs are enzymes that cleave components of the basement membrane underlying the endothelial cell wall. Non-limiting examples of MMPs include MMP-2, MMP-9, and MMP-1. In some embodiments, the engineered nucleic acid is configured to produce an inhibitor of a molecule (e.g., a protein) that inhibits MMPs. For example, the engineered nucleic acid may be configured to express an inhibitor of membrane type 1 MMP (MT1-MMP) (e.g., an RNAi molecule) or TIMP metallopeptidase inhibitor 1 (TIMP-1).

[0111] In some embodiments, the homing molecule is, for example, a ligand that binds to selectins 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).

[0112] The term "homing molecules" also encompasses transcription factors that regulate the production of molecules that improve / enhance cell homing.

[0113] In some embodiments, the homing molecule includes an antibody, such as an anti-integrin alpha-4, beta-7, or anti-MAdCAM.

[0114] Manipulated nucleic acids Provided herein are engineered nucleic acids encoding at least one chimeric protein, for example, a membrane-cleavable chimeric protein having the formula SC-MT or MT-CS as described herein. Provided herein are engineered nucleic acids encoding two or more chimeric proteins.

[0115] In certain embodiments described herein, the engineered nucleic acid encodes an expression cassette comprising a promoter and an exogenous polynucleotide sequence encoding a membrane-cleavable chimeric protein oriented N-terminus to C-terminus, having the formula: SC-MT or MT-CS, where S refers to a secreted effector molecule, C refers to a protease cleavage site, and MT refers to a cell membrane anchoring domain. The promoter is operably ligated to the exogenous polynucleotide sequence, and SC-MT or MT-CS is configured to be expressed as a single polypeptide.

[0116] In certain embodiments described herein, the engineered nucleic acid encodes an expression cassette comprising a promoter and an exogenous polynucleotide sequence encoding a membrane-cleavable chimeric protein having the protein of interest (e.g., any of the effector molecules described herein). The promoter is operably ligated to the exogenous polynucleotide sequence, and the membrane-cleavable chimeric protein is configured to be expressed as a single polypeptide.

[0117] "Engineered nucleic acids" are nucleic acids that do not occur naturally. However, it should be understood that while engineered nucleic acids are unnatural as a whole, they may contain naturally occurring nucleotide sequences. In some embodiments, engineered nucleic acids contain nucleotide sequences from different organisms (e.g., different species). For example, in some embodiments, engineered nucleic acids include mouse nucleotide sequences, bacterial nucleotide sequences, human nucleotide sequences, and / or viral nucleotide sequences. The term "engineered nucleic acids" includes recombinant nucleic acids and synthetic nucleic acids. "Recombinant nucleic acids" refer to molecules constructed by linking nucleic acid molecules and, in some embodiments, capable of replicating in living cells. "Synthetic nucleic acids" refer to molecules that are amplified or synthesized chemically or by other means. Synthetic nucleic acids include those that are chemically modified or otherwise modified, but can base-pair with natural nucleic acid molecules. Modifications include, but are not limited to, one or more modified nucleotide linkages and unnatural nucleic acids. The modifications are described in further detail in U.S. Patent No. 6,673,611 and U.S. Patent Application Publication 2004 / 0019001, each of which is incorporated herein by reference in whole. The modified internucleotide links may be phosphorodithioates or phosphorothioate links. Non-natural nucleic acids may be locked nucleic acids (LNAs), peptide nucleic acids (PNAs), glycol nucleic acids (GNAs), phosphorodiamidate morpholino oligomers (PMOs or "morpholino"), and threose nucleic acids (TNAs). Non-natural nucleic acids are described in further detail in International Application WO 1998 / 039352, U.S. Patent Application Publication 2013 / 0156849, and U.S. Patents 6,670,461; 5,539,082; 5,185,444, each of which is incorporated herein by reference in whole. Recombinant nucleic acids and synthetic nucleic acids also include molecules resulting from any of the aforementioned replications. The engineered nucleic acids in this disclosure may be encoded by a single molecule (e.g., contained within the same plasmid or other vector) or by multiple different molecules (e.g., multiple different, independently replicating molecules). The engineered nucleic acids may be isolated nucleic acids.Isolated nucleic acids include, but are not limited to, cDNA polynucleotides, RNA polynucleotides, RNAi oligonucleotides (e.g., siRNA, miRNA, antisense oligonucleotides, shRNA, etc.), mRNA polynucleotides, circular plasmids, linear DNA fragments, vectors, minicircles, ssDNA, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and oligonucleotides.

[0118] The engineered nucleic acids described herein can be produced using standard molecular biology methods (e.g., see 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 (e.g., see Gibson, D. Get al., Nature Methods, 343-345, 2009; and Gibson, D. Get al., Nature Methods, 901-903, 2010, each of which is incorporated herein by reference). GIBSON ASSEMBLY® typically uses three enzymatic activities in a single-tube reaction: 5' exonuclease, γ-extension activity of DNA polymerase, and DNA ligase activity. The 5' exonuclease activity bites back the 5' end sequence, exposing the complementary sequence for annealing. The polymerase activity then fills the gap on the annealed region. The DNA ligase then seals the nick, covalently linking 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 percentage of correct assembly. In some embodiments, the engineered nucleic acid constructs are produced using IN-FUSION® cloning (Clontech).

[0119] promoter Generally, in all embodiments described herein, the engineered nucleic acid encoding one or more membrane-cleavable chimeric proteins encodes an expression cassette including a promoter. In some embodiments, the engineered nucleic acid (e.g., engineered nucleic acid including an expression cassette) includes a promoter operably ligated to nucleotide sequences (e.g., exogenous polynucleotide sequences) encoding at least two different proteins. For example, the engineered nucleic acid may include a promoter operably ligated to nucleotide sequences 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 acid includes a promoter operably ligated to nucleotide sequences encoding one, two, three, four, five, six, seven, eight, nine, ten, or more different proteins. In some embodiments, the engineered nucleic acid (e.g., engineered nucleic acid including an expression cassette) includes a promoter operably ligated to nucleotide sequences (e.g., exogenous polynucleotide sequences) encoding at least two membrane-cleavable chimeric proteins. For example, the engineered nucleic acid may include a promoter operably ligated to nucleotide sequences 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 membrane-cleavable chimeric proteins. In some embodiments, the engineered nucleic acid includes a promoter operably ligated to nucleotide sequences encoding one, two, three, four, five, six, seven, eight, nine, ten, or more membrane-cleavable chimeric proteins.

[0120] A “promoter” refers to a regulatory region of a nucleic acid sequence that controls the initiation and rate of the rest of the transcription of that sequence. A promoter may also contain a small region to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can be bound. A promoter may be constitutive, inductive, repressive, tissue-specific, or any combination thereof. A promoter drives the expression or transcription of the nucleic acid sequence it regulates. In this specification, a promoter is considered to be “operably linked” to control (drive) the transcription initiation and / or expression of the nucleic acid sequence it regulates, provided that it is in the correct functional location and orientation relative to that sequence.

[0121] A promoter may be one that is naturally associated with a gene or sequence, such that it may be obtained by isolating a 5' non-coding sequence located upstream of the coding segment of a given gene or sequence. Such a promoter may be referred to as “endogenous.” In some embodiments, the coding nucleic acid sequence may be placed under the control of a recombinant or heterologous promoter, which refers to a promoter that is not typically 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 “native,” such as those containing different elements and / or mutations of different transcriptional regulatory regions that modify expression through genetic engineering methods known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, sequences may be produced using nucleic acid amplification techniques, including recombinant cloning and / or polymerase chain reaction (PCR) (see, e.g., U.S. Patents 4,683,202 and 5,928,906).

[0122] The promoter of an engineered nucleic acid may be an “inducible promoter,” which refers to a promoter characterized by modulating (e.g., initiating or activating) transcriptional activity in the presence of a signal, when influenced by or contacted by a signal. This signal may be an endogenous or usually exogenous state (e.g., light), a compound (e.g., a chemical or non-chemical compound), or a protein (e.g., a cytokine) that contacts the inducible promoter in a manner that is active in modulating transcriptional activity from the inducible promoter. Activation of transcription may include directly acting on the promoter to drive transcription, or indirectly acting on the promoter by inactivating a repressor that prevents the promoter from driving transcription. Conversely, inactivation of transcription may include directly acting on the promoter to prevent transcription, or indirectly acting on the promoter by activating a repressor that then acts on the promoter.

[0123] A promoter is “responsive” to or “modulated” by a signal if, in the presence of a local tumor condition (e.g., inflammation or hypoxia), or any such condition or signal, transcription from the promoter is activated, inactivated, increased, or decreased. In some embodiments, the promoter includes a response element, which is a short sequence of DNA within the promoter region that binds from the promoter to a specific molecule (e.g., a transcription factor) that modulates (regulates) gene expression. Response elements that may be used in accordance with this disclosure include, but are not limited to, the phloretin-adjustable 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 (IRE) (Han, K. 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: This includes 9503-9509 (as incorporated herein by reference). Other response elements are incorporated herein. Response elements may also include 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 homozygous molecules. Tandem repeats may be labeled as 2×, 3×, 4×, 5×, etc., to indicate the number of repeats present.

[0124] Table 5A lists non-limiting examples of responsive promoters (also referred to as "inducible promoters"), such as the TGF-beta responsive promoter, which illustrate the promoter and transcription factor design, as well as the effects of the inducer molecule toward the transcription factor (TF) and transgene transcription (T) (B, binding; D, dissociation; nd, undetermined) (A, activation; DA, inactivation; DR, repression) (see Horner, M. & Weber, W. FEBS Letters 586 (2012) 20784-2096m and the references cited therein). Other non-limiting examples of components of inducible promoters include those presented in Table 5B.

[0125] (Table 5A) Examples of responsive promoters TIFF0007865603000033.tif222159TIFF0007865603000034.tif185159

[0126] (Table 5B) Exemplary components of an inducible promoter TIFF0007865603000035.tif139159

[0127] Other non-exclusive 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 the modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer), the phosphoglycerate kinase (PGK) promoter, the splenic fociforming virus (SFFV) promoter, the Simian virus 40 (SV40) promoter, and the ubiquitin C (UbC) promoter (see Table 5C).

[0128] (Table 5C) Exemplary constitutive promoters TIFF0007865603000036.tif42159TIFF0007865603000037.tif245159TIFF0007865603000038.tif245159TIFF0007865603000039.tif245159TIFF0007865603000040.tif245159TIFF0007865603000041.tif170159

[0129] The promoter may be a tissue-specific promoter. Generally, tissue-specific promoters direct the transcription of nucleic acids (e.g., modified nucleic acids encoding chimeric proteins, such as membrane-cleavable chimeric proteins with formulas SC-MT or MT-CS) so that their expression is restricted to a specific cell type, organelle, or tissue. Tissue-specific promoters include, but are not limited to, albumin (liver-specific, Pinkert et al., (1987)), lymphocyte-specific promoters (Calame and Eaton, 1988), specific promoters for T cell receptors (Winoto and Baltimore, (1989)), and immunoglobulins (Banerji et al., (1983); Queen and Baltimore, 1983), neuron-specific promoters (e.g., nerve filament promoter; Byrne and Ruddle, 1989), pancreas-specific promoters (Edlund et al., (1985)), or mammary gland-specific promoters (milky white promoter, U.S. Patent No. 4,873,316 and European Patent Publication No. 264,166), as well as developmentally regulated promoters, such as the mouse hock promoter (Kessel and Gruss, Science 249:374-379 (1990)), or α-fetoprotein promoters (Campes and This includes Tilghman, Genes Dev. 3:537-546 (1989), and the contents of each of these are fully incorporated herein by reference. Promoters may be constitutive in each specific cell type, organelle, or tissue.Tissue-specific promoters and / or regulatory elements may also include the hepatic fatty acid-binding (FAB) protein gene specific to colon epithelial cells; the insulin gene specific to pancreatic cells; the transfiletin, alpha-1-antitrypsin, plasminogen activator inhibitor type 1 (PAI-I), apolipoprotein AI, and LDL receptor genes specific to hepatocytes; the myelin basic protein (MBP) gene specific to oligodendrocytes; the glial fibrillary acidic protein (GFAP) gene specific to glial cells; a promoter from OPSIN specific for targeting the eye; and a neuron-specific enolase (NSE) promoter specific to nerve cells. Examples of tissue-specific promoters, but not limited to, include the promoter for creatine kinase, which has been used to direct immunoglobulin heavy chain or light chain promoters for expression in muscle and cardiac tissue as well as 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 L7-alpha hydroylase (CYP-7) promoter, beta-galactosidase alpha-2,6 sialylcansferase 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 prostatic secretion protein 94 (SPS 94) promoter, the prostate-specific antigen complex promoter, and the human glandular kallikrein gene promoter (hgt-1). Exemplary tissue-specific expression elements for gastric tissue include, but are not limited to, the human H+ / K+-ATPase alpha subunit promoter.Exemplary tissue-specific expression elements for the pancreas include, but are not limited to, the pancreatitis-associated protein promoter (PAP), the elastase 1 transcription enhancer, the pancreas-specific amylase and elastase enhancer promoters, and the 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 neuronal filament 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) promoter, the human CD2 promoter and its 3' transcription enhancer, and the human NK and T cell-specific activation (NKG5) promoter. Exemplary tissue-specific expression elements for the colon include, but are not limited to, the pp60c-src tyrosine kinase promoter, the organ-specific neoantigen (OSN) promoter, and the colon-specific antigen-P promoter. An example of a tissue-specific expression element for mammary cells is, for example, the human alpha-lactalbumin promoter. Exemplary tissue-specific expression elements for the lungs include, but are not limited to, the cystic fibrosis transmembrane conductance regulator (CFTR) gene promoter.

