Protein payload release

Abstract

This article describes chimeric proteins, specifically membrane-cleavable chimeric systems. It also discusses nucleic acids, cells, and related methods.

Description

Prior Technology

[0001] Cell-based therapeutic platforms offer promising avenues for the treatment of various diseases. One such promising platform is CAR-T based therapy for the treatment of cancer. In view of its promise, improved cell-based therapies are needed. One active area of ​​exploration is engineering cell-based therapies to produce and / or secrete effector molecules, such as cytokines, a process known as armoring that enhances cell-based therapies. For example, unarmored CAR-T therapy does not work well in solid tumors, and armor can affect the entire cancer immune cycle and enhance CAR-T activity. However, uncontrolled or unregulated armor strategies can negatively impact therapy, such as off-target effects and toxicity in individuals. Accordingly, additional methods of controlling and modulating the armor of cell-based therapies, such as modulating the production and / or secretion of payload effector molecules, are needed.

Content of invention

[0003] In some embodiments, provided herein are cell-based therapy platforms that involve the regulated armor of cell-based therapy, such as the regulated secretion of payload effector molecules. Also provided herein, in some embodiments, are cell-based combination immunotherapies involving modulated armor for targeted therapy of cancers such as ovarian, breast, colon, lung, and pancreatic cancers.

[0004] However, the therapies provided herein can limit the systemic toxicity of armor. For example, the immunotherapies provided herein can be tumor-specific and effective while limiting systemic toxicity and / or other off-target effects due to armor. These therapies deliver proteins of interest, such as immunomodulatory effector molecules, in a regulated manner, including regulation of secretion kinetics, cell state specificity, and cell or tissue specificity. Optimizing the design of delivery vehicles to improve the overall function of cell-based therapies, such as cancer therapies, including but not limited to membrane cleavage sites for immunomodulatory effector molecules, promoters, linkers, signal peptides, delivery methods, Optimization of combination, regulation and sequence.

[0005] Non-limiting examples of effector molecules encompassed by the present disclosure include cytokines, antibodies, chemokines, nucleotides, peptides, enzymes, and oncolytic viruses. For example, cells can be engineered to express and secrete in a regulated manner at least one, two, three or more of the following effector molecules: 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 antibodies, anti-TGFβ antibodies, anti-TNFR2, MIP1α (CCL3), MIP1β (CCL5), CCL21, CpG oligodeoxynucleotides, and antitumor peptides (e.g., antimicrobial peptides with antitumor activity, see e.g. Gaspar, D. et al. Front Microbiol. 2013; 4: 294; Chu, H. et al. PLoS One. 2015; 10(5): e0126390 and website:aps.unmc.edu / AP / main.php).

[0006] Provided herein are engineered nucleic acids comprising an expression cassette comprising a promoter and an exogenous polynucleotide sequence encoding a membrane-cleavable chimeric protein from the N-terminus to The C-terminus is oriented and has the following formula: S - C - MT or MT - C - S, where S contains the secretable effector molecule, C contains the protease cleavage site, and MT contains the cell membrane tethering domain, where the promoter is operably linked to an exogenous polynucleotide sequence, and wherein S-C-MT or MT-C-S is configured to behave as a single polypeptide.

[0007] Also provided herein is a membrane cleavable chimeric protein that is oriented from N-terminus to C-terminus and has the following formula: S-C-MT or MT-C-S, wherein S comprises a secretable effector molecule and C comprises a protease cleavage sites, and the MT comprises a cell membrane tether domain in which S-C-MT or MT-C-S is configured to behave as a single polypeptide.

[0008] Also provided herein is an isolated cell comprising an engineered nucleic acid, wherein the engineered nucleic acid comprises an expression cassette comprising a promoter and an exogenous polynucleotide sequence encoding a membrane-cleavable chimeric protein , the membrane-cleavable chimeric protein is oriented from N-terminus to C-terminus and has the following formula: S - C - MT or MT - C - S, where S contains the secretable effector molecule, C contains the protease cleavage site, and MT contains A cell membrane tether domain, wherein the promoter is operably linked to an exogenous polynucleotide sequence, and wherein the S-C-MT or MT-C-S is configured to represent a single polypeptide.

[0009] Also provided herein is an isolated cell comprising a membrane cleavable chimeric protein, wherein the membrane cleavable chimeric protein is oriented from the N-terminus to the C-terminus and has the following formula: S-C-MT or MT-C-S, wherein S comprises a secretable effector molecule, C comprises a protease cleavage site, and MT comprises a cell membrane tether domain, and wherein S - C - MT or MT - C - S is configured to behave as a single polypeptide.

[0010] Also provided herein is a method of inducing membrane-tethered effector molecule release, comprising: a) providing a cell, wherein the cell comprises a membrane-bound protease and a membrane-cleavable chimeric protein, the membrane-cleavable chimeric protein extending from the N-terminus to The C-terminus is oriented and has the following formula: S - C - MT or MT - C - S, where S contains the secretable effector molecule and C contains a homologous protease cleavage site for a membrane-bound protease, where S - C - MT or MT - C-S is configured to express a single polypeptide; and b) culturing the cell under conditions suitable for expression of a membrane-bound protease and a membrane-cleavable chimeric protein, wherein after expression, the membrane-cleavable chimeric protein is tethered to The cell membrane of the cell, and wherein, after expression, the membrane-bound protease cleavage membrane can cleave the cognate membrane-bound protease cleavage site of the chimeric protein, thereby releasing the secretable effector molecule from the cell membrane.

[0011] In some aspects, the promoter is a constitutive promoter. In some aspects, 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 aspects, the promoter is an inducible promoter. In some aspects, the inducible promoter comprises a minimal promoter and a responsive element 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 elements, TCF-LEF response element promoter fusions, hypoxia response elements, SMAD binding elements, STAT3 binding sites, inducer molecule responsive promoters and tandem repeats thereof.

[0012] In some aspects, the promoter is a synthetic promoter. In some aspects, a synthetic promoter comprises an Activation Condition Controlled Polypeptide- (ACP-) binding domain sequence and a promoter sequence. In some aspects, 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, API response element, TCF - LEF response element promoter fusion, hypoxia response element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducer molecule responsive promoters and tandem repeats thereof. In some aspects, the ACP binding domain comprises one or more zinc finger binding sites. In some aspects, a synthetic promoter can be regulated by an activation-controlling polypeptide (ACP) that binds to the ACP-binding domain of the synthetic promoter.

[0013] In some aspects, the ACP is a transcriptional regulator. In some aspects, ACP is a transcriptional repressor. In some aspects, the ACP is a transcriptional activator. In some aspects, the ACP further comprises a repressor protease and one or more cognate cleavage sites for the repressor protease. In some aspects, the ACP further comprises the hormone binding domain of the estrogen receptor (ERT2 domain).

[0014] In some aspects, the ACP is a transcription factor. In some aspects, the transcription factor is a zinc finger-containing transcription factor. In some aspects, the ACP comprises a DNA-binding zinc finger protein domain (ZF protein domain) and a transcriptional effector domain. In some aspects, ZF protein domains are modular in design and consist of zinc finger arrays (ZFAs). In some aspects, the ZF protein domain comprises 1 to 10 ZFAs.

[0015] In some aspects, the effector domain is selected from the group consisting of: a herpes simplex virus protein 16 (VP16) activation domain; an activation domain comprising four tandem copies of VP16, i.e. a VP64 activation domain ; p65 activation domain of NFκB; Epstein-Barr virus R transactivator (Rta) activation domain; triple activation comprising VP64, p65 and Rta activation domain (VPR activation domain) Histone acetyltransferase (HAT) core domain of human E1A-associated protein p300 (p300 HAT core activation domain); Krüppel-associated box (KRAB) repression domain; Repressor element silencing transcription factor (REST) ​​repression domain; the WRPW motif of the hairy-related basic helix-loop-helix repressor protein, known as the WRPW repressor domain; DNA (cytosine-5) - methyltransferase 3B (DNMT3B) repression domain; and HP1α chromoshadow repression domain.

[0016] In some aspects, one or more cognate cleavage sites for the repressor protease are located between the ZF protein domain and the effector domain. In some aspects, the repressor protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3). In some aspects, the cognate cleavage site comprises an NS3 protease cleavage site. In some aspects, the NS3 protease cleavage site comprises an NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, or NS5A / NS5B junction cleavage site. In some aspects, NS3 protease can be blocked by protease inhibitors. In some aspects, the protease inhibitor is selected from the group consisting of simeprevir, danoprevir, asunaprevir, ciluprevir, wave Boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and vociprevir (voxiloprevir).

[0017] In some aspects, ACP is capable of nuclear localization following binding of the ERT2 domain to tamoxifen or a metabolite thereof. In some aspects, the tamoxifen metabolite is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen Fen (endoxifen).

[0018] In some aspects, the ACP further comprises a degron domain, and wherein the degron domain is operably linked to the ACP. In some aspects, the degron domain is derived from a degron selected from the group consisting of: HCV NS4 degron, PEST (two copies of residues 277-307 of human IκBα), GRR (human p105 residues 352-408 of yeast Cdc34), DRR (residues 210-295 of yeast Cdc34), SNS (tandem repeat of SP2 and NB (SP2-NB-SP2 of influenza A or influenza B), RPB (residues of yeast RPB four copies of bases 1688-1702), SPmix (the tandem repeat of SP1 and SP2 (SP2-SP1-SP2-SP1-SP2 of the influenza A virus M2 protein), NS2 (residues 79- 93), ODC (residues 106-142 of ornithine decarboxylase), Nek2A, mouse ODC (residues 422-461), mouse ODC_DA (residues 422-461 of mODC, including D433A and D434A point mutation), APC / C degron, COP1 E3 ligase binding degron motif, CRL4-Cdt2 binding PIP degron, actinfilin binding degron, KEAP1 binding degron, KLHL2 and KLHL3 binding degradation Determinants, MDM2-binding motifs, N-degrons, hydroxyproline modification in hypoxic signaling, plant hormone-dependent SCF-LRR binding degrons, SCF ubiquitin ligase-binding phosphorylation degrons (SCF ubiquitin ligase binding phosphodegron), phytohormone-dependent SCF-LRR-binding degrons, DSGxxS phosphate-dependent degrons, Siah binding motifs, SPOP SBC docking motifs, and PCNA binding PIP boxes. In some aspects, degradation The determinant domain comprises a cereblon (CRBN) polypeptide substrate domain capable of binding to CRBN in response to an immunomodulatory drug (IMiD) thereby promoting ubiquitin pathway-mediated ACP degradation. In some aspects, CRBN The 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 its drug-inducible binding Fragments of CRBN. In some aspects, the CRBN polypeptide receptor domain is a chimeric fusion product of a native CRBN polypeptide sequence. In some aspects, the CRBN polypeptide receptor domain has a combination of FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 175) IKZF3 / ZFP91 / IKZF3 chimeric fusion product of amino acid sequence. In some aspects, the IMiD is an FDA approved drug. In some aspects, the IMiD is selected from the group consisting of thalidomide, lenalidomide, and pomalidomide. In some aspects, the degron domain is N-terminal to the repressor protease, C-terminal to the repressor protease, N-terminal to the ZF protein domain, C-terminal to the ZF protein domain, N-terminal to the effector domain, or C-terminus of the effector domain.

[0019] In some aspects, the promoter is a tissue-specific promoter.

[0020] In some aspects, the secretable effector molecule comprises a signal peptide or signal anchor sequence. In some aspects, the signal peptide comprises a native signal peptide native to the secretable effector molecule. In some aspects, the signal peptide comprises a non-native signal peptide, or the signal anchor sequence comprises a non-native signal anchor sequence that is not native to the secretable effector molecule. In some aspects, 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 fluorescence Gaussia luciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, serum albumin preprotein, azurecin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, CXCL12, IL21, CD8, NKG2D, TNFR2 and GMCSF.

[0021] In some aspects, the secretable effector molecule is selected from a therapeutic class, wherein the therapeutic class is selected from the group consisting of: cytokines, chemokines, homing molecules, growth factors, coactivating molecules , tumor microenvironment regulators, ligands, antibodies, peptides and enzymes. In some aspects, the interleukin is selected from the group consisting of: IL-1-β, 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-γ and TNF-α. In some aspects, the secretable effector molecule comprises an IL-15, IL-12, or IL-12p70 fusion protein. In some aspects, the secretable effector molecule comprises IL-15. In some aspects, the secretable effector molecule comprises IL-15 having the amino acid sequence of SEQ ID NO: 199. In some aspects, the secretable effector molecule comprises IL-15 and IL-15Rα sushi domain. In some aspects, the secretable effector molecule comprises an IL-15 / IL-15Rα sushi domain fusion protein having the amino acid sequence of SEQ ID NO: 202. In some aspects, the secretable effector molecule consists of IL-15 and an IL-15Rα sushi domain. In some aspects, the secretable effector molecule comprises IL-12. In some aspects, the secretable effector molecule comprises an IL-12p70 fusion protein. In some aspects, the secretable effector molecule comprises an IL-12p70 fusion protein having the amino acid sequence of SEQ ID NO:203. In some aspects, the secretable effector molecule consists of IL-12. In some aspects, the secretable effector molecule consists of an IL-12p70 fusion protein. In some aspects, the secretable effector molecule is IL-15. In some aspects, the chemoattractant is selected from the group consisting of CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1. In some aspects, the homing molecule is selected from the group consisting of: anti-integrin α4, β7; anti-MAdCAM; SDF1; and MMP-2. In some aspects, the growth factor is selected from the group consisting of: FLT3L and GM-CSF. In some aspects, the coactivating molecule is selected from the group consisting of 4-1BBL and CD40L. In some aspects, the tumor microenvironment modulator is selected from the group consisting of adenosine deaminase, TGFβ inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2. In some aspects, the TGFβ inhibitor is selected from the group consisting of anti-TGFβ peptides, anti-TGFβ antibodies, TGFb-TRAP, and combinations thereof. In some aspects, 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-phosphatidylseramine acid antibody, anti-CD27 antibody, anti-TNFα antibody, anti-TREM1 antibody and anti-TREM2 antibody. In some aspects, the VEGF inhibitor comprises an anti-VEGF antibody, an anti-VEGF peptide, or a combination thereof. In some aspects, the secretable effector molecule is an effector molecule of human origin.

[0022] In some aspects, the protease cleavage site is selected from the group consisting of a type 1 transmembrane protease cleavage site, a type II transmembrane protease cleavage site, a GPI anchored protease cleavage site, an ADAM8 protease cleavage site 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 , ADAM28 protease cleavage site, ADAM30 protease cleavage site, ADAM33 protease cleavage site, BACE1 protease cleavage site, BACE2 protease cleavage site, SIP protease cleavage site, MT1-MMP protease cleavage site, MT3-MMP protease cleavage site site, MT5-MMP protease cleavage site, furin protease cleavage site, PCSK7 protease cleavage site, matriptase protease cleavage site, protease-2 protease cleavage site, MMP9 Protease cleavage site and NS3 protease cleavage site. In some aspects, the protease cleavage site is cleavable by a protease selected from the group consisting of type 1 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, protease protease, protease-2 protease, MMP9 protease, and NS3 protease.

[0023] In some aspects, the protease cleavage site is cleavable by ADAM17 protease. In some aspects, the protease cleavage site comprises a first region having the amino acid sequence of PRAE (SEQ ID NO: 176). In some aspects, the protease cleavage site comprises a second region having the amino acid sequence of KGG (SEQ ID NO: 177). In some aspects, the first region is positioned N-terminal to the second region. In some aspects, the protease cleavage site comprises the amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 178), wherein X1 is A, Y, P, S or F, and wherein X2 is V, L, S, I, Y, T or A. In some aspects, 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 aspects, the protease cleavage site comprises the amino acid sequence of PRAEAVKGG (SEQ ID NO: 179). In some aspects, the protease cleavage site comprises the amino acid sequence of PRAEALKGG (SEQ ID NO: 180). In some aspects, the protease cleavage site comprises the amino acid sequence of PRAEYSKGG (SEQ ID NO: 181). In some aspects, the protease cleavage site comprises the amino acid sequence of PRAEPIKGG (SEQ ID NO: 182). In some aspects, the protease cleavage site comprises the amino acid sequence of PRAEAYKGG (SEQ ID NO: 183). In some aspects, the protease cleavage site comprises the amino acid sequence of PRAESSKGG (SEQ ID NO: 184). In some aspects, the protease cleavage site comprises the amino acid sequence of PRAEFTKGG (SEQ ID NO: 185). In some aspects, the protease cleavage site comprises the amino acid sequence of PRAEAAKGG (SEQ ID NO: 186). In some aspects, the protease cleavage site comprises the amino acid sequence of DEPHYSQRR (SEQ ID NO: 187). In some aspects, the protease cleavage site comprises the amino acid sequence of PPLGPIFNPG (SEQ ID NO: 188). In some aspects, the protease cleavage site comprises the amino acid sequence of PLAQAYRSS (SEQ ID NO: 189). In some aspects, the protease cleavage site comprises the amino acid sequence of TPIDSSFNPD (SEQ ID NO: 190). In some aspects, the protease cleavage site comprises the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191). In some aspects, the protease cleavage site comprises the amino acid sequence of ITQGLAVSTISSFF (SEQ ID NO: 198).

[0024] In some aspects, the cell membrane tether domain comprises a transmembrane-intracellular domain or a transmembrane domain. In some aspects, the transmembrane-intracellular and / or transmembrane domains are derived from PDGFR-β, CD8, CD28, CD3ζ chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1 , LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1 or BTLA. In some aspects, the cell membrane tether domain comprises a cell surface receptor or a cell membrane-associated portion thereof.