[0130] In some embodiments, the promoter of this disclosure is modulated by signals within the tumor microenvironment. The tumor microenvironment is thought to modulate the promoter if, in the presence of the tumor microenvironment, the promoter activity increases or decreases by at least 10% compared to the promoter activity in the absence of the tumor microenvironment. In some embodiments, the promoter activity increases or decreases by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to the promoter activity in the absence of the tumor microenvironment. For example, promoter activity increases or decreases 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 promoter activity in the absence of the tumor microenvironment.

[0131] In some embodiments, promoter activity is increased or decreased by at least twofold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100 times) compared to promoter activity in the absence of the tumor microenvironment. For example, promoter activity is increased or decreased by at least threefold, at least fivefold, at least tenfold, at least twentyfold, at least fiftyfold, or at least 100 times compared to promoter activity in the absence of the tumor microenvironment. In some embodiments, promoter activity 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 times compared to promoter activity in the absence of the tumor microenvironment.

[0132] In some embodiments, the promoters of this 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 cause inflammation (e.g., levels of inflammatory cytokines increase under hypoxia). In some embodiments, the promoter activated under hypoxia is operably linked to a nucleotide encoding a chimeric protein that reduces the expression of inflammatory cytokine activity, thus reducing inflammation caused by hypoxia. In some embodiments, the promoter activated under hypoxia includes a hypoxia-responsive element (HRE). A "Hypoxia-responsive element (HRE)" is a response element that responds to hypoxia-inducible factors (HIF). In some embodiments, the HRE includes a consensus motif NCGTG (where N is either A or G).

[0133] Activated Conditionally Regulated 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 a system is also referred to herein as an "ACP-responsive promoter." Generally, an ACP promoter system includes a first expression cassette encoding an activation-conditional control polypeptide (ACP) and a second expression cassette encoding an ACP-responsive promoter operably ligated to an exogenous polynucleotide sequence, such as a membrane-cleavable chimeric protein as described herein or any other protein of interest (e.g., a protease). In some embodiments, the first and second expression cassettes are each encoded by separate engineered nucleic acids. In other embodiments, the first and second expression cassettes are encoded by the same engineered nucleic acid. The ACP-responsive promoter can be operably ligated to a nucleotide sequence encoding a single protein or multiple proteins of interest.

[0134] The promoter of the ACP promoter system, for example, a promoter that drives ACP expression, or a promoter sequence of an ACP-responsive promoter may include any of the promoter sequences described herein (see “Promoter” above). The ACP-responsive promoter may be derived from minP, NFκB 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-responsive element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducer molecule-responsive promoter, and their tandem repeats. In some embodiments, the ACP-responsive promoter includes a minimal promoter.

[0135] In some embodiments, the ACP-binding domain includes one or more zinc finger binding sites. In some embodiments, the ACP-responsive promoter includes a minimal promoter, and the ACP-binding domain includes one or more zinc finger binding sites. The ACP-binding domain may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more zinc finger binding sites. In some embodiments, the transcription factor is a zinc finger-containing transcription factor. In some embodiments, the zinc finger-containing transcription factor is a synthetic transcription factor. In some embodiments, the ACP-binding domain includes one or more zinc finger binding sites, and the ACP has a DNA-binding zinc finger protein domain (ZF protein domain). In some embodiments, the ACP includes a DNA-binding zinc finger protein domain (ZF protein domain) and an effector domain. In some embodiments, the ACP-binding domain includes one or more zinc finger binding sites, and the ACP has a DNA-binding zinc finger protein domain (ZF protein domain) and an effector domain. In some embodiments, the ZF protein domain is a modular design and consists of a zinc finger array (ZFA). The zinc finger array contains multiple zinc finger protein motifs linked together. Each zinc finger motif binds to a different nucleic acid motif. This results in a ZFA with specificity to any desired nucleic acid sequence, for example, a ZFA with desired specificity to an ACP-binding domain having a specific zinc finger binding site composition and / or configuration. The ZF motifs may be directly adjacent to each other or separated by a mobile linker sequence. In some embodiments, the ZFA is an array, string, or chain of ZF motifs arranged in tandem. The ZFA may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 zinc finger motifs.ZFAs may have zinc finger motifs of 1-10, 1-15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, or 5-15. ZF protein domains may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more ZFAs. A ZF domain may have ZFAs 1-10, 1-15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, or 5-15. In some embodiments, the ZF protein domain contains 1-10 ZFAs. In some embodiments, the ZF protein domain contains at least one ZFA. In some embodiments, the ZF protein domain contains at least two ZFAs. In some embodiments, the ZF protein domain contains at least three ZFAs. In some embodiments, the ZF protein domain contains at least four ZFAs. In some embodiments, the ZF protein domain contains at least five ZFAs. In some embodiments, the ZF protein domain contains at least ten ZFAs.

[0136] In some embodiments, ACP is a transcription modulator. In some embodiments, ACP is a transcription repressor. In some embodiments, ACP is a transcription activator. In some embodiments, ACP is a transcription factor. In some embodiments, ACP comprises a DNA-binding domain and a transcription effector domain. In some embodiments, the DNA-binding domain comprises a tetracycline (or derivative thereof) repressor (TetR) domain. In some embodiments, ACP is the antigen-recognition receptor of this disclosure.

[0137] ACP may also further include effector domains, such as transcription effector domains. For example, a transcription effector domain may be the effector or activator domain of a transcription factor. Transcription factor activation domains are also known as transactivation domains and act as skeletal domains of proteins, such as transcription coregulators, that act to activate or repress gene transcription. Any suitable transcription effector domain may be used in ACP, but is not limited to: the herpes simplex virus protein 16 (VP16) activation domain; the VP64 activation domain, which consists of four tandem copies of VP16; the p65 activation domain of NFκB; the Epstein-Barr virus R transactivator (Rta) activation domain; the tripartite activator containing 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-related protein p300 (known as the p300HAT core activation domain); the Kruppel-related box (KRAB) repressor domain; the repressor element silencing transcription factor (REST) ​​repressor domain; and the WRPW of the Hairy-related basic helix-loop-helix repressor protein. (Sequence ID 224) Motif (This motif is WRPW) (Sequence ID 224) The domains include the following, which are known as repressive domains: the DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repressive domain; and the HP1 alpha-chromoshadow repressive domain, or any combination thereof.

[0138] In some embodiments, the effector domain is a transcription effector domain selected from the following: herpes simplex virus protein 16 (VP16) activation domain; VP64 activation domain, which is an activation domain consisting of four tandem copies of VP16; p65 activation domain of NFκB; Epstein-Barr virus R transactivator (Rta) activation domain; tripartite activator containing VP64, p65, and Rta activation domains (the tripartite activator is known as the VPR activation domain); histone acetyltransferase (HAT) core domain of human E1A-related protein p300 (known as the p300HAT core activation domain); Kruppel-related box (KRAB) repressor domain; repressor element silencing transcription factor (REST) ​​repressor domain; WRPW of Hairy-related basic helix-loop-helix repressor protein. (Sequence ID 224) Motif (This motif is WRPW) (Sequence ID 224) These are known repression domains: the DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain and the HP1 alpha-chromoshadow repression domain.

[0139] In some embodiments, ACP is a small molecule (e.g., drug)-inducible polypeptide. For example, in some embodiments, ACP may be induced by tetracycline (or its derivatives) and include a TetR domain and a VP16 effector domain. In some embodiments, ACP includes an estrogen receptor variant, such as ERT2, and may be modulated by tamoxifen or its metabolites (e.g., 4-hydroxy-tamoxifen [4-OHT], N-desmethyltamoxifen, tamoxifen-N-oxide, or endoxifen) through tamoxifen-regulated nuclear localization.

[0140] In some embodiments, ACP is a small molecule (e.g., drug)-inducible polypeptide containing an inhibitory protease and one or more homogeneous cleavage sites of the inhibitory protease. In some embodiments, the inhibitory protease is active in the absence of a specific drug (cleaving the homogeneous cleavage site) and inactive in the presence of a specific drug (not cleaving the homogeneous cleavage site). In some embodiments, the specific drug is a protease inhibitor. In some embodiments, the protease inhibitor specifically inhibits a given inhibitory protease of this disclosure. The inhibitory protease may be any of the proteases described herein that can be inactivated in the presence or absence of a specific drug (see “Protease Cleavage Sites” above for exemplary inhibitory proteases, homogeneous cleavage sites, and protease inhibitors).

[0141] In some embodiments, ACP has a degron domain (see "Degron Systems and Domains" above for exemplary degron sequences). The degron domain may be in any order or position relative to the individual domains of ACP. For example, the degron domain may be at the N-terminus of an inhibitory protease, the C-terminus of an inhibitory protease, the N-terminus of a ZF protein domain, the C-terminus of a ZF protein domain, the N-terminus of an effector domain, or the C-terminus of an effector domain.

[0142] Multi-cistron systems and multiple promoter systems In some embodiments, the engineered nucleic acid (e.g., engineered nucleic acid including an expression cassette) is configured to produce multiple chimeric proteins. For example, the nucleic acid may be configured to produce 2 to 20 different chimeric proteins.In some embodiments, nucleic acids are 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 It is configured to produce chimeric proteins of 9, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, or 19-20.In some embodiments, the nucleic acid is configured to produce chimeric proteins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0143] In some embodiments, the engineered nucleic acid may be multicistronic, i.e., two or more distinct polypeptides (e.g., multiple chimeric proteins) may 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 may be linked to a nucleotide sequence encoding a second chimeric protein (e.g., first gene:linker:second gene, 5' to 3' direction). The linker may encode a 2A ribosome skipping element, e.g., T2A. Other 2A ribosome skipping elements include, but are not limited to, E2A, P2A, and F2A. The 2A ribosome skipping element enables the production of distinct polypeptides encoded by the first and second genes during translation. The linker may encode a cleavable linker polypeptide sequence, e.g., a Fulin cleavage site or a TEV cleavage site, where, following expression, the cleavable linker polypeptide is cleaved to produce distinct polypeptides encoded by the first and second genes. The cleavable linker may include polypeptide sequences that further facilitate cleavage, such as mobile linkers (e.g., Gly-Ser-Gly sequences).

[0144] Linkers can encode intra-sequence ribosome entry sites (IRESs), ensuring that separate polypeptides encoded by the first and second genes are produced during translation. Linkers can also encode splice acceptors, such as viral splice acceptors.

[0145] The linker may be a combination of linkers, such as a furin-2A linker, capable of producing separate polypeptides through 2A ribosome skipping, followed by further cleavage of the furin site allowing for complete removal of the 2A residue. In some embodiments, the linker combination may include a furin sequence, a mobile 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 this disclosure is a furin-Gly-Ser-Gly-T2A fusion polypeptide.

[0146] Generally, a multicistron system can express any number of genes or parts thereof using any number or combination of linkers (e.g., the engineered nucleic acid can encode first, second, and third chimeric proteins, each separated by a linker so that separate polypeptides are encoded by the first, second, and third chimeric proteins).

[0147] The engineered nucleic acid can express genes from multiple ORFs using multiple promoters, that is, two or more distinct mRNA transcripts can be produced from a single engineered nucleic acid. For example, a first promoter can be operably ligated to a polynucleotide sequence encoding a first chimeric protein, and a second promoter can be operably ligated to a polynucleotide sequence encoding a second chimeric protein. In general, any number of chimeric proteins can be expressed using any number of promoters. In some embodiments, at least one of the ORFs expressed from multiple promoters may be multicistronic.

[0148] As used herein, "linker" may refer to a polypeptide that links a first polypeptide sequence and a second polypeptide sequence, the multi-cistron linker described above, or an additional promoter operably linked to the additional ORF described above.

[0149] Manipulated cells Provided herein are engineered cells that produce membrane-cleavable chimeric proteins, and methods for producing engineered cells. Generally, engineered cells of the Disclosure may be engineered to express the chimeric proteins provided herein, for example, membrane-cleavable chimeric proteins having the formulas SC-MT or MT-CS as described herein. These cells are referred to herein as “engineered cells.” These cells typically contain engineered nucleic acids that do not occur naturally. In some embodiments, cells are engineered to contain a chimeric protein, for example, a nucleic acid containing a promoter operably ligated to a nucleotide sequence encoding a membrane-cleavable chimeric protein. Engineered cells of the Disclosure may contain engineered nucleic acids that are incorporated into the cell’s genome. Engineered cells may also contain engineered nucleic acids that can be expressed without being incorporated into the cell’s genome, for example, modified by a transient expression system, such as a plasmid or mRNA.

[0150] This disclosure also encompasses the additive and synergistic relationships between chimeric proteins and the engineered cells from which they are produced. In some embodiments, cells are engineered to produce at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) chimeric proteins, for example, at least two membrane-cleavable chimeric proteins. In other embodiments, cells are engineered to produce at least one chimeric protein having an effector molecule not naturally produced by the cell. Such an effector molecule may, for example, complement the function of an effector molecule naturally produced by the cell.

[0151] In some embodiments, cells are engineered to express the extracellular domain of an anti-CD3 and / or anti-CD28 agonist that is anchored to the membrane.

[0152] In some embodiments, cells (e.g., immune cells or stem cells) are engineered to produce multiple chimeric proteins. For example, cells may be engineered to produce 2 to 20 different chimeric proteins, such as 2 to 20 different membrane-cleavable chimeric proteins.In some embodiments, 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, 8~19, 8 ~18, 8~17, 8~16, 8~15, 8~14, 8~13, 8~12, 8~11, 8~10, 8~9, 9~20, 9~19, 9~18, 9~17, 9~16, 9~15, 9~14, 9~13, 9~12, 9~11, 9~10, 10~20, 10~19, 10~18, 10~17, 10~16, 10~15, 10~14, 10~13, 10~12, 10~11, 11~20, 11~19, 11~18, 11~17, 11~16, 11~15, 11~14, 11~13, 11~12, 12~20, 12~19, Cells engineered to produce chimeric proteins of 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, or 19-20.In some embodiments, cells are manipulated to produce chimeric proteins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0153] In some embodiments, the engineered cell contains one or more engineered nucleic acids encoding a promoter operably ligated to a nucleotide sequence encoding a chimeric protein. In some embodiments, the cell is engineered to contain multiple engineered nucleic acids, for example, at least two engineered nucleic acids, each encoding a promoter operably ligated to a nucleotide sequence encoding at least one (e.g., 1, 2, or 3) chimeric protein. For example, the cell may be engineered to contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten engineered nucleic acids, each encoding a promoter operably ligated to a nucleotide sequence encoding at least one (e.g., 1, 2, or 3) chimeric protein. In some embodiments, the cell is engineered to contain two, three, four, five, six, seven, eight, nine, ten, or more engineered nucleic acids, each encoding a promoter operably ligated to a nucleotide sequence encoding at least one (e.g., 1, 2, or 3) chimeric protein. The manipulated cell may include a manipulated nucleic acid encoding at least one of the linkers described above, for example, a polypeptide linking a first polypeptide sequence to a second polypeptide sequence, one or more multicistron linkers described above, one or more additional promoters operably linked to an additional ORF, or a combination thereof.

[0154] In some embodiments, cells (e.g., immune cells or stem cells) are engineered to express a protease. In some embodiments, cells are engineered to express a heterologous protease. In some embodiments, cells are engineered to express a heterologous protease, such as a heterologous protease that cleaves the protease cleavage site of a membrane-cleavable chimeric protein, in addition to cells that express a chimeric protein. In some embodiments, the engineered cells contain one or more engineered nucleic acids encoding a promoter operably ligated to a nucleotide sequence encoding a protease, such as a heterologous protease. Proteases and protease cleavage sites are described in more detail in the section titled “Protease Cleavage Sites” herein.

[0155] In some embodiments, cells (e.g., immune cells or stem cells) are engineered to produce at least one homing molecule. "Homing" refers to the active navigation (migration) of a cell to a target site (e.g., a cell, a tissue (e.g., a tumor), or an organ). "Homing molecule" refers to a molecule that directs a cell toward the target site. In some embodiments, the homing molecule functions to recognize and / or initiate an interaction between the engineered cell and the target site. Non-exclusive examples of homing molecules include CXCR1, CCR9, CXCR2, CXCR3, CXCR4, CCR2, CCR4, FPR2, VEGFR, IL6R, CXCR1, CSCR7, and PDGFR.

[0156] In some embodiments, the homing molecule is a chemokine receptor (a cell surface molecule that binds to a chemokine). Non-limiting examples of chemokine receptors that may be produced by the manipulated cells of this disclosure include: CXC chemokine receptors (e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7), CC chemokine receptors (CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, and CCR11), CX3C chemokine receptors (e.g., CX3CR1 that binds to CX3CL1), and XC 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, cells are engineered to produce CXCL8, CXCL9, and / or CXCL10 (which promotes T cell recruitment), CCL3 and / or CXCL5, and CCL21 (which promote Th1 recruitment and polarization). In some embodiments, cells are engineered to produce CXCR4.

[0157] In some embodiments, cells (e.g., immune cells or stem cells) are engineered to produce G protein-coupled receptors (GPCRs) that detect N-formylated oligopeptides (including, but not limited to, FPR2 and FPRL1).

[0158] In some embodiments, cells (e.g., immune cells or stem cells) are engineered to produce receptors that detect interleukins (including, but not limited to, IL6R).

[0159] In some embodiments, cells (e.g., immune cells or stem cells) are engineered to produce receptors (including, but not limited to, FGFR, PDGFR, EGFR, and VEGF family receptors, including, but not limited to, VEGF-C and VEGF-D) that detect growth factors secreted from other cells, tissues, or tumors.

[0160] In some embodiments, cells (e.g., immune cells or stem cells) are engineered to produce one or more integrins. The cells of the Disclosure may be engineered to produce any combination of integrin α and β subunits. The α subunit of an integrin may, but are not limited to, ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, IGTA7, ITGA8, ITGA9, IGTA10, IGTA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGA2B, ITGAX. The β subunit of an integrin may, but are not limited to, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, and ITGB8.

[0161] In some embodiments, cells (e.g., immune cells or stem cells) are engineered to produce one or more matrix metalloproteinases (MMPs). Non-limiting examples of MMPs include MMP-2, MMP-9, and MMP-1. In some embodiments, cells are engineered to produce inhibitors of molecules (e.g., proteins) that inhibit MMPs. For example, cells may be engineered to express inhibitors of membrane type 1 MMPs (MT1-MMPs) (e.g., RNAi molecules) or TIMP metallopeptidase inhibitor 1 (TIMP-1).

[0162] In some embodiments, cells (e.g., immune cells or stem cells) are engineered to produce ligands that bind to selectins (e.g., hematopoietic cell E- / L-selectin ligand (HCELL), Dykstran et al., Stem Cells. 2016 Oct;34(10):2501-2511) on the endothelium of a target tissue, for example.

[0163] The term "homing molecules" also encompasses transcription factors that regulate the production of molecules that improve / enhance cell homing.

[0164] Furthermore, provided herein are cells engineered to produce multiple chimeric proteins, at least two of which modulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, at least one (e.g., one, two, three, four, five or more) chimeric protein comprises an effector molecule that stimulates at least one immunostimulatory mechanism in the tumor microenvironment or inhibits at least one immunosuppressive mechanism in the tumor microenvironment. In some embodiments, at least one (e.g., one, two, three, four, five or more) chimeric protein comprises an effector molecule that inhibits at least one immunosuppressive mechanism in the tumor microenvironment, and at least one chimeric protein (e.g., one, two, three, four, five or more) inhibits at least one immunosuppressive mechanism in the tumor microenvironment. In yet other embodiments, at least two (e.g., two, three, four, five or more) chimeric proteins comprise an effector molecule that stimulates at least one immunostimulatory mechanism in the tumor microenvironment. In yet another embodiment, at least two (e.g., one, two, three, four, five or more) chimeric proteins include effector molecules that inhibit at least one immunosuppressive mechanism in the tumor microenvironment.

[0165] In some embodiments, cells (e.g., immune cells or stem cells) are engineered to produce at least one chimeric protein containing effector molecules that stimulate T cell signaling, activity, and / or recruitment. In some embodiments, cells are engineered to produce at least one chimeric protein containing effector molecules that stimulate antigen presentation and / or processing. In some embodiments, cells are engineered to produce at least one chimeric protein containing effector molecules that stimulate natural killer cell-mediated cytotoxic signaling, activity, and / or recruitment. In some embodiments, cells are engineered to produce at least one chimeric protein containing effector molecules that stimulate dendritic cell differentiation and / or maturation. In some embodiments, cells are engineered to produce at least one chimeric protein containing effector molecules that stimulate immune cell recruitment. In some embodiments, cells are engineered to produce at least one chimeric protein containing effector molecules that stimulate M1 macrophage signaling, activity, and / or recruitment. In some embodiments, cells are engineered to produce at least one chimeric protein containing an effector molecule that stimulates Th1 polarization. In some embodiments, cells are engineered to produce at least one chimeric protein containing an effector molecule that stimulates stromal degradation. In some embodiments, cells are engineered to produce at least one chimeric protein containing an effector molecule that stimulates immunostimulatory metabolite production. In some embodiments, cells are engineered to produce at least one chimeric protein containing an effector molecule that stimulates type I interferon signaling. In some embodiments, cells are engineered to produce at least one chimeric protein containing an effector molecule that inhibits negative co-stimulatory signaling. In some embodiments, cells are engineered to produce at least one chimeric protein containing an effector molecule that inhibits pro-apoptotic signaling (e.g., via TRAIL) in anti-tumor immune cells. In some embodiments, cells are engineered to produce T-regulating (T reg) Cells are engineered to produce at least one chimeric protein containing an effector molecule that inhibits cell signaling, activity, and / or recruitment. In some embodiments, cells are engineered to produce at least one chimeric protein containing an effector molecule that inhibits tumor checkpoint molecules. In some embodiments, cells are engineered to produce at least one chimeric protein containing an effector molecule that activates interferon gene stimulator (STING) signaling. In some embodiments, cells are engineered to produce at least one chimeric protein containing an effector molecule that inhibits bone marrow-derived suppressor cell signaling, activity, and / or recruitment. In some embodiments, cells are engineered to produce at least one chimeric protein containing an effector molecule that degrades immunosuppressive factors / metabolites. In some embodiments, cells are engineered to produce at least one chimeric protein containing an effector molecule that inhibits vascular endothelial growth factor signaling. In some embodiments, cells are engineered to produce at least one chimeric protein containing effector molecules that directly kill tumor cells (e.g., granzymes, perforins, oncolytic viruses, cytolytic peptides, and enzymes, such as antitumor antibodies that induce ADCC).

[0166] In some embodiments, at least one chimeric protein comprises effector molecules that stimulate 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; macrophage signaling; stromal degradation; immunostimulatory metabolite production; or type I interferon signaling; and at least one chimeric protein The proteins include effector molecules that inhibit negative co-stimulatory signaling, inhibit pro-apoptotic signaling of anti-tumor immune cells, inhibit regulatory T (Treg) cell signaling, activity, and / or recruitment, inhibit tumor checkpoint molecules, activate interferon gene stimulator (STING) signaling, inhibit bone marrow-derived suppressor cell signaling, activity, and / or recruitment, degrade immunosuppressive factors / metabolites, inhibit vascular endothelial growth factor signaling, or directly kill tumor cells.

[0167] In some embodiments, cells (e.g., immune cells or stem cells) are manipulated to produce at least one chimeric protein containing an effector molecule selected from IL-12, IFN-β, IFN-γ, IL-2, IL-15, IL-7, IL-36γ, IL-18, IL-1β, OX40 ligand, and CD40L. Exemplary immune checkpoint molecules that can be targeted for blockade or inhibition include, but are not limited to, CTLA-4, 4-1BB(CD137), 4-1BBL(CD137L), PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4 (belonging to the CD2 family of molecules and expressed on all NK, γδT, and memory CD8+(αβ)T cells), CD160 (also referred to as BY55), and CGEN-15049. Immune checkpoint inhibitors include antibodies, antigen-binding fragments thereof, or other binding proteins that bind to one or more of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4, CD160, and CGEN-15049 to block or inhibit their activity. Exemplary checkpoint inhibitors include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-LAG-3 antibodies, anti-TIM-3 antibodies, anti-TIGIT antibodies, anti-VISTA antibodies, anti-KIR antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-HVEM antibodies, anti-BTLA antibodies, anti-GAL9 antibodies, anti-A2AR antibodies, anti-phosphatidylserine antibodies, anti-CD27 antibodies, anti-TNFα antibodies, anti-TREM1 antibodies, and anti-TREM2 antibodies.Exemplary immune checkpoint inhibitors include pembrolizumab (anti-PD-1; MK-3475 / Keytruda®- Merck), nivolumab (anti-PD-1; Opdivo®- BMS), pidilizumab (anti-PD-1 antibody; CT-011- Teva / CureTech), AMP224 (anti-PD-1; NCI), avelumab (anti-PD-L1; Bavencio®- Pfizer), durvalumab (anti-PD-L1; MEDI4736 / Imfinzi®- Medimmune / AstraZeneca), atezolizumab (anti-PD-L1; Tecentriq®- Roche / Genentech), and BMS-936559 (anti-PD-L1 - This includes BMS, tremelimumab (anti-CTLA-4; Medimmune / AstraZeneca), ipilimumab (anti-CTLA-4; Yervoy®-BMS), lirirumab (anti-KIR; BMS), and monalizumab (anti-NKG2A; Innate Pharma / AstraZeneca).

[0168] In some embodiments, cells (e.g., immune cells or stem cells) are manipulated to produce at least one chimeric protein comprising an effector molecule selected from IL-12, IFN-β, IFN-γ, IL-2, IL-15, IL-7, IL-36γ, IL-18, IL-1β, OX40 ligand, and CD40L; and / or at least one checkpoint inhibitor selected from anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, and anti-IL-35 antibody; and / or at least one chimeric protein comprising an effector molecule selected from MIP1α(CCL3), MIP1β(CCL5), and CCL21; and / or at least one chimeric protein comprising an effector molecule selected from CpG oligodeoxyribonucleotides; and / or at least one chimeric protein comprising an effector molecule selected from microbial peptides.

[0169] In some embodiments, cells (e.g., immune cells or stem cells) are engineered to produce at least one chimeric protein comprising IFN-β and an effector molecule selected from cytokines, antibodies, chemokines, nucleotides, peptides, enzymes, and interferon gene stimulants (STINGs). In some embodiments, cells are engineered to produce IFN-β and at least one cytokine or receptor / ligand (e.g., IL-12, IFN-γ, IL-2, IL-15, IL-7, IL-36γ, IL-18, IL-1β, OX40-ligand, and / or CD40L).