[0025] In some aspects, the cell membrane tether domain comprises a post-translational modification tag, or a motif capable of post-translational modification to modify the chimeric protein to include a post-translational modification tag, wherein the post-translational modification tag is capable of binding to the cell membrane association. In some aspects, the post-translational modification tag comprises a lipid anchor domain, optionally wherein the lipid anchor domain is selected from the group consisting of a GPI lipid anchor, a myristoylation tag, and a palmitoylation tag.

[0026] In some aspects, when expressed in a cell, the secreted effector molecule is tethered to the cell membrane of the cell. In some aspects, the secretable effector molecule is released from the cell membrane when expressed in a cell expressing a protease capable of cleaving the protease cleavage site. In some aspects, the protease expressed on the cell membrane is endogenous to the cell. In some aspects, the protease is selected from the group consisting of Type 1 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, Protein Lyase protease, Protease-2 protease and MMP9 protease. In some aspects, the protease is ADAM17 protease.

[0027] In some aspects, the protease expressed on the cell membrane is heterologous to the cell. In some aspects, the protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3). In some aspects, the protease cleavage site comprises an NS3 protease cleavage site. In some aspects, the NS3 protease cleavage site comprises an NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, or NS5A / NS5B junction cleavage site. In some aspects, proteases can be blocked by protease inhibitors. In some aspects, the protease inhibitor is selected from the group consisting of simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, pariravir Gecarevir, Telaprevir, Glaprevir, Gelcarevir, and Vociprevir. In some aspects, the expression and / or localization of proteases can be modulated. In some aspects, expression and / or localization are regulated by the cell state of the cell.

[0028] In some aspects, the engineered nucleic acid is a single- or double-stranded nucleic acid selected from the group consisting of: DNA, cDNA, RNA, mRNA, and naked plastids.

[0029] In some aspects, the isolated cell line is 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 cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor infiltrating lymphocytes (TIL), innate lymphoid cells, mast cells, eosinophils, basophils Sex spheres, neutrophils, bone marrow cells, macrophages, monocytes, dendritic cells, red blood cells, platelet cells, human embryonic stem cells (ESC), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSC) , induced pluripotent stem cells (iPSC) and iPSC-derived cells. In some aspects, the isolated cells are natural killer (NK) cells.

[0030] In some aspects, the isolated cells are autologous. In some aspects, the isolated cells are allogeneic. In some aspects, the isolated cell line is a tumor cell 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, pancreas tumor cells, prostate tumor cells, skin tumor cells, thyroid tumor cells and uterine tumor cells.

[0031] In some aspects, the isolated cell line is engineered via transduction with an oncolytic virus.

[0032] In some aspects, the isolated cells further comprise a protease capable of cleaving the protease cleavage site. In some aspects, the protease is an endogenous protease. In some aspects, the endogenous protease is selected from the group consisting of: Type 1 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, PCSK7 Protease, Protease Protease, Protease-2 Protease, and MMP9 Protease. In some aspects, the endogenous protease is ADAM17 protease. In some aspects, the protease is a heterologous protease. In some aspects, the heterologous protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3). In some aspects, the protease is expressed on the cell membrane of the isolated cell. In some aspects, a protease is capable of cleaving a protease cleavage site. In some aspects, cleavage of the protease cleavage site releases a secretable effector molecule from the cell membrane of the isolated cell.

[0033] In some aspects, the protease cleavage site comprises a first region having the amino acid sequence of PRAE (SEQ ID NO: 176). In some aspects, the protease cleavage site comprises a second region having the amino acid sequence of KGG (SEQ ID NO: 177). In some aspects, the first region is positioned N-terminal to the second region. In some aspects, the protease cleavage site comprises the amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 178), wherein X1 is A, Y, P, S or F, and wherein X2 is V, L, S, I, Y, T or A. In some aspects, 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 aspects, the protease cleavage site comprises the amino acid sequence of PRAEAVKGG (SEQ ID NO: 179). In some aspects, the protease cleavage site comprises the amino acid sequence of PRAEALKGG (SEQ ID NO: 180). In some aspects, the protease cleavage site comprises the amino acid sequence of PRAEYSKGG (SEQ ID NO: 181). In some aspects, the protease cleavage site comprises the amino acid sequence of PRAEPIKGG (SEQ ID NO: 182). In some aspects, the protease cleavage site comprises the amino acid sequence of PRAEAYKGG (SEQ ID NO: 183). In some aspects, the protease cleavage site comprises the amino acid sequence of PRAESSKGG (SEQ ID NO: 184). In some aspects, the protease cleavage site comprises the amino acid sequence of PRAEFTKGG (SEQ ID NO: 185). In some aspects, the protease cleavage site comprises the amino acid sequence of PRAEAAKGG (SEQ ID NO: 186). In some aspects, the protease cleavage site comprises the amino acid sequence of DEPHYSQRR (SEQ ID NO: 187). In some aspects, the protease cleavage site comprises the amino acid sequence of PPLGPIFNPG (SEQ ID NO: 188). In some aspects, the protease cleavage site comprises the amino acid sequence of PLAQAYRSS (SEQ ID NO: 189). In some aspects, the protease cleavage site comprises the amino acid sequence of TPIDSSFNPD (SEQ ID NO: 190). In some aspects, the protease cleavage site comprises the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191). In some aspects, the protease cleavage site comprises the amino acid sequence of ITQGLAVSTISSFF (SEQ ID NO: 198).

[0034] In some aspects, the isolated cells further comprise an antigen recognition receptor. In some aspects, the antigen recognition receptor recognizes an antigen 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, dense junction Protein 18.2, cMet, CSPG4, CTLA, DLK1, DLL3, DR5, EGFR, ENPP3, EpCAM, EphA2, Ephrin A4, ETBR, FGFR2, FGFR3, FRα, FRb, GCC, GD2, GFRa4, gpA33, 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, Connexin 4, NKG2D, NOTCH3, NY ESO 1, Ovarin, P-cadherin, pan-Erb2, PSCA, PSMA, PTK7, ROR1, S Aures, SCT, SLAMF7, SLITRK6, SSTR2, STEAP1, Survivin, TDGF1, TIM1, TROP2 and WT1. In some aspects, an antigen recognition receptor comprises an antigen binding domain. In some aspects, the antigen binding domain comprises an antibody, an antigen binding fragment of an antibody, a F(ab) fragment, a F(ab') fragment, a single chain variable fragment (scFv), or a single domain antibody (sdAb). In some aspects, the antigen binding domain comprises a single chain variable fragment (scFv). In some aspects, a scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some aspects, VH and VL are separated by a peptide linker. In some aspects, the scFv comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. In some aspects, the antigen recognizing receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0035] In some aspects, the antigen recognizes a receptor CAR. In some aspects, the CAR comprises one or more intracellular signaling domains, and the one or more intracellular signaling domains are selected from the group consisting of: CD3ζ chain intracellular signaling domain, CD97 intracellular signaling structure 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 Signaling domain, DAP12 intracellular signaling domain and MyD88 intracellular signaling domain. In some aspects, the CAR comprises a transmembrane domain, and the transmembrane domain is selected from the group consisting of: CD8 transmembrane domain, CD28 transmembrane domain, CD3ζ chain transmembrane domain, CD4 transmembrane structure 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 structure Domain and BTLA transmembrane domain. In some aspects, the CAR comprises a spacer between the antigen binding domain and the transmembrane domain.

[0036] Also provided herein are compositions comprising any of the isolated cells described herein and a pharmaceutically acceptable carrier.

[0037] Also provided herein is a method of treating an individual in need thereof comprising administering a therapeutically effective dose of any of the isolated cells described herein or any of the compositions described herein. In some aspects, the isolated cells are derived from an individual. In some aspects, the isolated cells are allogeneic to the individual. In some aspects, the method further comprises administering a checkpoint inhibitor. In some aspects, 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-TNFa antibody, anti-TREM1 antibody and anti-TREM2 antibody. In some aspects, the method further comprises administering an anti-CD40 antibody.

[0038] Also provided herein are lipid-based constructs comprising any of the engineered nucleic acids described herein, or any of the expression vectors described herein, or any of the membrane-cleavable chimeric proteins described herein. In some aspects, lipid-based structures comprise extracellular vesicles, lipid nanoparticles, micelles, or liposomes. In some aspects, the extracellular vesicle is selected from the group consisting of nanovesicles and exosomes. In some aspects, lipid-based structures comprise lipid nanoparticles or micelles. In some aspects, lipid-based structures comprise liposomes.

[0039] Also provided herein are compositions comprising any of the lipid-based structures described herein and a pharmaceutically acceptable carrier.

[0040] Also provided herein is a method of treating a subject in need thereof comprising administering a therapeutically effective amount of any of the lipid-based structures described herein or any of the compositions described herein. In some aspects, administering comprises systemic administration. In some aspects, lipid-based structures enable the engineering of individual cells. In some aspects, the method further comprises administering a checkpoint inhibitor. In some aspects, 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-TNFa antibody, anti-TREM1 antibody and anti-TREM2 antibody. In some aspects, the method further comprises administering an anti-CD40 antibody.

[0041] Also provided herein are nanoparticles comprising any of the engineered nucleic acids described herein or any of the membrane-cleavable chimeric proteins described herein. In some aspects, nanoparticles comprise inorganic materials.

[0042] Also provided herein are compositions comprising any of the nanoparticles described herein.

[0043] Also provided herein is a method of treating an individual in need thereof, the method comprising administering a therapeutically effective dose of any nanoparticle described herein or any composition described herein. In some aspects, administering comprises systemic administration. In some aspects, nanoparticles can engineer the cells of an individual. In some aspects, the method further comprises administering a checkpoint inhibitor. In some aspects, 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-TNFa antibody, anti-TREM1 antibody and anti-TREM2 antibody. In some aspects, the method further comprises administering an anti-CD40 antibody.

[0044] Also provided herein are viruses engineered to contain any of the engineered nucleic acids described herein or any of the expression vectors described herein. In some aspects, the virus is selected from the group consisting of lentivirus, retrovirus, oncolytic virus, adenovirus, adeno-associated virus (AAV), and virus-like particle (VLP).

[0045] Also provided herein are compositions comprising any of the engineered viruses described herein and a pharmaceutically acceptable carrier.

[0046] Also provided herein are methods of treating an individual in need thereof comprising administering a therapeutically effective dose of any engineered virus described herein or any composition described herein. In some aspects, administering comprises systemic administration. In some aspects, the engineered virus infects the individual's cells and expresses the expression cassette. In some aspects, the method further comprises administering a checkpoint inhibitor. In some aspects, 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-TNFa antibody, anti-TREM1 antibody and anti-TREM2 antibody. In some aspects, the method further comprises administering an anti-CD40 antibody.

Implementation

[0048] CROSS-REFERENCE TO RELATED APPLICATIONS

[0049] This application claims the benefit of U.S. Provisional Application No. 63 / 193,004, filed May 25, 2021, and U.S. Provisional Application No. 63 / 109,812, filed November 4, 2020, which Each of these applications is hereby incorporated by reference in its entirety for all purposes.

[0050] Provided herein are chimeric proteins (or engineered nucleic acids encoding chimeric proteins) having the formula S-C-MT or MT-C-S, oriented from N-terminus to C-terminus and appearing as a single polypeptide. S refers to secretable effector molecule. C refers to the protease cleavage site. MT refers to the cell membrane tether domain. Membrane cleavable chimeric proteins are engineered such that secretion of effector molecules can be regulated in a protease-dependent manner. In particular, membrane cleavable chimeric proteins are engineered such that the secretion of effector molecules can be regulated as part of a "membrane cleavable" system in which a protease cleavage site ("C") and a cell membrane tether domain (" MT") allows regulation of secretion of effector molecules in a protease-dependent manner. Without wishing to be bound by theory, components of the membrane cleavage system present in membrane cleavable chimeric proteins typically regulate secretion by means of the following cellular processes: - MT: The cell membrane tether domain contains a transmembrane domain (or transmembrane- intracellular domain) that directs the cellular transport of the chimeric protein, allowing the protein to insert into or otherwise associate with the cell membrane ("tether") -C: expressed in the chimeric protein and localized to Once in the cell membrane, the protease cleavage site directs cleavage of the chimeric protein, allowing release ("secretion") of the effector molecule into the extracellular space. Typically, a protease cleavage site is specific for a protease, including sites engineered to be specific for a protease. Protease cleavage sites can be selected or engineered for optimal protein expression, cell type-specific cleavage, cell state-specific cleavage, and / or loading with desired kinetics (e.g., membrane-bound chimeric protein levels versus secreted cleavage and release

[0051] In some aspects, provided herein are membrane cleavable chimeric proteins (or encoding membrane cleavable chimeric proteins) having a protein of interest (e.g., any effector molecule described herein), a protease cleavage site, and a cell membrane tether domain. engineered nucleic acids for chimeric proteins).

[0052] "Effector molecule" refers to a molecule (eg, a nucleic acid, such as DNA or RNA, or a protein (polypeptide) or peptide) that binds to another molecule and modulates the biological activity of the molecule to which it binds. For example, effector molecules can be used as ligands to increase or decrease enzyme activity, gene expression, or cellular signaling. Thus, in some embodiments, effector molecules modulate (activate or inhibit) different immunomodulatory mechanisms. An effector molecule can also indirectly regulate a second downstream molecule by directly binding to and modulating the molecule.

[0053] In certain embodiments described herein (e.g., generally, for all membrane cleavable chimeric proteins described herein), the effector molecule is a secretable effector molecule (e.g., as used herein The membrane-cleavable chimeric protein has the formula S-C-MT or MT-C-S, referred to as "S"). Non-limiting examples of effector molecules include cytokines, chemokines, enzymes that regulate metabolite levels, growth factors, coactivating molecules, modulators of the tumor microenvironment, ligands, peptides, enzymes, antibodies, regulatory cytokines, Antibody or decoy molecules, homing molecules and / or integrins.

[0054] The term "modulation" encompasses maintenance of biological activity, inhibition (partial or complete) of biological activity and stimulation / activation (partial or complete) of biological activity. The term also encompasses reducing or increasing (eg, enhancing) a biological activity. When tumor-mediated immunosuppressive mechanisms mediated by one effector molecule (eg, stimulation of T cell signaling) differ from tumor-mediated immunosuppressive mechanisms mediated by another effector molecule (eg, stimulation of antigen presentation and / or processing) , it is believed that the two different effector molecules "regulate different mechanisms of tumor-mediated immunosuppression".

[0055] Modulation by effector molecules can be direct or indirect. Direct modulation occurs when an effector molecule binds to another molecule and modulates the activity of that molecule. Indirect modulation occurs when an effector molecule binds to another molecule, modulates the activity of that molecule, and as a result of this modulation, the activity of a further molecule (to which the effector molecule is not bound) is modulated.

[0056] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in an increase in immunostimulation and / or anti-tumor immune response (e.g., systemically or in the tumor microenvironment) by at least 10 % (for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). For example, modulation of tumor-mediated immunosuppressive mechanisms can result in at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 70% increase in immune stimulation and / or anti-tumor immune response At least 80%, at least 90%, at least 100%. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in a 10-20%, 10-30%, 10-40%, 10-50%, 10-60% increase in immune stimulation and / or anti-tumor immune response %, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70% %, 20-80%, 20-90%, 20-100%, 20-200%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100% or 50-200% %. It should be understood that immune stimulation and / or an "increase" of an anti-tumor immune response, e.g., systemically or in the tumor microenvironment, is relative to the immune stimulation and / or anti-tumor immune response that would otherwise occur in the absence of one or more effector molecules. antitumor immune response.

[0057] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in an increase in immunostimulation and / or anti-tumor immune response (e.g., systemically or in the tumor microenvironment) by at least 2 Times (eg, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 25 times, 50 times, or 100 times). For example, modulation of tumor-mediated immunosuppressive mechanisms can result in at least a 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold increase in immune stimulation and / or anti-tumor immune response. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in a 2-10-fold, 2-20-fold, 2-30-fold, 2-40-fold, 2-50-fold increase in immune stimulation and / or anti-tumor immune response times, 2-60 times, 2-70 times, 2-80 times, 2-90 times or 2-100 times.

[0058] Non-limiting examples of immunostimulatory and / or anti-tumor immune mechanisms include T cell signaling, activation, and / or recruitment; antigen presentation and / or processing; natural killer cell-mediated cytotoxic signaling, activation, and / or recruitment ; dendritic cell differentiation and / or maturation; immune cell recruitment; pro-inflammatory macrophage signaling, activity and / or recruitment; matrix degradation; production of immunostimulatory metabolites; Stimulator of Interferon Gene (STING) signaling (which increases IFN secretion and Th1 polarization, promoting anti-tumor immune response) and / or type I interferon signaling. An effector molecule may stimulate at least one (one or more) of the above-mentioned immunostimulatory mechanisms, thereby resulting in an increase in the immunostimulatory response. The aforementioned immune stimulation can be assessed, for example, using in vitro assays for T cell proliferation or cytotoxicity, in vitro antigen presentation assays, expression assays (e.g., for specific markers), and / or cell secretion assays (e.g., cytokines) And / or changes in anti-tumor immune mechanisms.

[0059] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in at least a 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 200%). For example, modulation of tumor-mediated immunosuppressive mechanisms can result in at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% reduction of the immunosuppressive response %, at least 100%. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in a 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70% reduction in the immunosuppressive response , 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80% , 20-90%, 20-100%, 20-200%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100%, or 50-200%. It is to be understood that a "reduction" of an immunosuppressive response, eg, systemically or in the tumor microenvironment, is relative to an immunosuppressive response that would otherwise occur in the absence of the effector molecule or molecules.