[0170] In some embodiments, cells (e.g., immune cells or stem cells) are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule (e.g., "S" in formula SC-MT or MT-CS) is a cytokine, chemokine, homing molecule, growth factor, co-activating molecule, tumor microenvironment modifier, ligand, antibody, peptide, or enzyme. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is a cytokine. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is a chemokine. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is a homing molecule. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is a growth factor. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is a co-activating molecule. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is a tumor microenvironment modifier. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is a ligand. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is an antibody. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is a peptide. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is an enzyme.

[0171] In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule (e.g., "S" in formula SC-MT or MT-CS) is IL-1-beta, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-12p70 fusion protein, IL-15, IL-17A, IL-18, IL-21, IL-22, type I interferon, or TNF-alpha. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-CXCL11 fusion protein, CCL19, CXCL9, or XCL1. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is anti-integrin alpha 4, beta 7, anti-MAdCAM, SDF1, or MMP-2. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is 4-1BBL or CD40L. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is adenosine deaminase, a TGF beta inhibitor, an immune checkpoint inhibitor, a VEGF inhibitor, or HPGE2. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is anti-TGF beta peptide, anti-TGF beta antibody, TGFb-TRAP, or a combination thereof. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is an anti-VEGF antibody, an anti-VEGF peptide, or a combination thereof.In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is an anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIM-3 antibody, anti-TIGIT antibody, anti-VISTA antibody, anti-KIR antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-GAL9 antibody, anti-A2AR antibody, anti-phosphatidylserine antibody, anti-CD27 antibody, anti-TNFa antibody, anti-TREM1 antibody, or anti-TREM2 antibody.

[0172] In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule (e.g., "S" in formula SC-MT or MT-CS) comprises IL-15. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is a fusion of IL-15 and the sushi domain of IL-15Rα. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector consists of IL-15. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered to produce one or more additional effector molecules. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered to produce one or more additional secreted effector molecules. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered to produce cytokines, chemokines, homing molecules, growth factors, coactivating molecules, tumor microenvironment modifiers, ligands, antibodies, polynucleotides, peptides, or enzymes. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered to produce IL-12, IFN-γ, IL-2, IL-7, IL-36γ, IL-18, IL-1β, OX40 ligand, or CD40L. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered to produce IL-12.In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered to produce IFN-γ. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered to produce IL-2. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered to produce IL-7. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered to produce IL-36γ. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered to produce IL-18. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered to produce IL-1β. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered to produce OX40 ligand. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered to produce CD40L.

[0173] In some embodiments, cells are manipulated to produce at least one membrane-cleavable chimeric protein, and the cells are further manipulated to produce one or more additional membrane-cleavable chimeric proteins.

[0174] In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule (e.g., "S" in formula SC-MT or MT-CS) is IL-15, and the cells are further engineered to produce one or more additional membrane-cleavable chimeric proteins. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered for additional membrane-cleavable chimeric proteins, where the additional secreted effector molecule produces cytokines, chemokines, homing molecules, growth factors, coactivating molecules, tumor microenvironment modifiers, ligands, antibodies, polynucleotides, peptides, or enzymes. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered for an additional membrane-cleavable chimeric protein, where the additional secreted effector molecule is IL-12, IFN-γ, IL-2, IL-7, IL-36γ, IL-18, IL-1β, OX40 ligand, or CD40L. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered for an additional membrane-cleavable chimeric protein, where the additional secreted effector molecule is IL-12. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered for an additional membrane-cleavable chimeric protein, where the additional secreted effector molecule is IFN-γ. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered to produce an additional membrane-cleavable chimeric protein, where the additional secreted effector molecule is IL-2.In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered for an additional membrane-cleavable chimeric protein, where the additional secreted effector molecule is IL-7. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered for an additional membrane-cleavable chimeric protein, where the additional secreted effector molecule is IL-36γ. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered for an additional membrane-cleavable chimeric protein, where the additional secreted effector molecule is IL-18. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered for an additional membrane-cleavable chimeric protein, where the additional secreted effector molecule is IL-1β. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered for an additional membrane-cleavable chimeric protein, where the additional secreted effector molecule is OX40 ligand. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL-15, and the cells are further engineered for an additional membrane-cleavable chimeric protein, where the additional secreted effector molecule is CD40L.

[0175] Cells can also be further manipulated to express additional proteins in addition to the chimeric proteins described herein (e.g., membrane-cleavable chimeric proteins having the formulas SC-MT or MT-CS described herein), the protein of interest, or effector molecules. Cells can be further manipulated to express one or more antigen-recognition receptors. Examples of antigens that can be targeted by one or more antigen-recognizing receptors include, but are not limited to, 5T4, ADAM9, AFP, AXL, B7-H3, B7-H4, B7-H6, BCMA, C4.4, CA6, Cadherin 3, Cadherin 6, CCR4, CD19, CD20, CD22, CD123, CD133, CD138, CD142, CD166, CD25, CD30, CD33, CD352, CD37, CD38, CD44, CD56, CD66e, CD70, CD71, CD74, CD79b, CD80, CEA, CEACAM5, Claudin 18.2, cMet, CSPG4, CTLA, and DLK1. , DLL3, DR5, EGFR, ENPP3, EpCAM, EphA2, EphrinA4, ETBR, FGFR2, FGFR3, FLT3, FR alpha, FRb, GCC, GD2, GFRa4, gpA33, GPC2, GPC3, gpNBM, GPRC5, HER2, IL-13R, IL-13Ra , IL-13Ra2, IL-8, IL-15, IL1RAP, Integrin aV, KIT, L1CAM, LAMP1, Lewis Y, LeY, LIV-1, LRRC, LY6E, MCSP, Mesothelin, MUC1, MUC16, MUC1C, NaPi2B, Nectin 4, NKG2D, NOTCH3, NY Includes ESO 1, ovalin, p-cadherin, pan-Erb2, PSCA, PSMA, PTK7, ROR1, S Aures, SCT, SLAMF7, SLITRK6, SSTR2, STEAP1, Survivin, TDGF1, TIM1, TROP2, and WT1.

[0176] Antigen-recognition receptors may include an antigen-binding domain, such as an antibody, an antigen-binding fragment of an antibody, an F(ab) fragment, an F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb). Antigen-recognition receptors may include scFv. scFv may include a heavy-chain variable domain (VH) and a light-chain variable domain (VL), which can be separated by a peptide linker. For example, scFv may have the structure VH-L-VL or VL-L-VH, where VH is the heavy-chain variable domain, L is the peptide linker, and VL is the light-chain variable domain.

[0177] Antigen-recognition receptors may be chimeric antigen receptors (CARs). CARs may have one or more intracellular signaling domains, such as the CD3 zeta chain intracellular signaling domain, CD97 intracellular signaling domain, CD11a-CD18 intracellular signaling domain, CD2 intracellular signaling domain, ICOS intracellular signaling domain, CD27 intracellular signaling domain, CD154 intracellular signaling domain, CD8 intracellular signaling domain, OX40 intracellular signaling domain, 4-1BB intracellular signaling domain, CD28 intracellular signaling domain, ZAP40 intracellular signaling domain, CD30 intracellular signaling domain, GITR intracellular signaling domain, HVEM intracellular signaling domain, DAP10 intracellular signaling domain, DAP12 intracellular signaling domain, MyD88 intracellular signaling domain, fragments thereof, combinations thereof, or combinations of fragments thereof. CARs may have transmembrane domains, such as CD8 transmembrane domains, CD28 transmembrane domains, CD3 zeta chain transmembrane domains, CD4 transmembrane domains, 4-1BB transmembrane domains, OX40 transmembrane domains, ICOS transmembrane domains, CTLA-4 transmembrane domains, PD-1 transmembrane domains, LAG-3 transmembrane domains, 2B4 transmembrane domains, BTLA transmembrane domains, fragments thereof, combinations thereof, or combinations of fragments thereof. CARs may have a spacer region between the antigen-binding domain and the transmembrane domain.

[0178] Antigen-recognition receptors can be T cell receptors (TCRs).

[0179] Manipulated cell types Furthermore, what is provided herein are engineered cells. Cells may be engineered to contain any of the engineered nucleic acids described herein (e.g., any of the engineered nucleic acids encoding membrane-cleavable chimeric proteins described herein). Cells may be engineered to have any of the characteristics of any of the engineered cells described herein. In certain embodiments, what is provided herein are cells engineered to produce one or more chimeric proteins, where one or more chimeric proteins are membrane-cleavable chimeric proteins having the formula SC-MT or MT-CS as described herein. In certain embodiments, what is provided herein are cells engineered to produce two or more chimeric proteins. In certain embodiments, what is provided herein are cells engineered to produce two or more chimeric proteins as described herein, where each chimeric protein is a protein or effector molecule of a different purpose. In certain embodiments, what is provided herein are cells that have been engineered to produce any of the chimeric proteins described herein and to produce different effector molecules or proteins of interest (e.g., homing molecules, antigen receptors, etc.) separately.

[0180] The manipulated cells may include, but are not limited to, immune cells, T cells, CD8+ T cells, CD4+ T cells, gamma delta (γδ) T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, or dendritic cells. The manipulated cells may be T cells. The manipulated cells may be NK cells.

[0181] The manipulated cells may include, but are not limited to, stem cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), or iPSC-derived cells.

[0182] Manipulated cells may be tumor-derived cells. Examples of tumor cells include, but are not limited to, bladder tumor cells, brain tumor cells, breast tumor cells, cervical tumor cells, colorectal tumor cells, esophageal tumor cells, glioma cells, kidney tumor cells, liver tumor cells, lung tumor cells, melanoma cells, ovarian tumor cells, pancreatic tumor cells, prostate tumor cells, skin tumor cells, thyroid tumor cells, and uterine tumor cells.

[0183] Cells can be manipulated to produce chimeric proteins using methods known to those skilled in the art. For example, cells can be transduced to manipulate tumors. In one embodiment, cells are transduced using a virus.

[0184] In certain embodiments, cells are transduced using oncolytic viruses. Examples of oncolytic viruses include, but are not limited to, oncolytic herpes simplex virus, oncolytic adenovirus, oncolytic measles virus, oncolytic influenza virus, oncolytic Indianabecyclovirus, oncolytic Newcastle disease virus, oncolytic vaccinia virus, oncolytic poliovirus, oncolytic myxoma virus, oncolytic reovirus, oncolytic mumps virus, oncolytic maraba virus, oncolytic rabies virus, oncolytic rotavirus, oncolytic hepatitis virus, oncolytic rubella virus, oncolytic dengue virus, oncolytic chikungunya virus, oncolytic respiratory syncytial virus, oncolytic lymphocytic choriomeningitis virus, oncolytic morbillivirus, oncolytic lentivirus, oncolytic replication retrovirus, oncolytic rhabdovirus, oncolytic seneca-valley virus, oncolytic sindobisvirus, and any variants or derivatives thereof.

[0185] The virus may be a recombinant virus comprising any of the oncolytic viruses described herein and encoding another transgene that codes for one or more chimeric proteins, such as any of the engineered nucleic acids described herein.

[0186] Furthermore, provided herein are engineered red blood cells. Red blood cells may be engineered to contain any of the engineered nucleic acids described herein. Red blood cells may be engineered to have any of the characteristics of any of the engineered cells described herein. In certain embodiments, provided herein are red blood cells engineered to produce one or more of the chimeric proteins described herein. In certain embodiments, provided herein are red blood cells engineered to produce two or more of the chimeric proteins described herein.

[0187] Furthermore, provided herein are engineered platelet cells. Platelet cells can be engineered to contain any of the engineered nucleic acids described herein. Platelet cells can be engineered to have any of the characteristics of any of the engineered cells described herein. In certain embodiments, provided herein are platelet cells engineered to produce one or more of the chimeric proteins described herein. In certain embodiments, provided herein are platelet cells engineered to produce two or more of the chimeric proteins described herein.

[0188] Furthermore, provided herein are engineered bacterial cells. Bacterial cells can be engineered to contain any of the engineered nucleic acids described herein. Bacterial cells can be engineered to have any of the characteristics of any of the engineered cells described herein. In certain embodiments, provided herein are bacterial cells engineered to produce two or more chimeric proteins described herein. Bacterial cells can be engineered to produce one or more mammalian chimeric proteins. Bacterial cells can be engineered to produce two or more mammalian chimeric proteins. Examples of bacterial cells include, but are not limited to, Clostridium beigerinchi, Clostridium sporogenes, Clostridium novi, Escherichia coli, Pseudomonas erginosa, Listeria monocytogenes, Salmonella tiphyllum, and Salmonella choleraesuis.

[0189] The manipulated cells may be human cells. The manipulated cells may be human primary cells. The manipulated primary cells may be tumor-infiltrating primary cells. The manipulated primary cells may be primary T cells. The manipulated primary cells may be hematopoietic stem cells (HSCs). The manipulated primary cells may be natural killer (NK) cells. The manipulated primary cells may be any somatic cells. The manipulated primary cells may be mesotherapy stem cells (MSCs). Human cells (e.g., immune cells) may be manipulated to contain any of the manipulated nucleic acids described herein. Human cells (e.g., immune cells) may be manipulated to have any of the characteristics of the manipulated cells described herein. In certain embodiments, what is provided herein is human cells (e.g., immune cells) manipulated to produce one or more of the chimeric proteins described herein. In certain embodiments, what is provided herein is human cells (e.g., immune cells) manipulated to produce two or more of the chimeric proteins described herein.