[0060] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in at least a 2-fold (eg, 2-fold) reduction in immunosuppressive responses (eg, systemically or in the tumor microenvironment) , 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 25 times, 50 times or 100 times). For example, modulation of tumor-mediated immunosuppressive mechanisms can result in at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold reduction in the immunosuppressive response. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in a 2-10 fold, 2-20 fold, 2-30 fold, 2-40 fold, 2-50 fold, 2-60 fold reduction in the immunosuppressive response , 2-70 times, 2-80 times, 2-90 times or 2-100 times.

[0061] 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, tumor checkpoint molecules Production / maintenance, myeloid-derived suppressor cell signaling, activity and / or recruitment, immunosuppressive factor / metabolite production and / or vascular endothelial growth factor signaling. An effector molecule can inhibit at least one (one or more) of the above-mentioned immunosuppressive mechanisms, thereby resulting in a reduction of the immunosuppressive response. Changes in the aforementioned immunosuppressive mechanisms can be assessed, for example, by analyzing: increased T cell proliferation and / or increased IFNγ production (negative co-stimulatory signal, Treg cell signaling and / or MDSC); Annexin V / PI flow cytometry Staining (pro-apoptotic signaling); flow staining (tumor checkpoint molecule production / maintenance) for expression (e.g., PDL1 expression); ELISA, LUMINEX®, RNA via qPCR, enzymatic assays, e.g., IDO tryptamine Acid catabolism (immunosuppressive factor / metabolite production); and phosphorylation of PI3K, Akt, p38 (VEGF signaling).

[0062] In some embodiments, the effector molecules function additively: the effects of the two effector molecules may, for example, be equal to the sum of the effects of the two effector molecules functioning individually. In other embodiments, the effector molecules function synergistically: the effect of two effector molecules may, for example, be greater than the combined function of the two effector molecules.

[0063] Effector molecules that modulate tumor-mediated immunosuppressive mechanisms and / or alter the tumor microenvironment may be, for example, secreted factors (eg, cytokines, chemokines, antibodies, and / or modulating extracellular mechanisms involved in the immune system or decoy receptors), inhibitors (e.g., antibodies, antibody fragments, ligand TRAP, and / or small blocking peptides), intracellular factors that control cell state (e.g., microRNAs that modulate cell state to enhance pro-inflammatory properties and / or transcription factors), factors packaged into extracellular bodies (e.g., microRNAs, cytosolic factors, and / or extracellular factors), surface-displayed factors (e.g., checkpoint inhibitors, TRAIL) and / or metabolic genes (eg, enzymes that produce / regulate or degrade metabolites or amino acids).

[0064] In some embodiments, at least one effector molecule stimulates immunostimulatory mechanisms in the tumor microenvironment and / or inhibits immunosuppressive mechanisms in the tumor microenvironment.

[0065] In some embodiments, at least one effector molecule (a) stimulates T cell signaling, activation and / or recruitment, (b) stimulates antigen presentation and / or processing, (c) stimulates natural killer cell-mediated cellular Toxic signaling, activity and / or recruitment, (d) stimulation of dendritic cell differentiation and / or maturation, (e) stimulation of immune cell recruitment, (f) stimulation of pro-inflammatory macrophage signaling, activity and / or recruitment or inhibition of anti-inflammatory Inflammatory macrophage signaling, activity and / or recruitment, (g) stimulation of matrix degradation, (h) stimulation of immunostimulatory metabolite production, (i) stimulation of type I interferon signaling, (j) inhibition of negative costimulatory signaling, ( k) Inhibition of pro-apoptotic signaling of anti-tumor immune cells, (l) inhibition of T regulatory (Treg) cell signaling, activity and / or recruitment, (m) inhibition of tumor checkpoint molecules, (n) stimulation of interferon gene stimulators (STING) signaling, (o) inhibiting myeloid-derived suppressor cell signaling, activity and / or recruitment, (p) degrading immunosuppressive factors / metabolites, (q) inhibiting vascular endothelial growth factor signaling, and / or (r) directly kill tumor cells.

[0066] In some embodiments, effector molecules may be selected from the following non-limiting classes of molecules: interleukins, antibodies, chemokines, nucleotides, peptides, and enzymes. Non-limiting examples of the foregoing classes of effector molecules are listed in Table 1, and specific sequences encoding exemplary effector molecules are listed in Table 2. The effector molecules can be human, such as those listed in Table 1 or Table 2, or the human equivalents of the murine effector molecules listed in Table 1 or Table 2. Effector molecules can be of human origin, such as endogenous human effector molecules, or modified and / or optimized for function (e.g., codon-optimized for improved performance, modified for improved stability, or in other Modified at the signal sequence) effector molecules (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, such as codon optimization for a particular species (e.g., human, mouse, bacteria, etc.) .

[0067] In some embodiments, the effector molecule comprises interleukin 12 (IL-12), eg, p35 and p40 as a dimer generally referred to in the art as IL12p70. In some embodiments, the first effector molecule comprises an IL12p70 fusion protein. In some embodiments, the IL12p70 fusion protein is a human IL12p70 fusion protein. In some embodiments, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 203.

[0068] In some embodiments, the effector molecule comprises interleukin 15 (IL-15). In some embodiments, the effector molecule consists of IL-15 (see, eg, SEQ ID NO: 199). In some embodiments, the effector molecule comprises a fusion protein comprising IL-15 and the extracellular portion of IL-15 receptor alpha (IL-15Rα), such as the sushi domain as set forth in SEQ ID NO: 201 . An exemplary IL-15 / IL-15Rα sushi domain fusion is provided as SEQ ID NO: 202. Table 1. Exemplary Effector Molecules Effector name category Function Anti-CD40 or CD40 ligand Agonist antibody Stimulates B cells and antigen-presenting cells. Flt3 ligand agonist Stimulate bone marrow cells and antigen presenting cells CXCL10-11 fusion chemokines Attract T cells TGFb blocking peptide antagonist peptide Inhibits TGFb pathway, TME modulator Adenosine deaminase (ADA) TME modulator Degradation of inhibitory adenosine in the TME Kyneurinase TME modulator Degradation of kyneurine HPGE2 TME modulator Degradation of PGE2 CXCL13 chemokines attract B cells Anti-PD-1 / PD-L1 Agonist antibody remove checkpoint anti-CTLA-4 Agonist antibody remove checkpoint anti-VEGF antagonist antibody Neutralizes immunosuppressive / angiogenic factors Anti-TNFα antagonist antibody Neutralizes interleukins / pro-tumor factors Anti-IL-10 antagonist antibody Neutralizes immunosuppressive cytokines Anti-SDF1 / CXCL12 antagonist antibody Neutralizes tumor-promoting chemokine (TβRII)2 well Capture trap Neutralizes immunosuppressive cytokines CCL21 chemokines Attract white blood cells / NK CCL1 chemokines Attract white blood cells / NK CCL17 chemokines Attract white blood cells / NK CCL19 chemokines Attract white blood cells / NK CCL21 chemokines Attract white blood cells / NK CCL20 chemokines Attract white blood cells / NK CCL21a chemokines Attract white blood cells / NK MIP1b (CCL5) chemokines Attract white blood cells / NK CXCL10 chemokines Attract white blood cells / NK CXCL11 chemokines Attract white blood cells / NK CCL2 chemokines attract monocytes MIP-1α (CCL3) chemokines Attract white blood cells / NK XCL1 chemokines Attract white blood cells / NK IFNβ Cytokines T cell response, tumor cell killing IFNγ Cytokines T cell response, tumor cell killing IL-12 Cytokines T cells, NK cells IL-1β Cytokines T cells, NK cells IL-15 Cytokines Stimulate T cells and NK IL-2 Cytokines Stimulate T cells and NK IL-21 Cytokines Stimulate T-cells IL-24 Cytokines Stimulate T-cells IL36-γ Cytokines Stimulate T-cells IL-7 Cytokines Stimulate T-cells IL-22 Cytokines Stimulate T-cells IL-18 Cytokines Stimulate T-cells granzyme / perforin enzyme directly kill tumor cells OX86 (anti-OX40) Ligand Stimulate T-cells anti-TGFβ neutralizing antibody Neutralizes immunosuppressive cytokines TRAIL Receptor / ligand directly kill tumor cells FASL (CD49L) Receptor / ligand directly kill tumor cells OX40-L Receptor / ligand Stimulate T-cells cGAS secreted molecule stimulate antigen presenting cells 41BBL secreted molecule co-activation of T cells CD40L secreted molecule Stimulate T-cells GM-CSF secreted molecule mononuclear growth factor STING secreted molecule stimulate antigen presenting cells HAC-V "Microbody"_PD1 antagonist antibody suppress checkpoint wxya Prodrug Converted to cytotoxic molecules after activation CpG / nucleotide Nucleotide STING agonist Table 2: Exemplary Effector Molecule Sequences IL-12 (human) (SEQ ID NO: 56) ATGTGCCATCAGCAGCTTGTCATATCTTGGTTTTCACTTGTATTCCTGGCCAGCCCTTTGGTTGCGATCTGGGAGCTCAAGAAGGATGTGTACGTTGTAGAGCTGGACTGGTACCCCGATGCTCCCGGTGAGATGGTCGTTTTGACATGTGACACTCCAGAAGAGGACGGTATTACGTGGACTCTGGACCAGTCCTCCGAAGTTCTTGGTTCT GGTAAGACTCTGACTATCCAGGTGAAAGAATTTGGGGATGCGGGACAATACACATGCCACAAGGGAGGCGAGGTGTTGTCTCATAGTTTGCTGCTTCTCCACAAGAAAGAGGATGGAATCTGGAGCACCGACATACTCAAGGATCAAAAGGAACCCAAAAATAAGACATTTCTGCGATGTGAGGCTAAGAACTATAGTGGCCGCTTCACTTGTTGGTG GCTGACTACCATCAGCACAGATCTCACGTTTTTCAGTAAAAAGTAGTAGAGGTTCAAGTGATCCTCAAGGGGTAACGTGCGGTGCTGCAACACTGTCTGCTGAACGCGTAAGAGGAGATAATAAGGAGTACGAGTATTCCGTAGAATGCCAAGAGGACAGTGCTTGTCCTGCGGCCGAGGAGTCTCTCCCCAATAGAAGTGATGGTGGACGCGGTGCATAAACTGA AATATGAGAACTACACAAAGCAGTTTTTTATAAGAGATATCATCAAGCCCGATCCGCCGAAGAATTTGCAACTTAAACCGCTTAAAAACTCACGCCAGGTTGAAGTATCCTGGGAGTATCCGGATACATGGTCAACACCACACAGCTATTTTTCCCTTACCTTCTGTGTGCAGGTCCAAGGGAAGAGCAAAAGGGAGAAGAAGGACAGGGTATTCACTGATAAAAC TTCCGCGACGGTCATCTGCCGAAAAAACGCTAGTATATCTGTACGGGCGCAGGATAGGTACTATAGTTCTTCTTGGTCTGAGTGGGCCTCAGTTCCGTGCTCTGGGGGAGGAAGTGGAGGAGGGTCCGGCGGTGGAAGCGGGGGAGGGAGTCGCAACTTGCCAGTGGCTACACCAGATCCAGGCATGTTCCATGTCTGCATCATTCCCA GAATCTCCTGAGAGCGGTGTCAAATATGCTCCAAAAAGCGAGACAAACACTGGAATTTTACCCGTGTACCAGTGAGGAGATTGATCACGAGGACATAACCAAGGACAAGACCTCAACTGTAGAAGCGTGTTTGCCGCTGGAGTTGACTAAGAATGAGTCCTGCCTCAATTCCAGAGAAACTTCATTCATTACTAACGGCAGTTGTCTTGCATCCCGGAAAACGTCCT TTATGATGGCCCTTTGCCTTAGTTCAATTTACGAGGATCTTAAAATGTATCAAGTGGAGTTTAAAACCATGAATGCTAAACTTCTTATGGACCCCAAACGACAAATTTTTCTGGATCAGAATATGCTTGCCGTGATAGACGAACTCATGCAGGCGCTTAATTTTAACTCCGAAACAGTTCACAAAAAATCTAGCCTTGAAGAACCTGATTTTTATAAAACGAAGATTA AACTGTGTATCCTGCTGCATGCCTTTCGCATCCGAGCTGTCACAATCGATAGGGTTATGTCCTACCTTAACGCGAGCtaG IL-12p70 (human; codon optimized; signal sequence in bold) (SEQ ID NO: 57) ATGTGCCATCAGCAACTCGTCATCTCCTGGTTCTCCCTTGTGTTCCTCGCTTCCCCTCTGGTCGCCATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAGCTGGATTGGTACCCGGACGCCCCTGGAGAAATGGTCGTGCTGACTTGCGATACGCCAGAAGAGGACGGCATAACCTGGACCCTGGATCAGAGCTCCGAGGTGCTCGGA