[0190] Manipulated cells can be isolated from a subject (self), such as a subject known or suspected of having cancer. Methods of cell isolation are known to those skilled in the art and include, but are not limited to, sorting techniques based on cell surface marker expression, such as FACS sorting, positive isolation techniques, and negative isolation, magnetic isolation, and combinations thereof. Manipulated cells may be allogeneic with respect to the subject being treated. Allogeneic modified cells can be HLA-matched to the subject being treated. Manipulated cells may be cultured cells, such as ex vivo cultured cells. Manipulated cells may be ex vivo cultured cells, such as primary cells isolated from a subject. Cultured cells can be cultured with one or more cytokines.

[0191] Furthermore, provided herein are methods for culturing the manipulated cells described herein. Methods for culturing manipulated cells as described herein are known. Those skilled in the art will recognize that the culture conditions depend on the specific manipulated cells of interest. Those skilled in the art will also recognize that the culture conditions depend on the specific downstream use of the manipulated cells, for example, the subsequent administration of the manipulated cells to a subject.

[0192] Methods for manipulating cells Also provided herein are compositions and methods for manipulating cells to produce one or more target proteins or effector molecules (e.g., membrane-cleavable chimeric proteins having the formulas SC-MT or MT-CS as described herein).

[0193] Generally, cells are engineered to produce a protein or effector molecule of interest by introducing (i.e., delivering) one or more proteins or effector molecules of interest into the cytosol and / or nucleus of the cell, for example, a polynucleotide encoding a chimeric protein as described herein, which includes the protein or effector molecule of interest. For example, the polynucleotide encoding one or more chimeric proteins may be any engineered nucleic acid encoding a membrane-cleavable chimeric protein having the formula SC-MT or MT-CS as described herein. Delivery methods include, but are not limited to, virus-mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, sonication, and cell membrane deformation by physical means. Those skilled in the art will understand that the choice of delivery method may depend on the specific cell type to be engineered.

[0194] Virus-borne delivery Cells can be manipulated using viral vector-based delivery platforms. Generally, cells are manipulated by viral vector-based delivery platforms through introduction (i.e., delivery) into host cells. For example, a viral vector-based delivery platform can manipulate cells by introducing one of the manipulated nucleic acids described herein (e.g., one of the exogenous polynucleotide sequences encoding chimeric proteins described herein, e.g., membrane-cleavable chimeric proteins having the formula SC-MT or MT-CS described herein, and / or one of the expression cassettes described herein, comprising a promoter and an exogenous polynucleotide sequence encoding a chimeric protein in the N-terminal to C-terminal direction). While a viral vector-based delivery platform can be a nucleic acid, as such, manipulated nucleic acids can also include nucleic acids derived from manipulated viruses. Such nucleic acids derived from manipulated viruses can also be referred to as recombinant viruses or manipulated viruses.

[0195] A viral vector-based delivery platform may encode two or more engineered nucleic acids, genes, or transgenes within the same nucleic acid. For example, an engineered virus-derived nucleic acid, e.g., a recombinant virus or engineered virus, may encode one or more transgenes, including, but not limited to, an engineered nucleic acid described herein that encodes one or more chimeric proteins described herein. One or more transgenes encoding one or more chimeric proteins may be configured to express one or more chimeric proteins and / or other proteins of interest. In addition to one or more transgenes (e.g., transgenes encoding one or more chimeric proteins and / or other proteins of interest), a viral vector-based delivery platform may encode one or more genes referred to as cis-acting elements or genes, e.g., viral genes required for viral infectivity and / or viral production (e.g., capsid proteins, envelope proteins, viral polymerases, viral transcriptases, etc.).

[0196] A viral vector-based delivery platform may include two or more viral vectors, for example, separate viral vectors encoding engineered nucleic acids, genes, or transgenes, as described herein and referred to as transactive elements or genes. For example, a helper-dependent viral vector-based delivery platform may provide additional genes required for viral infectivity and / or viral production on one or more additional separate vectors, in addition to a vector encoding one or more chimeric proteins and / or other proteins of interest. One viral vector may deliver two or more engineered nucleic acids, for example, one vector delivering engineered nucleic acids configured to produce two or more chimeric proteins and / or other proteins of interest. Two or more viral vectors may deliver two or more engineered nucleic acids, for example, two or more vectors delivering one or more engineered nucleic acids configured to produce one or more chimeric proteins and / or other proteins of interest. The number of viral vectors used may depend on the packaging capacity of the viral vector-based vaccine platform mentioned above, but those skilled in the art can select a suitable number of viral vectors.

[0197] Generally, any of the viral vector-based systems can be used for the in vitro production of molecules, such as chimeric proteins, effector molecules, and / or other proteins of interest as described herein, or for the in vivo delivery of engineered nucleic acids encoding one or more chimeric proteins and / or other proteins of interest in in vivo and ex vivo gene therapy procedures. The selection of a suitable viral vector-based system depends on various factors, such as cargo / payload size, immunogenicity of the viral system, target cells of interest, intensity and timing of gene expression, and other factors recognized by those skilled in the art.

[0198] A viral vector-based delivery platform may be an RNA-based virus or a DNA-based virus. Exemplary viral vector-based delivery platforms include, but are not limited to, herpes simplex virus, adenovirus, measles virus, influenza virus, Indiana beshiclovirus, Newcastle disease virus, vaccinia virus, poliovirus, myxoma virus, reovirus, mumps virus, maraba virus, rabies virus, rotavirus, hepatitis virus, rubella virus, dengue virus, chikungunya virus, respiratory syncytial virus, lymphocytic choriomeningitis virus, morbillivirus, lentivirus, replication retrovirus, rhabdovirus, seneca-valley virus, sindobis virus, and any variants or derivatives thereof.Other exemplary viral vector-based delivery platforms have been described in the Art, including, but are not limited to, vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or lentiviruses, including second, third, or hybrid second / third generation lentiviruses and any generation of recombinant lentiviruses designed to target specific cell types or receptors (e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1): 45-61, Sakuman et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3): 603-18, Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43 (1): 682-690; see Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery, J. Virol. (1998) 72 (12): 9873-9880).

[0199] This sequence may be preceded by one or more sequences that target intracellular compartments. Upon introduction (i.e., delivery) into host cells, infected cells (i.e., engineered cells) can express the chimeric protein and / or other proteins of interest. Useful vaccinia vectors and methods in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). The BCG vector is described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for the introduction (i.e., delivery) of engineered nucleic acids, such as Salmonella cyfi vectors and similar, will be apparent to those skilled in the art from the description herein.

[0200] Viral vector-based delivery platforms may be viruses that target cells, but are referred to herein as oncolytic viruses. Examples of oncolytic viruses include, but are not limited to, oncolytic herpes simplex virus, oncolytic adenovirus, oncolytic measles virus, oncolytic influenza virus, oncolytic Indianabecyclovirus, oncolytic Newcastle disease virus, oncolytic vaccinia virus, oncolytic poliovirus, oncolytic myxoma virus, oncolytic reovirus, oncolytic mumps virus, oncolytic marabavirus, oncolytic rabies virus, oncolytic rotavirus, oncolytic hepatitis virus, oncolytic rubella virus, oncolytic dengue virus, oncolytic chikungunya virus, oncolytic respiratory syncytial virus, oncolytic lymphocytic choriomeningitis virus, oncolytic morbillivirus, oncolytic lentivirus, oncolytic replication retrovirus, oncolytic rhabdovirus, oncolytic seneca-valley virus, oncolytic sindobisvirus, and any variants or derivatives thereof. Any of the oncolytic viruses described herein may be recombinant oncolytic viruses comprising one or more transgenes (e.g., engineered nucleic acids) encoding one or more chimeric proteins and / or other proteins of interest. The transgenes encoding one or more chimeric proteins and / or other proteins of interest may be configured to express the chimeric proteins and / or other proteins of interest.

[0201] Viral vector-based delivery platforms can be retroviral-based. Generally, retroviral vectors consist of cis-acting long terminal repeats with packaging capabilities for exogenous sequences up to 6–10 kb. A minimum cis-acting LTR is sufficient for vector replication and packaging, which is then used to incorporate one or more manipulated nucleic acids (e.g., transgenes encoding one or more chimeric proteins and / or other proteins of interest) into target cells to provide persistent transgene expression. Retrovirus-based delivery systems include, but are not limited to, delivery systems based on mouse leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (e.g., Buchscher et al., J.Virol.66:2731-2739 (1992); Johann et al., J.Virol.66:1635-1640 (1992); Sommnerfelt et al., Virol.176:58-59 (1990); Wilson et al., J.Virol.63:2374-2378 (1989); Miller et al., J.Virol.65:2220-2224 (1991); see PCT / US94 / 05700). Other retrovirus systems include the Phoenix retrovirus system.

[0202] Viral vector-based delivery platforms can be lentiviral-based. Generally, lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce high viral titers. Lentiviral-based delivery platforms can be HIV-based, such as the ViraPower system (ThermoFisher) or the pLenti system (Cell Biolabs). Lentiviral-based delivery platforms can also be SIV or FIV-based. Other exemplary lentivirus-based delivery platforms are described in more detail in U.S. Patents Nos. 7,311,907; 7,262,049; 7,250,299; 7,226,780; 7,220,578; 7,211,247; 7,160,721; 7,078,031; 7,070,993; 7,056,699; and 6,955,919, each incorporated herein by reference for all purposes.

[0203] Viral vector-based delivery platforms can be adenovirus-based. Generally, adenovirus-based vectors allow for very high transduction efficiency in many cell types, do not require cell division, achieve high titer and expression levels, and can be produced in large quantities in relatively simple systems. Generally, adenoviruses can be used for transient expression of transgenes in infected cells because adenoviruses typically do not integrate into the host genome. Adenovirus-based delivery platforms are described in more detail in Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655, each incorporated herein by reference for all purposes. Other exemplary adenovirus-based delivery platforms are described in more detail in U.S. Patents 5,585,362; 6,083,716, 7,371,570; 7,348,178; 7,323,177; 7,319,033; 7,318,919; and 7,306,793, and International Patent Application WO96 / 13597, each incorporated herein by reference for all purposes.

[0204] The viral vector-based delivery platform may be adeno-associated virus (AAV) based. Adeno-associated virus (AAV) vectors may be used to transduce cells with engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). It can be used for the in vitro production of target proteins, such as chimeric proteins and / or effector molecules described herein, or for the in vivo delivery of engineered nucleic acids encoding one or more chimeric proteins and / or other target proteins in in vivo and ex vivo gene therapy procedures (e.g., West et al., Virology 160:38-47 (1987); U.S. Patent Nos. 4,797,368; 5,436,146; 6,632,670; 6,642,051; 7,078,387; 7,314,912; 6,498,244; 7,906,111; U.S. Patent Publications US 2003-0138772, US 2007 / 0036760, and US 2009 / 0197338; Gao, et al.) al., J. Virol, 78(12):6381-6388 (June 2004); Gao, et al, Proc Natl Acad Sci USA, 100(10):6081-6086 (May 13, 2003); and international patent applications WO 2010 / 138263 and WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994), each incorporated herein by reference for all purposes.Exemplary methods for constructing recombinant AAV vectors are described in more detail in U.S. Patent No. 5,173,414; Tratschin et ah, Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et ah, Mol. Cell, Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:64666470 (1984); and Samuiski et ah, J. Virol. 63:03822-3828 (1989), each incorporated herein by reference for all purposes. Generally, AAV-based vectors contain a capsid protein having an amino acid sequence corresponding to one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.Rh10, AAV11, or their variants. In specific examples, an AAV-based vector may have a capsid protein with an amino acid sequence corresponding to AAV2. In specific examples, an AAV-based vector may have a capsid protein with an amino acid sequence corresponding to AAV8.

[0205] AAV vectors can be engineered to have either an exogenous polynucleotide sequence having formula:SC-MT or MT-CS, encoding a membrane-cleavable chimeric protein as described herein.

[0206] Viral vector-based delivery platforms can be virus-like particle (VLP) platforms. Generally, VLPs are constructed by producing viral structural proteins and purifying the resulting viral particles. After purification, the cargo / payload (e.g., any of the engineered nucleic acids described herein) is then ex vivo encapsulated within the purified particles. Thus, VLP production maintains the separation of the nucleic acids encoding the viral structural proteins and those encoding the cargo / payload. The viral structural proteins used in VLP production can be produced in a variety of expression systems, including mammalian, yeast, insect, bacterial, or in vivo translational expression systems. The purified viral particles can be denatured and reformed in the presence of the desired cargo using methods known to those skilled in the art to produce VLPs. VLP production is described in more detail in Seow et al. (Mol Ther. 2009 May; 17(5): 767-777), which is incorporated herein by reference for all purposes.

[0207] Viral vector-based delivery platforms can be modified to target (i.e., infect) a range of cells, a narrow subset of cells, or specific cells. Generally, the tropism of the virus is determined by the envelope protein selected for the viral vector-based delivery platform. The virus used in a viral vector-based delivery platform can be pseudotyped to target specific cells of interest. A viral vector-based delivery platform can be generalized and capable of infecting a range of cells. For example, a generalized viral vector-based delivery platform may contain the VSV-G envelope. A viral vector-based delivery platform can be amphoteric and capable of infecting mammalian cells. Therefore, those skilled in the art can select the appropriate tropism, pseudotype, and / or envelope protein for targeting the desired cell type.