AGCGGAAAGACCCTGACCATTCAAGTCAAGGAGTTCGGCGACGCGGGCCAGTACACTTGCCACAAGGGTGGCGAAGTGCTGTCCCACTCCCTGCTGCTGCTGCCACAAGAAAGAGGATGGAATCTGGTCCACTGACATCCTCAAGGACCAAAAAGAACCGAAGAACAAGACCTTCCTCCGCTGCGAAGCCAAGAACTACAGCGGTCGGTTCACCTGTTGGTG GCTGACGACAATCTCCACCGACCTGACTTTCTCCGTGAAGTCGTCACGGGGATCAAGCGATCCTCAGGGCGTGACCTGTGGAGCCGCCACTCTGTCCGCCGAGAGAGTCAGGGGAGACAACAAGGAATATGAGTACTCCGTGGAATGCCAGGAGGACAGCGCCTGCCCTGCCGCGGAAGAGTCCCTGCCTATCGAGGTCATGGTCGATGCCGT GCATAAGCTGAAATACGAGAACTACACTTCCTCCTTCTTTATCCGCGACATCATCAAGCCTGACCCCCCCAAGAACTTGCAGCTGAAGCCACTCAAGAACTCCCGCCAAGTGGAAGTGTCTTGGGAATATCCAGACACTTGGAGCACCCCGCACTCATACTTCTCGCTCACTTTCTGTGTGCAAGTGCAGGGAAAGTCCAAACGGGAGAAGAAAGACCGGGTGTTC ACCGACAAAACCTCCGCCACTGTGATTTGTCGGAAGAACGCGTCAATCAGCGTCCGGGCGCAGGATAGATACTACTCGTCCTCCTGGAGCGAATGGGCCAGCGTGCCTTGTTCCGGTGGCGGATCAGGCGGAGGTTCAGGAGGAGGCTCCGGAGGAGGTTCCCGGAACCTCCCTGTGGCAACCCCCGACCCTGGAATGTTCCCGTGCCTACACCACTCC CAAAACCTCCTGAGGGCTGTGTCGAACATGTTGCAGAAGGCCCGCCAGACCCTTGAGTTTCTACCCCTGCACCTCGGAAGAAATTGATCACGAGGACATCACCAAGGACAAGACCTCGACCGTGGAAGCCTGCCTGCCGCTGGAACTGACCAAGAACGAATCGTGTCTGAACTCCCGCGAGACAAGCTTTTATCACTAACGGCAGCTGCCTGGCGTCGAGAAA GACCTCATTCATGATGGCGCTCTGTCTTTTCCTCGATCTACGAAGATCTGAAGATGTATCAGGTCGAGTTCAAAGACCATGAACGCCAAGCTGCTCATGGACCCGAAGCGGCAGATCTTCCTGGACCAGAATATGCTCGCCGTGATTGATGAACTGATGCAGGCCCTGAATTTCAACTCCGAGACTGTGCCTCAAAAAGTCCAGCCTGGAAGAACCGGACTTCTACAAG ACCAAGATCAAGCTGTGCATCCTGTTGCACGCTTTCCGCATTCGAGCCGTGACCATTGACCGCGTGATGTCCTACCTGAACGCCAGT IL-12 (mouse) (SEQ ID NO: 58) ATGTGTCCACAGAAGCTGACAATAAGTTGGTTTGCCATTGTCCTCCTGGTGAGCCCACTCATGGCAATGTGGGAACTCGAAAAGGATGTCTACGTGGTAGAAGTAGATTGGACTCCAGACGCGCCAGGGGAGACAGTGAATTTGACATGTGACACACCAGAAGAAGATGACATTACATGGACATCTGACCAACGCCATGGCGTAATAGGGAGTGGGA AAACACTCACGATCACAGTTAAAGAGTTCTTGGATGCTGGTCAATATACTTGCCATAAAGGCGGCGAGACACTCAGCCACTCACATTTGCTTTTGCATAAAAAGAGAATGGCATTTGGAGCACTGAAATACTTAAGAACTTTAAGAACAAGACATTTCTCAAGTGTGAGGCCCCTAATTACAGCGGCAGGTTCACGTGCTCATGGCTGGTCCAGCGCAACATGG ACCTCAAGTTTAACATAAAATTCTTCTTCCTCTTCACCTGACTCCAGAGCTGTTACTTGCGGCATGGCTTCTCTGAGCGCAGAAAAAGTAACGTTGGATCAAAGAGACTACGAAAAGTACTCTGTTTCTTGTCAAAGAGGATGTTACGTGCCCGACGGCCGAAGAAACGCTTCCAATTGAACTCGCGTTGGAAGCTCGCCAACAAAAACAGTAGAAAACTACAGTACAAGC TTCTTTATACGGGATATAATTAAACCCGATCCCCCCAAGAACTTGCAAATGAAACCACTTAAGAACAGCCAGGTGGAAGTTTCCTGGGAGTATCCAGACTCATGGAGTACTCCTCACAGCTATTTTTCTCTGAAATTCTTTGTAAGGATACAACGGAAGAAAGAGAAGATGAAAGAGACCGAGGAGGGTTGTAATCAGAAGGGAGCGTTTCTCGTGGAGAAAACG TCTACCGAAGTCCAATGTAAAGGTGGCAATGTGTGCGTCCAAGCTCAGGATAGATACTATAATTCAAGTTGCTCCAAGTGGGCCTGTGTTCCATGCCGCGTTCGGAGCGGGGGAGGTAGCGGAGGAGGTAGTGGGGGTGGGTCAGGAGGAGGGAGTCGAGTTATTCCCGGTGTCAGGCCCCGCACGCTGCTTGAGCCAGAGTCGCAACCTCC TTAAGACAACAGATGACATGGTGAAAACAGCACGCGAAAAGCTTAAACACTACTCTTGTACGGCGGAGGATATTGATCACGAGGATATTACCCGAGACCAAACTAGCACTTTGAAAACCTGTCTGCCCCTTGAACTTCATAAAAATGAGAGCTGTCTGGCTACACGAGAGACGTCAAGTACGACTAGGGGCAGCTGTCTCCCGCCGCAAAAGACAAGCCTCATGAT GACGCTCTGTTTGGGTTCCATTTACGAGGACTTGAAAATGTATCAAACGGAGTTCCAGGCTATAAATGCGGCGTTGCAGAACCATAACCATCAACAATTATACTTGATAAAGGCATGTTGGTGGCGATTGATGAACTCATGCAGAGTCTCAATCACAACGGGGAAACGTTGAGACAGAAACCCCCAGTCGGTGAAGCGGACCCATATCGAGTAAAA ATGAAGCTCTGCATTCTGCTTCACGCATTCAGCACTAGAGTTGTTACCATCAACCGGGTAATGGGATATCTCTCCAGTGCGtaG IL21 (human; codon-optimized; signal sequence in bold) (SEQ ID NO: 59) ATGGAACGCATTGTGATCTGCCTGATGGTCATCTTCCTGGGCACCTTAGTGCACAAGTCGAGCAGCCAGGGACAGGACAGGCACATGATTAGAATGCGCCAGCTCATCGATATCGTGGACCAGTTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTCCTGCCGGCCCCCGAAGATGTGGAAACCAATTGCGAATGGTCGGCATTTTCCTGCTTTCA AAAGGCACAGCTCAAGTCCGCTAACACCGGGAACAACGAACGGATCATCAACGTGTCCATCAAAAAGCTGAAGCGGAAGCCTCCTCCACCAACGCCGGACGGAGGCAGAAGCATAGGCTGACTTGCCCGTCATGCGACTCCTACGAGAAGAAGCCGCCGAAGGAGTTCCTGGAGCGGTTCAAGTCGCTCCTGCAAAAGATGATTCATCAGCACCTGTCC TCCCGGACTCATGGGTCTGAGGATTCA IL-12p70_T2A_IL21 (human; codon optimized; signal sequence in bold) (SEQ ID NO: 60) ATGTGCCATCAGCAACTCGTCATCTCCTGGTTCTCCCTTGTGTTCCTCGCTTCCCCTCTGGTCGCCATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAGCTGGATTGGTACCCGGACGCCCCTGGAGAAATGGTCGTGCTGACTTGCGATACGCCAGAAGAGGACGGCATAACCTGGACCCTGGATCAGAGCTCCGAGGTGCTCGGA AGCGGAAAGACCCTGACCATTCAAGTCAAGGAGTTCGGCGACGCGGGCCAGTACACTTGCCACAAGGGTGGCGAAGTGCTGTCCCACTCCCTGCTGCTGCTGCCACAAGAAAGAGGATGGAATCTGGTCCACTGACATCCTCAAGGACCAAAAAGAACCGAAGAACAAGACCTTCCTCCGCTGCGAAGCCAAGAACTACAGCGGTCGGTTCACCTGTTGGTG GCTGACGACAATCTCCACCGACCTGACTTTCTCCGTGAAGTCGTCACGGGGATCAAGCGATCCTCAGGGCGTGACCTGTGGAGCCGCCACTCTGTCCGCCGAGAGAGTCAGGGGAGACAACAAGGAATATGAGTACTCCGTGGAATGCCAGGAGGACAGCGCCTGCCCTGCCGCGGAAGAGTCCCTGCCTATCGAGGTCATGGTCGATGCCGT GCATAAGCTGAAATACGAGAACTACACTTCCTCCTTCTTTATCCGCGACATCATCAAGCCTGACCCCCCCAAGAACTTGCAGCTGAAGCCACTCAAGAACTCCCGCCAAGTGGAAGTGTCTTGGGAATATCCAGACACTTGGAGCACCCCGCACTCATACTTCTCGCTCACTTTCTGTGTGCAAGTGCAGGGAAAGTCCAAACGGGAGAAGAAAGACCGGGTGTTC ACCGACAAAACCTCCGCCACTGTGATTTGTCGGAAGAACGCGTCAATCAGCGTCCGGGCGCAGGATAGATACTACTCGTCCTCCTGGAGCGAATGGGCCAGCGTGCCTTGTTCCGGTGGCGGATCAGGCGGAGGTTCAGGAGGAGGCTCCGGAGGAGGTTCCCGGAACCTCCCTGTGGCAACCCCCGACCCTGGAATGTTCCCGTGCCTACACCACTCC CAAAACCTCCTGAGGGCTGTGTCGAACATGTTGCAGAAGGCCCGCCAGACCCTTGAGTTTCTACCCCTGCACCTCGGAAGAAATTGATCACGAGGACATCACCAAGGACAAGACCTCGACCGTGGAAGCCTGCCTGCCGCTGGAACTGACCAAGAACGAATCGTGTCTGAACTCCCGCGAGACAAGCTTTTATCACTAACGGCAGCTGCCTGGCGTCGAGAAA GACCTCATTCATGATGGCGCTCTGTCTTTTCCTCGATCTACGAAGATCTGAAGATGTATCAGGTCGAGTTCAAAGACCATGAACGCCAAGCTGCTCATGGACCCGAAGCGGCAGATCTTCCTGGACCAGAATATGCTCGCCGTGATTGATGAACTGATGCAGGCCCTGAATTTCAACTCCGAGACTGTGCCTCAAAAAGTCCAGCCTGGAAGAACCGGACTTCTACAAG