[0208] Lipid structure delivery system Manipulated nucleic acids (e.g., any of the manipulated nucleic acids described herein) can be introduced into cells using lipid-mediated delivery systems. Generally, lipid-mediated delivery systems use structures consisting of an outer lipid membrane enclosing an internal compartment. Examples of lipid-based structures include, but are not limited to, lipid-based nanoparticles, liposomes, micelles, exosomes, vesicles, extracellular vesicles, cells, or tissues. Lipid structure delivery systems can deliver cargo / payloads (e.g., any of the manipulated nucleic acids described herein) in vitro, in vivo, or ex vivo.

[0209] Lipid-based nanoparticles may include, but are not limited to, monolayer liposomes, multilayer liposomes, and lipid preparations. As used herein, “liposome” is a general term encompassing in vitro preparations of lipid media formed by encapsulating a desired cargo, e.g., engineered nucleic acids, e.g., any of the engineered nucleic acids described herein, etc., within a lipid shell or lipid aggregate. Liposomes may generally be characterized as having a vesicular structure with a phospholipid-containing bilayer membrane and an internal medium generally containing an aqueous composition. Liposomes may include, but are not limited to, emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, layered layers, and the like. Liposomes may be monolayer liposomes. Liposomes may be multilayer liposomes. Liposomes may be multi-vesicular liposomes. Liposomes may be positively charged, negatively charged, or neutrally charged. In certain embodiments, liposomes are neutrally charged. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids as well as sterols, such as cholesterol. The selection of lipids is generally guided by consideration of the desired purpose, e.g., criteria for in vivo delivery, such as liposome size, acid instability, and liposome stability in the bloodstream. A variety of methods are available for preparing liposomes, as described, for example, in Szokan et al., Ann. Rev. Biophys. Bioeng. 9; 467 (1980), U.S. Patents No. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369, each incorporated herein by reference for all purposes.

[0210] Multilayer liposomes are generally formed spontaneously when lipids containing phospholipids are suspended in an excess aqueous solution, and multiple lipid layers are separated by the aqueous medium. The water and dissolved solute are encapsulated in a closed structure between the lipid bilayers after the self-reconstitution of the lipid components. The desired cargo (e.g., polypeptides, nucleic acids, small molecule drugs, engineered nucleic acids, such as any of the engineered nucleic acids described herein, viral vectors, virus-based delivery systems, etc.) can be encapsulated within the aqueous interior of the liposome, attached to the liposome via linking molecules that associate with both the liposome and the polypeptide / nucleic acid, scattered within the lipid bilayer of the liposome, encapsulated within the liposome, complexed with the liposome, or otherwise associated with the liposome, enabling delivery to the target entity. Lipophilic molecules or molecules with lipophilic regions can also dissolve in or associate with the lipid bilayer.

[0211] Liposomes used in this embodiment can be prepared by different methods, as may be known to those skilled in the art. The preparation of liposomes is described in more detail in WO 2016 / 201323, international applications PCT / US85 / 01161 and PCT / US89 / 05040, and U.S. Patents 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; each is incorporated herein by reference for all purposes.

[0212] Liposomes may be cationic liposomes. Examples of cationic liposomes are described in more detail in U.S. Patents 5,962,016; 5,030,453; 6,680,068, U.S. Patent Application 2004 / 0208921, and International Patent Applications WO03 / 015757A1, WO04029213A2, and WO02 / 100435A1, each incorporated herein by reference in whole.

[0213] Lipid-mediated gene delivery methods are described, for example, in WO 96 / 18372; WO 93 / 24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Patent No. 5,279,833; Rose Patent No. 5,279,833; WO91 / 06309; and Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987), each incorporated herein by reference for all purposes.

[0214] Exosomes are small membrane vesicles of endocytosis that are released into the extracellular environment after fusion of the polyendoplasmic reticulum with the plasma membrane. Exosomes range in size from 30 to 100 nm in diameter. Their surfaces consist of a lipid bilayer from the donor cell membrane, they contain cytosol from the exosome-producing cell, and exhibit membrane proteins from the parent cell on their surface. Exosomes useful for nucleic acid delivery are known to those skilled in the art, for example, those described in more detail in U.S. Patent No. 9,889,210, which are incorporated herein by reference for all purposes.

[0215] As used herein, the term “extracellular vesicle” or “EV” refers to a cell-derived vesicle containing a membrane that encloses an internal space. Generally, extracellular vesicles include all membrane-bound vesicles having a diameter smaller than the cell from which they originate. Generally, extracellular vesicles range in diameter from 20 nm to 1000 nm and may contain a variety of high-molecular-weight cargoes, either within the internal space presented on the outer surface of the extracellular vesicle and / or across the membrane. Cargoes may include nucleic acids (e.g., any of the manipulated nucleic acids described herein), proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. Examples, but not limited to, include apoptotic bodies, cell fragments, cell-derived vesicles by direct or indirect manipulation (e.g., by continuous extrusion or treatment with alkaline solution), vesicled organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). Extracellular vesicles may originate from living or dead organisms, explanted tissues or organs, and / or cultured cells.

[0216] As used herein, the term “exosome” refers to a small, cell-derived vesicle (20–300 nm in diameter, more preferably 40–200 nm in diameter) that includes a membrane surrounding its internal space and is produced from a cell by direct plasma membrane budding or fusion of late endosomes with the plasma membrane. Exosomes contain lipids or fatty acids and polypeptides and may include a payload (e.g., therapeutic agents), a receiver (e.g., a targeting portion), polynucleotides (e.g., nucleic acids, RNA, or DNA, such as any of the engineered nucleic acids described herein), sugars (e.g., monosaccharides, polysaccharides, or glycans), or other molecules. Exosomes originate from the producing cell and can be isolated from the producing cell based on their size, density, biochemical parameters, or a combination thereof. Exosomes are a type of extracellular vesicle. Generally, exosome production / biosynthesis does not result in the destruction of the producing cell. The exosomes and their preparation are described in further detail in WO 2016 / 201323, which is incorporated herein by reference in its entirety.

[0217] As used herein, the term “nanopes” (also referred to as “microvesicles”) refers to small, cell-derived vesicles (20–250 nm in diameter, more preferably between 30–150 nm in diameter) that contain a membrane surrounding their internal space and are produced by a cell through direct or indirect manipulation, and that are not produced by the producing cell without such manipulation. Generally, nanovesicles are a subspecies of extracellular vesicles. Suitable manipulations of the producing cell include, but are not limited to, serial extrusion, treatment with alkaline solutions, sonication, or a combination thereof. Nanovesicle production may, in some cases, result in the destruction of the producing cell. Preferably, the nanovesicle population is substantially free of vesicles derived from the producing cell by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane. Nanovesicles contain lipids or fatty acids and polypeptides, and optionally include a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting portion), polynucleotides (e.g., nucleic acids, RNA, or DNA, such as any of the engineered nucleic acids described herein), sugars (e.g., monosaccharides, polysaccharides, or glycans), or other molecules. Once the nanovesicles are induced from the producing cells according to the said procedure, they can be isolated from the producing cells based on their size, density, biochemical parameters, or a combination thereof.

[0218] Lipid nanoparticles (LNPs) are generally synthetic lipid structures that form membranes and vesicle-like structures depending on the amphiphilic nature of the lipids (Riley 2017). Generally, these vesicles are absorbed into the membrane of target cells and deliver cargo / payload, such as either engineered nucleic acids or viral systems as described herein, by releasing the cargo into the cytosol. The lipids used in LNP formation can be cationic, anionic, or neutral. Lipids can be synthetic or of natural origin and, in some examples, biodegradable. Lipids can include fats, cholesterol, phospholipids, lipid conjugates including, but not limited to, polyethylene glycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat-soluble vitamins. Lipid compositions generally include defined mixtures of materials, such as cationic, neutral, anionic, and amphiphilic lipids. In some examples, functional chemical groups are provided, including certain lipids, to prevent LNP aggregation, prevent lipid oxidation, or facilitate the attachment of additional moieties. Lipid composition can affect overall LNP size and stability. In one example, the lipid composition includes dilinoleylmethyl-4-dimethylaminobutyrate (MC3) or an MC3-like molecule. MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as PEG or PEG-conjugated lipids, sterols, or neutral lipids. Also, LNPs can be further engineered or functionalized to facilitate targeting of specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity.

[0219] Micelles are generally spherical synthetic lipid structures formed using single-chain lipids, where the hydrophilic heads of the single-chain lipids form the outer layer or membrane and the hydrophobic tails of the single-chain lipids form the micelle core. Micelles typically refer to lipid structures that contain only a lipid monolayer. Micelles are described in more detail in Quader et al. (Mol Ther. 2017 Jul 5; 25(7): 1501-1513), which is incorporated herein by reference for all purposes.

[0220] Nucleic acid vectors, such as expression vectors directly exposed to serum, can have several undesirable consequences, including degradation of the nucleic acid by serum nucleases or off-target stimulation of the immune system by free nucleic acids. Similarly, viral delivery systems directly exposed to serum can induce undesirable immune responses and / or neutralization of the viral delivery system. Therefore, encapsulation of engineered nucleic acids and / or viral delivery systems can be used to avoid degradation while also avoiding potential off-target effects. In certain examples, engineered nucleic acids and / or viral delivery systems are completely encapsulated within a delivery medium, for example, within the aqueous interior of a LNP. Encapsulation of engineered nucleic acids and / or viral delivery systems within an LNP can be carried out by techniques well known to those skilled in the art, such as droplet generation performed on microfluidic mixing and microfluidic droplet generation devices. Such devices include, but are not limited to, standard T-junction devices or flow-focusing devices. In one example, a desired lipid formulation, such as MC3 or an MC3-like composition, is supplied to a droplet-generating device in parallel with an engineered nucleic acid or viral delivery system and any other desired drug, so as to completely encapsulate the delivery vector and the desired drug within the MC3 or MC3-like based LNPs. In one example, the droplet-generating device can control the size range and size distribution of the produced LNPs. For example, the LNPs may have sizes in the range of 1 to 1000 nanometers in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nanometers. Following droplet generation, the delivery medium (e.g., engineered nucleic acid and / or viral delivery system) encapsulating the cargo / payload can be further processed or manipulated to prepare them for administration.

[0221] Nanoparticle delivery Nucleic acids that have been engineered (e.g., any of the engineered nucleic acids described herein) can be delivered using nanomaterials. The nanomaterial vehicle can importantly be made of non-immunogenic materials and generally can avoid inducing immunity against the delivery vector itself. These materials include, but are not limited to, lipids (as previously described), inorganic nanomaterials, and other polymeric materials. Nanomaterial particles are described in detail in Riley et al. (Recent Advances in Nanomaterials for Gene Delivery - A Review. Nanomaterials 2017, 7(5), 94), which is incorporated herein by reference for all purposes.

[0222] Genome editing system A genome editing system can be used to manipulate a host genome to encode one or more engineered nucleic acids, such as an engineered nucleic acid encoding a chimeric protein (e.g., any of the membrane-cleavable chimeric proteins having the formula S-C-MT or MT-C-S described herein). Generally, a "genome editing system" refers to any system for integrating a foreign gene into the genome of a host cell. Genome editing systems include, but are not limited to, transposon systems, nuclease genome editing systems, and virus vector-based delivery platforms.

[0223] Transposon systems can be used to incorporate engineered nucleic acids, such as engineered nucleic acids encoding one or more chimeric proteins (e.g., any membrane-cleavable chimeric proteins having the formulas SC-MT or MT-CS as described herein), into the host genome. Transposons generally consist of a cargo / payload nucleic acid and a terminal inversion repeat (TIR) ​​adjacent to the transposase. Transposon systems can provide transposons in cis or trans orientation with a TIR-adjacent cargo. Transposon systems can be retrotransposon systems or DNA transposon systems. Generally, transposon systems randomly incorporate the cargo / payload (e.g., engineered nucleic acids) into the host genome. Examples of transposon systems include systems using transposons from the Tc1 / mariner transposon superfamily, such as the Sleeping Beauty transposon system, which are described in more detail in Hudecek et al. (Crit Rev Biochem Mol Biol. 2017 Aug;52(4):355-380) and U.S. Patents 6,489,458, 6,613,752, and 7,985,739, each of which is incorporated herein by reference for all purposes. Another example of a transposon system includes the PiggyBac transposon system, which are described in more detail in U.S. Patents 6,218,185 and 6,962,810, each of which is incorporated herein by reference for all purposes.

[0224] Nuclease genome editing systems can be used to manipulate the host genome to encode one or more manipulated nucleic acids, such as chimeric proteins (e.g., any of the membrane-cleavable chimeric proteins having the formulas SC-MT or MT-CS as described herein). While we do not wish to dwell on theory, generally speaking, nuclease-mediated gene editing systems used to introduce exogenous genes utilize the cell's innate DNA repair mechanisms, particularly the homologous recombination (HR) repair pathway. Simply put, following damage to genomic DNA (typically a double-strand break), the cell can resolve the damage by using another DNA source having identical or substantially identical sequences at both its 5' and 3' ends as a template during DNA synthesis to repair the damage. In natural contexts, HDR can use other chromosomes present in the cell as templates. In gene editing systems, exogenous polynucleotides are introduced into the cell and used as homologous recombination templates (HRT or HR templates). Generally, any additional exogenous sequences not naturally found on the chromosome, involving damage between the 5' and 3' complementary ends of an HRT (e.g., a gene or a portion of a gene), can be incorporated into a templated HDR within a given genomic locus. Thus, a typical HR template for a given genomic locus has a nucleotide sequence identical to the first region of the endogenous genomic target locus, a nucleotide sequence identical to the second region of the endogenous genomic target locus, and a nucleotide sequence encoding a cargo / payload nucleic acid (e.g., any of the engineered nucleic acids described herein, e.g., any of the engineered nucleic acids encoding one or more chimeric proteins (e.g., any of the membrane-cleavable chimeric proteins having the formula SC-MT or MT-CS as described herein)).

[0225] In some cases, HR templates can be linear. Examples of linear HR templates include, but are not limited to, linearized plasmid vectors, ssDNA, synthetic DNA, and PCR-amplified DNA. In certain cases, HR templates can be circular, such as plasmids. Circular templates may include supercoiled templates.

[0226] Identical or substantially identical sequences found at the 5' and 3' ends of the HR template are generally referred to as arms (HR arms) with respect to the introduced exogenous sequence. HR arms may be identical (i.e., 100% identical) to the region of the endogenous genomic target locus. In some cases, HR arms may be substantially identical to the region of the endogenous genomic target locus. While substantially identical HR arms can be used, it may be advantageous for HR arms to be identical because the efficiency of the HDR pathway may be affected by HR arms with less than 100% identity.

[0227] Each HR arm, i.e., the 5' and 3' HR arms, may be the same size or different sizes. The length of each HR arm may be greater than or equal to 50, 100, 200, 300, 400, or 500 base pairs. While HR arms can generally be of any length, practical considerations, such as the effect of HR arm length and overall template size on overall editing efficiency, may also be taken into account. Each HR arm may be identical or substantially identical to a region of an endogenous genomic target locus directly adjacent to the cleavage site. Each HR arm may be identical or substantially identical to a region of an endogenous genomic target locus directly adjacent to the cleavage site. Each HR arm may be identical or substantially identical to a region of an endogenous genomic target locus located within a certain distance from the cleavage site, for example, one base pair, less than or equal to 10 base pairs, less than or equal to 50 base pairs, or less than or equal to 100 base pairs.

[0228] Nuclease genome editing systems can cleave target genomic loci using a variety of nucleases, including, but not limited to, clustered and regularly arranged short palindromic repeat (CRISPR) family nucleases or derivatives thereof, transcription activator-like effector nucleases (TALENs) or derivatives thereof, zinc finger nucleases (ZFNs) or derivatives thereof, and homing endonucleases (HEs) or derivatives thereof.

[0229] CRISPR-mediated gene editing systems can be used to manipulate a host genome to encode one or more manipulated nucleic acids, such as chimeric proteins (e.g., degron-fusion chimeric proteins as described herein or membrane-cleavable chimeric proteins having the formulas SC-MT or MT-CS as described herein). CRISPR systems are described in more detail in M. Adli ("The CRISPR tool kit for genome editing and beyond," Nature Communications; volume 9 (2018), Article number: 1911), all of which are incorporated herein by reference. Generally, CRISPR-mediated gene editing systems include a CRISPR-associated (Cas) nuclease and RNA that directs cleavage to a specific target sequence. An exemplary CRISPR-mediated gene editing system is the CRISPR / Cas9 system, which consists of a Cas9 nuclease and RNA having a CRISPR RNA (crRNA) domain and a transactivating CRISPR (tracrRNA) domain. crRNA typically has two RNA domains: a guide RNA (gRNA) that directs specificity to a target sequence ("defined nucleotide sequence"), e.g., a genomic sequence, through base-pair hybridization; and an RNA domain that hybridizes to tracrRNA. TracrRNA can interact with nucleases (e.g., Cas9) to facilitate recruitment to genomic loci. The crRNA and tracrRNA polynucleotides may be separate polynucleotides. The crRNA and tracrRNA polynucleotides may also be a single polynucleotide, but are referred to as a single guide RNA (sgRNA). While the Cas9 system is exemplified herein, other CRISPR systems may be used, e.g., the Cpf1 / Cas12 or Cas13 systems.Nucleases may include their derivatives, such as Cas9 functional mutants, which generally mediate single-strand cleavage of defined nucleotide sequences, in contrast to the complete double-strand cleavage typically produced by the Cas9 enzyme, known as Cas9 "nickase" mutants.

[0230] Generally, components of a CRISPR system interact with each other to form ribonucleoprotein (RNP) complexes, which mediate sequence-specific cleavage. In some CRISPR systems, each component can be produced separately and used to form the RNP complex. In some CRISPR systems, each component can be produced separately in vitro and brought into contact with each other (i.e., "complexed") in vitro to form the RNP complex. The RNP produced in vitro can then be introduced (i.e., "delivered") into the cytosol and / or nucleus of a cell, for example, into the cytosol and / or nucleus of a T cell. The RNP complex produced in vitro can be delivered to cells by a variety of means, not limited to electroporation, lipid-mediated transfection, cell membrane deformation by physical means, lipid nanoparticles (LNPs), virus-like particles (VLPs), and sonication. In certain cases, the RNP complex produced in vitro can be delivered to cells using the Nucleofactor / Nucleofection® electroporation-based delivery system (Lonza®). Other electroporation systems include, but are not limited to, the MaxCyte electroporation system, the Miltenyi CliniMACS electroporation system, the Neon electroporation system, and the BTX electroporation system. CRISPR nucleases, such as Cas9, can be produced (i.e., synthesized and purified) in vitro using various protein production techniques known in the art. CRISPR RNAs, such as sgRNA, can be produced (i.e., synthesized and purified) in vitro using various RNA production techniques known to those skilled in the art, such as in vitro transcription or chemical synthesis.

[0231] RNP complexes produced in vitro can be complexed with nucleases and gRNAs in different ratios. RNP complexes produced in vitro can also be used in different amounts in CRISPR-mediated editing systems. For example, the total amount of RNP added can be adjusted depending on the number of cells to be edited, such as reducing the amount of RNP complex added when editing a large number of cells in the reaction.

[0232] In some CRISPR systems, each component (e.g., Cas9 and sgRNA) is encoded separately by a polynucleotide, and each polynucleotide can be introduced into the cell together or separately. In some CRISPR systems, each component is encoded by a single polynucleotide (i.e., a multi-promoter or multi-cistron vector; see the description of exemplary multi-cistron systems below) and can be introduced into the cell. Following the expression of the CRISPR components encoded by each polynucleotide in the cell (e.g., translation of nucleases and transcription of CRISPR RNA), an RNP complex can be formed in the cell and then directed for site-specific cleavage.

[0233] Some RNPs can be engineered to have a portion that facilitates the delivery of RNPs into the nucleus. For example, Cas9 nucleases may have a nuclear localization signaling (NLS) domain, and if the Cas9 RNP complex is delivered into the cell cytosol, or after Cas9 translation and subsequent RNP formation, the NLS can facilitate further transport of Cas9 RNPs into the nucleus.

[0234] The manipulated cells described herein can be manipulated using nonviral methods, for example, the nucleases and / or CRISPR-mediated gene editing systems described herein can be delivered to cells using nonviral methods. The manipulated cells described herein can be manipulated using viral methods, for example, the nucleases and / or CRISPR-mediated gene editing systems described herein can be delivered to cells using any of the viral methods, such as adenovirus, retrovirus, lentivirus, or other virus-based delivery methods described herein.

[0235] Some CRISPR systems can provide two or more CRISPR compositions, each designed to independently target the same gene or common genomic locus with two or more target nucleotide sequences. For example, two separate CRISPR compositions can be provided, directed to cleave at two different target nucleotide sequences within a specific distance of each other. Some CRISPR systems can provide two or more CRISPR compositions, each designed to independently target the reverse strand of the same gene or common genomic locus. For example, two separate CRISPR "nickase" compositions can be provided, directed to cleave at the same gene or common genomic locus on the reverse strand.

[0236] In general, the characteristics of the CRISPR-mediated editing systems described herein can be applied to other nuclease-based genome editing systems. A TALEN is an engineered site-specific nuclease, which consists of a DNA-binding domain of a TALE (transcriptional activator-like effector) and a catalytic domain of the restriction endonuclease Fokl. Different artificial TALENs can be created to target various nucleotide sequences by altering the amino acids present in the highly variable residue region of the monomer of the DNA-binding domain. The DNA-binding domain then directs the nuclease to the target sequence, creating a double-strand break. The TALEN-based systems are described in more detail in U.S. Patent No. 12 / 965,590; U.S. Patent No. 8,450,471; U.S. Patent No. 8,440,431; U.S. Patent No. 8,440,432; U.S. Patent No. 10,172,880; and U.S. Patent No. 13 / 738,381, all of which are incorporated herein by reference in their entirety. The TALEN-based editorial systems are described in more detail in U.S. Patent Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publications 2005 / 0064474; 2007 / 0218528; and 2005 / 0267061, all of which, in their entirety, are incorporated herein by reference for all purposes.

[0237] Other modified delivery systems Various additional means for introducing a manipulated nucleic acid (e.g., any of the manipulated nucleic acids described herein) into a cell or other target recipient entity, such as any of the lipid structures described herein.

[0238] Polynucleotides can be delivered to recipient entities using electroporation. Electroporation is a method of internalizing cargo / payloads into the internal compartments of a target cell or entity by applying an electric field to transiently permeate the outer membrane or shell of the target cell or entity. Generally, this method involves placing the cell or target entity between two electrodes in a solution containing the cargo of interest (e.g., any of the engineered nucleic acids described herein). The lipid membrane of the cell is then broken, i.e., permeated, by applying a transient set voltage, thereby allowing the cargo to enter the interior of the entity, for example, the cytoplasm of the cell. In the case of cells, at least a portion, but not the majority, of the cell remains viable. Cells and other entities can be electroporated in vitro, in vivo, or ex vivo. Electroporation conditions (e.g., cell number, cargo concentration, recovery conditions, voltage, time, volume, pulse type, pulse length, volume, cuvette length, electroporation solution composition, etc.) vary depending on several factors, including, but not limited to, the type of cells or other recipient entities, the cargo delivered, the desired internalization efficiency, and the desired viability. Optimization of such criteria is within the scope of the art of the art. Various devices and protocols can be used for electroporation. Examples include, but are not limited to, the Neon® transfection system, MaxCyte® Flow Electroporation® system, Lonza® Nucleofector® system, and Bio-Rad® electroporation system.

[0239] Other means for introducing engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) into cells or other target recipient entities include, but are not limited to, sonication, gene guns, hydrodynamic injection, and cell membrane deformation by physical means.

[0240] Compositions and methods for in vivo delivery of engineered mRNA, such as naked plasmids or mRNA, are described in detail in Kowalski et al. (Mol Ther. 2019 Apr 10; 27(4): 710-728) and Kaczmarek et al. (Genome Med. 2017; 9: 60), each of which is incorporated herein by reference for all purposes.

[0241] Delivery vehicle Also provided herein are compositions for delivering a cargo / payload (the "delivery vehicle").

[0242] The cargo can include a nucleic acid (e.g., any of the engineered nucleic acids described herein, such as any of the engineered nucleic acids encoding a membrane-disruptable chimeric protein having the formula S-C-MT or MT-C-S described herein), a protein, a carbohydrate, a lipid, a small molecule, and / or a combination thereof. The cargo can be any of the chimeric proteins provided herein (e.g., any of the membrane-disruptable chimeric proteins having the formula S-C-MT or MT-C-S described herein). The cargo can be a combination of two or more of the chimeric proteins described herein. The cargo can be a combination of a chimeric protein described herein and another cargo of interest, such as another protein, carbohydrate, lipid, small molecule, and / or a combination thereof.

[0243] The delivery medium may comprise any composition suitable for delivering cargo. The delivery medium may comprise any composition suitable for delivering proteins (e.g., any of the chimeric proteins described herein). The delivery medium may be any of the lipid structure delivery systems described herein. For example, the delivery medium may comprise lipid-based structures, but are not limited to lipid-based nanoparticles, liposomes, micelles, exosomes, vesicles, extracellular vesicles, cells, or tissues. The delivery medium may comprise any of the nanoparticles described herein, such as nanoparticles comprising lipids (as previously described), inorganic nanomaterials, and other polymer materials.

[0244] The delivery medium may be capable of delivering cargo to cells, for example, by delivering one of the chimeric proteins described herein to cells. The delivery medium may be configured to target specific cells, for example, by using a reorienting antibody for targeting specific cells. The delivery medium may be capable of delivering cargo to cells in vivo.

[0245] The delivery medium may be capable of delivering cargo to a tissue or tissue environment (e.g., a tumor microenvironment), or delivering one of the chimeric proteins described herein in vivo to a tissue or tissue environment. Delivering cargo may include secreting cargo, for example, one of the chimeric proteins described herein. Therefore, the delivery medium may be capable of secreting cargo, for example, one of the chimeric proteins described herein. The delivery medium may be capable of secreting cargo to a tissue or tissue environment (e.g., a tumor microenvironment), for example, one of the chimeric proteins described herein into the tissue or tissue environment. The delivery medium may be configured to target a specific tissue or tissue environment (e.g., a tumor microenvironment), for example, using a reorienting antibody to target a specific tissue or tissue environment.

[0246] Treatment methods Furthermore, provided herein are methods comprising delivering or administering engineered cells provided herein to a subject (e.g., a human subject) in vivo to produce at least one target protein (e.g., any of the chimeric proteins provided herein, such as a membrane-cleavable chimeric protein having the formula SC-MT or MT-CS described herein, or a secretory effector molecule provided herein after protease cleavage of a chimeric protein) produced by the engineered cells. Further provided herein are methods comprising delivering or administering engineered cells provided herein to a subject (e.g., a human subject) in vivo to produce at least two target proteins of interest, such as at least two of the chimeric proteins provided herein, such as a membrane-cleavable chimeric protein having the formula SC-MT or MT-CS described herein, produced by the engineered cells.

[0247] Further provided herein are methods for delivering or administering to a subject (e.g., a human subject) any of the delivery media described herein, for example, any of the delivery media described herein containing any of the target proteins described herein, or any of the chimeric proteins provided herein, for example, membrane-cleavable chimeric proteins having the formula SC-MT or MT-CS described herein. Further provided herein are methods for delivering to a subject (e.g., a human subject) any of the delivery media described herein, for example, any of the delivery media described herein containing two or more proteins of the chimeric proteins provided herein, for example, at least two, or any of the membrane-cleavable chimeric proteins having the formula SC-MT or MT-CS described herein.

[0248] In some embodiments, the engineered cells or delivery medium are administered via intravenous, intraperitoneal, intratracheal, subcutaneous, intratumoral, oral, anal, intranasal (e.g., encapsulated in delivery particles), or arterial (e.g., internal carotid artery) routes. Thus, the engineered cells or delivery medium may be administered systemically or locally (e.g., via TME or intratumoral administration). The engineered cells can be isolated from a subject, such as a subject known or suspected of having cancer. The engineered cells may be homogeneous with respect to the subject being treated. The homogeneous modified cells can be HLA-matched to the subject being treated. The delivery medium may be any of the lipid structure delivery systems described herein. The delivery medium may be any of the nanoparticles described herein.

[0249] The manipulated cells or delivery medium may be administered either alone or in combination with other therapies, in a manner dependent on the condition to be treated, and either concurrently or sequentially. For example, the manipulated cells or delivery medium may be administered in combination with one or more IMiDs described herein. FDA-approved IMiDs may be administered in their approved forms. In another embodiment, the manipulated cells or delivery medium may be administered in combination with checkpoint inhibitor therapy. Exemplary checkpoint inhibitors include, but are not limited to, anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIM-3 antibody, anti-TIGIT antibody, anti-VISTA antibody, anti-KIR antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-GAL9 antibody, anti-A2AR antibody, anti-phosphatidylserine antibody, anti-CD27 antibody, anti-TNFα antibody, anti-TREM1 antibody, and anti-TREM2 antibody. Exemplary immune checkpoint inhibitors include pembrolizumab (anti-PD-1; MK-3475 / Keytruda®- Merck), nivolumab (anti-PD-1; Opdivo®- BMS), pidilizumab (anti-PD-1 antibody; CT-011- Teva / CureTech), AMP224 (anti-PD-1; NCI), avelumab (anti-PD-L1; Bavencio®- Pfizer), durvalumab (anti-PD-L1; MEDI4736 / Imfinzi®- Medimmune / AstraZeneca), atezolizumab (anti-PD-L1; Tecentriq®- Roche / Genentech), and BMS-936559 (anti-PD-L1 - This includes BMS, tremelimumab (anti-CTLA-4; Medimmune / AstraZeneca), ipilimumab (anti-CTLA-4; Yervoy®-BMS), lirirumab (anti-KIR; BMS), and monalizumab (anti-NKG2A; Innate Pharma / AstraZeneca). In other cases, the manipulated cells or delivery medium may be administered in combination with a TGF-beta inhibitor, a VEGF inhibitor, or HPGE2.In another example, the manipulated cells or delivery medium can be administered in combination with an anti-CD40 antibody.

[0250] Some methods involve selecting subjects (or patient populations) who have tumors (or cancer) and treating them with engineered cells or delivery media that modulate tumor-mediated immunosuppressive mechanisms.

[0251] The manipulated cells or delivery media of this disclosure may, in some cases, be used to treat cancer, such as ovarian cancer. Other cancers are described herein. For example, manipulated cells may be used to treat bladder tumors, brain tumors, breast tumors, cervical tumors, colorectal tumors, esophageal tumors, gliomas, kidney tumors, liver tumors, lung tumors, melanomas, ovarian tumors, pancreatic tumors, prostate tumors, skin tumors, thyroid tumors, and / or uterine tumors. The manipulated cells or delivery media of this disclosure may be used to treat cancers involving tumors located in the peritoneal cavity of the subject.

[0252] The methods provided herein also include delivering a preparation of engineered cells or a delivery medium. In some embodiments, the preparation is a substantially pure preparation containing, for example, less than 5% (e.g., less than 4%, 3%, 2%, or 1%) of cells other than engineered cells. The preparation is 1 × 10⁻⁶ 5 From cells / kg to 1 × 10 7 The preparation of the manipulated cells or delivery medium may include cells / kg. The preparation of the manipulated cells or delivery medium may include a pharmaceutical composition having one or more pharmaceutically acceptable carriers. For example, the preparation of the manipulated cells or delivery medium may include any of the manipulated viruses, such as the manipulated AAV virus, or any of the manipulated viral vectors, such as the AAV vector described herein.

[0253] In vivo expression The methods provided herein also include in vivo delivery of compositions capable of producing manipulated cells, as described herein, for example, any of the manipulated nucleic acids described herein, which can be delivered to cells in vivo. Such compositions include any of the virus-mediated delivery platforms, any of the lipid structure delivery systems, any of the nanoparticle delivery systems, any of the genome editing systems, or any of the other manipulation delivery systems described herein that can manipulate cells in vivo.

[0254] The methods provided herein also include delivering in vivo a composition capable of producing any of the target proteins described herein, for example, a chimeric protein provided herein, for example, a membrane-cleavable chimeric protein having the formula SC-MT or MT-CS described herein. The methods provided herein also include delivering in vivo a composition capable of producing two or more of the target proteins described herein. The composition capable of in vivo production of the target protein includes, but is not limited to, any of the engineered nucleic acids described herein. The composition capable of in vivo production of the target protein may be naked mRNA or a naked plasmid.

[0255] Additional Embodiments 1. An engineered nucleic acid comprising an expression cassette comprising a promoter oriented from the N-terminus to the C-terminus and an exogenous polynucleotide sequence encoding a membrane-cleavable chimeric protein, wherein the formula is: Having SC-MT or MT-CS, During the ceremony, S contains secretory effector molecules, C includes the protease cleavage site, MT includes a cell membrane anchoring domain, In this modified nucleic acid, the promoter is operably ligated to an exogenous polynucleotide sequence, and in this modified nucleic acid, SC-MT or MT-CS is configured to be expressed as a single polypeptide. 2. The promoter is a constitutive promoter, which is the manipulated nucleic acid of Embodiment 1. 3. The constitutive promoter is selected from the group consisting of: CAG, HLP, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4...

Claims

1. A chimeric protein oriented from the N-terminus to the C-terminus, with formula: S-C-MT or MT-C-S It has, During the ceremony, S contains secretory effector molecules, C includes the protease cleavage site, and MT includes a cell membrane anchoring domain, Here, S-C-MT or MT-C-S is configured to be expressed as a single polypeptide. When expressed in cells expressing a protease capable of cleaving the aforementioned protease cleavage site, the secreted effector molecule is released from the cell membrane, and The aforementioned protease cleavage site includes the amino acid sequence of PRAEYSKGG (SEQ ID NO: 181), or the amino acid sequence of PRAEPIKGG (SEQ ID NO: 182), or the amino acid sequence of PRAEYKGG (SEQ ID NO: 183), or the amino acid sequence of PRAESSKGG (SEQ ID NO: 184), or the amino acid sequence of PRAEFTKGG (SEQ ID NO: 185), or the amino acid sequence of DEPHYSQRR (SEQ ID NO: 187), or the amino acid sequence of PPLGPIFNPG (SEQ ID NO: 188), or the amino acid sequence of PLAQAYRSS (SEQ ID NO: 189), or the amino acid sequence of TPIDSSFNPD (SEQ ID NO: 190), or the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191). Chimeric protein.

2. The chimeric protein according to claim 1, wherein the secreted effector molecule comprises a signal peptide or a signal anchor sequence.

3. The chimeric protein according to claim 1 or 2, wherein the secreted effector molecule is selected from a therapeutic class, and the therapeutic class is selected from the group consisting of cytokines, chemokines, homing molecules, growth factors, coactivating molecules, tumor microenvironment modifiers, ligands, antibodies, peptides, and enzymes.

4. The chimeric protein according to any one of claims 1 to 3, wherein the secreted effector molecule comprises an IL-15, IL-12, or IL-12p70 fusion protein.

5. The chimeric protein according to any one of claims 1 to 4, wherein the protease cleavage site is cleavable by ADAM17 protease.

6. The chimeric protein according to any one of claims 1 to 5, wherein the cell membrane anchoring domain includes a transmembrane-intracellular domain or a transmembrane domain.

7. The chimeric protein according to any one of claims 1 to 6, wherein the cell membrane anchoring domain includes a post-translational modification tag, or a post-translational modificationable motif for modifying the chimeric protein to include a post-translational modification tag, and the post-translational modification tag is capable of associating with a cell membrane.

8. a. When expressed in a cell, the secretory effector molecule is anchored to the cell membrane of the cell and / or b. The protease expressed on the cell membrane is endogenous to the cell and / or c. The protease is ADAM17 protease. The chimeric protein according to any one of claims 1 to 7.

9. An engineered nucleic acid comprising an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a chimeric protein according to any one of claims 1 to 8.

10. An expression vector comprising the manipulated nucleic acid described in claim 9.

11. Isolated cells comprising a chimeric protein according to any one of claims 1 to 8, an engineered nucleic acid according to claim 9, or an expression vector according to claim 10.

12. The isolated cells according to claim 11, wherein the cells are selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, bone marrow cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells.

13. The isolated cell according to claim 11 or claim 12, wherein the cell further comprises a protease capable of cleaving the protease cleavage site.

14. The isolated cells according to any one of claims 11 to 13, wherein the cells further comprise an antigen recognition receptor.

15. A composition comprising a chimeric protein according to any one of claims 1 to 8, an engineered nucleic acid according to claim 9, an expression vector according to claim 10, or an isolated cell according to any one of claims 11 to 14, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.

16. Use of a therapeutically effective dose of any isolated cells according to any one of claims 11 to 14 or any of the compositions according to claim 15 for the preparation of a pharmacopoeia for treating a subject that requires it.

17. Methods for inducing the release of membrane-tethered effector molecules, including the following: (a) To provide the cells according to any one of claims 11 to 14, and (b) Culturing the cells under conditions suitable for the expression of the membrane-bound protease and the chimeric protein, Upon expression, the chimeric protein is anchored to the cell membrane of the cell, and Upon expression, the membrane-bound protease cleaves the homologous membrane-bound protease cleavage site of the chimeric protein, thereby releasing the secreted effector molecule from the cell membrane. thing.

18. The chimeric protein according to claim 1, wherein the signal peptide comprises a natural signal peptide that is natural for the secreted effector molecule, or the signal peptide comprises a non-natural signal peptide, or the signal anchor sequence comprises a non-natural signal anchor sequence that is non-natural for the secreted effector molecule.

19. The chimeric protein according to claim 18, wherein the non-natural signal peptide or the non-natural signal anchor sequence is selected from the group consisting of IL-12, IL-2, optimized IL-2, trypsinogen-2, Gaussian alciferase, CD5, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azulocidine preprotein, osteonectin, CD33, IL-6, IL-8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpine E1, GRO-Alpha, CXCL12, IL-21, CD8, NKG2D, TNFR2, and GMCSF.

20. (i) The cytokine is selected from the group consisting of IL-1-beta, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-12p70 fusion protein, IL-15, IL-17A, IL-18, IL-21, IL-22, type I interferon, interferon-gamma, and TNF-alpha. (ii) The chemokine is selected from the group consisting of CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1. (iii) The homing molecule is selected from the group consisting of anti-integrin alpha 4, beta 7; anti-MAdCAM; SDF1; and MMP-2. (iv) The growth factor is selected from the group consisting of FLT3L and GM-CSF. (v) The co-activating molecule is selected from the group consisting of 4-1BBL and CD40L, or (vi) The tumor microenvironment modifying factor is selected from the group consisting of adenosine deaminase, TGF beta inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2. The chimeric protein according to claim 3.

21. (i) The TGF beta inhibitor is selected from the group consisting of anti-TGF beta peptide, anti-TGF beta antibody, TGF b-TRAP, and combinations thereof. (ii) The immune checkpoint inhibitor is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIM-3 antibody, anti-TIGIT antibody, anti-VISTA antibody, anti-KIR antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-GAL9 antibody, anti-A2AR antibody, anti-phosphatidylserine antibody, anti-CD27 antibody, anti-TNFa antibody, anti-TREM1 antibody, and anti-TREM2 antibody, or (iii) The VEGF inhibitor comprises an anti-VEGF antibody, an anti-VEGF peptide, or a combination thereof. The chimeric protein according to claim 20.

22. The chimeric protein according to claim 6, wherein the transmembrane-intracellular domain and / or transmembrane domain are 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, and / or the cell membrane anchoring domain comprises a cell surface receptor or its cell membrane binding portion.

23. The chimeric protein according to claim 7, wherein the post-translational modification tag comprises a lipid anchor domain, and the lipid anchor domain is selected from the group consisting of a GPI lipid anchor, a myristoylation tag, and a palmitoylation tag.

24. The manipulated nucleic acid according to claim 9, wherein the promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, a tissue-specific promoter, and a synthetic promoter.

25. The isolated cell according to claim 13, wherein the protease is an endogenous protease, and the endogenous protease is ADAM17 protease.

26. The isolated cell according to claim 14, wherein the antigen recognition receptor is CAR.

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