ACCAAGATCAAGCTGTGCATCCTGTTGCACGCTTTCCGCATTCGAGCCGTGACCATTGACCGCGTGATGTCCTACCTGAACGCCAGTAGACGGAAACGCGGAAGCGGAGAGGGCAGAGGCTCGCTGCTTACATGCGGGGACGTGGAAGAGAACCCCGGTCCGATGGAACGCATTGTGATCTGCCTGATGGTCATCTTCCTGGGCACCTTAGTGCA CAAGTCGAGCAGCCAGGGACAGGACAGGCACATGATTAGAATGCGCCAGCTCATCGATATCGTGGACCAGTTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTCCTGCCGGCCCCCGAAGATGTGGAAACCAATTGCGAATGGTCGGCATTTTCCTGCTTTCAAAAGGCACAGCTCAAGTCCGCTAACACCGGGAACAACGAACGGATCATCAACGTGTCCAT CAAAAAAGCTGAAGCGGAAGCCTCCCCTCCACCAACGCCGGACGGAGGCAGAAGCATAGGCTGACTTGCCCGTCATGCGACTCCTACGAGAAGAAGCCGCCGAAGGAGTTCCTGGAGCGGTTCAAGTCGCTCCTGCAAAAGATGATTCATCAGCACCTGTCCTCCCGGACTCATGGGTCTGAGGATTCA IL-12_2A_CCL21a (human) (SEQ ID NO: 61) ATGTGCCATCAGCAGCTTGTCATATCTTGGTTTTCACTTGTATTCCTGGCCAGCCCTTTGGTTGCGATCTGGGAGCTCAAGAAGGATGTGTACGTTGTAGAGCTGGACTGGTACCCCGATGCTCCCGGTGAGATGGTCGTTTTGACATGTGACACTCCAGAAGAGGACGGTATTACGTGGACTCTGGACCAGTCCTCCGAAGTTCTTGGTTCT GGTAAGACTCTGACTATCCAGGTGAAAGAATTTGGGGATGCGGGACAATACACATGCCACAAGGGAGGCGAGGTGTTGTCTCATAGTTTGCTGCTTCTCCACAAGAAAGAGGATGGAATCTGGAGCACCGACATACTCAAGGATCAAAAGGAACCCAAAAATAAGACATTTCTGCGATGTGAGGCTAAGAACTATAGTGGCCGCTTCACTTGTTGGTG GCTGACTACCATCAGCACAGATCTCACGTTTTTCAGTAAAAAGTAGTAGAGGTTCAAGTGATCCTCAAGGGGTAACGTGCGGTGCTGCAACACTGTCTGCTGAACGCGTAAGAGGAGATAATAAGGAGTACGAGTATTCCGTAGAATGCCAAGAGGACAGTGCTTGTCCTGCGGCCGAGGAGTCTCTCCCCAATAGAAGTGATGGTGGACGCGGTGCATAAACTGA AATATGAGAACTACACAAAGCAGTTTTTTATAAGAGATATCATCAAGCCCGATCCGCCGAAGAATTTGCAACTTAAACCGCTTAAAAACTCACGCCAGGTTGAAGTATCCTGGGAGTATCCGGATACATGGTCAACACCACACAGCTATTTTTCCCTTACCTTCTGTGTGCAGGTCCAAGGGAAGAGCAAAAGGGAGAAGAAGGACAGGGTATTCACTGATAAAAC TTCCGCGACGGTCATCTGCCGAAAAAACGCTAGTATATCTGTACGGGCGCAGGATAGGTACTATAGTTCTTCTTGGTCTGAGTGGGCCTCAGTTCCGTGCTCTGGGGGAGGAAGTGGAGGAGGGTCCGGCGGTGGAAGCGGGGGAGGGAGTCGCAACTTGCCAGTGGCTACACCAGATCCAGGCATGTTCCATGTCTGCATCATTCCCA GAATCTCCTGAGAGCGGTGTCAAATATGCTCCAAAAAGCGAGACAAACACTGGAATTTTACCCGTGTACCAGTGAGGAGATTGATCACGAGGACATAACCAAGGACAAGACCTCAACTGTAGAAGCGTGTTTGCCGCTGGAGTTGACTAAGAATGAGTCCTGCCTCAATTCCAGAGAAACTTCATTCATTACTAACGGCAGTTGTCTTGCATCCCGGAAAACGTCCT TTATGATGGCCCTTTGCCTTAGTTCAATTTACGAGGATCTTAAAATGTATCAAGTGGAGTTTAAAACCATGAATGCTAAACTTCTTATGGACCCCAAACGACAAATTTTTCTGGATCAGAATATGCTTGCCGTGATAGACGAACTCATGCAGGCGCTTAATTTTAACTCCGAAACAGTTCACAAAAAATCTAGCCTTGAAGAACCTGATTTTTATAAAACGAAGATTA AACTGTGTATCCTGCTGCATGCCTTTCGCATCCGAGCTGTCACAATCGATAGGGTTATGTCCTACCTTAACGCGAGCCGGCGCAAGAGGGGTTCCGGAGAGGGAAGGGGTAGTCTGCTCACCTGCGGCGATGTTGAAGAAAATCCTGGTCCCATGGCGCAAAGTCTGGCTCTTTCACTCCTGATCCTGGTCTTGGCCTTCGGGATTCCGAG GACCCAAGGAAGTGATGGTGGCGCCCAAGATTGTTGCCTTAAATACAGCCAGCGGAAAATACCCGCGAAAGTGGTCAGGAGTTATAGAAAACAGGAGCCTTCCCTGGGTTGTAGTATCCCCGCCATACTTTTCCTCCCGAGAAAACGGAGCCAGGCCGAACTGTGCGCTGACCCTAAGGAACTTTGGGTGCAACAACTTATGCAACACCTGGATAAGACAC CTTCTCCTCAAAAGCCAGCTCAGGGCTGCCGAAAAGATAGAGGCGCCTCAAAAACCGGAAAAAAGGGCAAAGGTTTCTAAAGGATGTAAGCGGACTGAACGCTCTCAAACGCCTAAAGGGCCGtaG IL-12_2A_CCL21a (mouse) (SEQ ID NO: 62) ATGTGTCCACAGAAGCTGACAATAAGTTGGTTTGCCATTGTCCTCCTGGTGAGCCCACTCATGGCAATGTGGGAACTCGAAAAGGATGTCTACGTGGTAGAAGTAGATTGGACTCCAGACGCGCCAGGGGAGACAGTGAATTTGACATGTGACACACCAGAAGAAGATGACATTACATGGACATCTGACCAACGCCATGGCGTAATAGGGAGTGGGA AAACACTCACGATCACAGTTAAAGAGTTCTTGGATGCTGGTCAATATACTTGCCATAAAGGCGGCGAGACACTCAGCCACTCACATTTGCTTTTGCATAAAAAGAGAATGGCATTTGGAGCACTGAAATACTTAAGAACTTTAAGAACAAGACATTTCTCAAGTGTGAGGCCCCTAATTACAGCGGCAGGTTCACGTGCTCATGGCTGGTCCAGCGCAACATGG ACCTCAAGTTTAACATAAAATCTTCTTCCTCTTCACCTGACTCCAGAGCTGTTACTTGCGGCATGGCTTCTCTGAGCGCAGAAAAAGTAACGTTGGATCAAAGAGACTACGAAAAGTACTCTGTTTCTTGTCAAGAGGATGTTACGTGCCCGACGGCCGAAGAAACGCTTCCAATTGAACTCGCGTTGGAAGCTCGCCAACAAAAACAAGTGAAAACTACAGTACAAGC TTCTTTATACGGGATATAATTAAACCCGATCCCCCCAAGAACTTGCAAATGAAACCACTTAAGAACAGCCAGGTGGAAGTTTCCTGGGAGTATCCAGACTCATGGAGTACTCCTCACAGCTATTTTTCTCTGAAATTCTTTGTAAGGATACAACGGAAGAAAGAGAAGATGAAAGAGACCGAGGAGGGTTGTAATCAGAAGGGAGCGTTTCTCGTGGAGAAAACG TCTACCGAAGTCCAATGTAAAGGTGGCAATGTGTGCGTCCAAGCTCAGGATAGATACTATAATTCAAGTTGCTCCAAGTGGGCCTGTGTTCCATGCCGCGTTCGGAGCGGGGGAGGTAGCGGAGGAGGTAGTGGGGGTGGGTCAGGAGGAGGGAGTCGAGTTATTCCCGGTGTCAGGCCCCGCACGCTGCTTGAGCCAGAGTCGCAACCTCC TTAAGACAACAGATGACATGGTGAAAACAGCACGCGAAAAGCTTAAACACTACTCTTGTACGGCGGAGGATATTGATCACGAGGATATTACCCGAGACCAAACTAGCACTTTGAAAACCTGTCTGCCCCTTGAACTTCATAAAAATGAGAGCTGTCTGGCTACACGAGAGACGTCAAGTACGACTAGGGGCAGCTGTCTCCCGCCGCAAAAGACAAGCCTCATGAT GACGCTCTGTTTGGGTTCCATTTACGAGGACTTGAAAATGTATCAAACGGAGTTCCAGGCTATAAATGCGGCGTTGCAGAACCATAACCATCAACAATTATACTTGATAAAGGCATGTTGGTGGCGATTGATGAACTCATGCAGAGTCTCAATCACAACGGGGAAACGTTGAGACAGAAACCCCCAGTCGGTGAAGCGGACCCATATCGAGTAAAA ATGAAGCTCTGCATTCTGCTTCACGCATTCAGCACTAGAGTTGTTACCATCAACCGGGTAATGGGATATCTCTCCAGTGCGCGGCGCAAGAGGGGTTCCGGAGAGGGAAGGGGTAGTCTGCTCACCTGCGGCGATGTTGAAGAAAATCCTGGTCCCATGGCGCAAATGATGACCCTTTCCCTGCTGAGTCTTGTCCTCGCGCTCTGCATCCCGTG GACGCAGGGGTCTGATGGGGGGGGCCAAGACTGTTGCCTGAAGTATTCACAAAAAAGATACCGTACTCTATTGTCAGAGGGTACAGGAAGCAAGAACCCTCCTTGGGTTGCCCTATACCAGCAATTCTTTTCTCCCCACGCAAGCATTCCAAACCAGAACTGTGTGCGAACCCCGAGGAGGGTTGGGTACAGAACTTGATGCGAAGGCTTGACCAGCC CCCAGCCCCTGGCAAGCAGTCACCTGGGTGCAGAAAAAACAGAGGTACTTCAAAGAGCGGCAAGAAAGGCAAAGGGAGTAAAGGATGTAAAAGAACGGAGCAGACCCAGCCTTCACGAGGCtaG CCL21a_2A_IL-12 (mouse) (SEQ ID NO: 63) ATGGCGCAAATGATGACCCTTTCCCTGCTGAGTCTTGTCCTCGCGCTCTGCATCCCGTGGACGCAGGGGTCTGATGGGGGGGCCAAGACTGTTGCCTGAAGTATTCACAAAAAAGATACCGTACTCTATTGTCAGAGGGTACAGGAAGCAAGAACCCTCCTTGGGTTGCCCTATACCAGCAATTCTTTTTCCCCCACGCAAGCATTCCAAACCAG AACTGTGTGCGAACCCCGAGGAGGGTTGGGTACAGAACTTGATGCGAAGGCTTGACCAGCCCCCAGCCCCTGGCAAGCAGTCACCTGGGTGCAGAAAAAACAGAGGTACTTCAAAGAGCGGCAAGAAAGGCAAAGGGAGTAAAGGATGTAAAAGAACGGAGCAGACCCAGCCTTCACGAGGCCGGCGCAAGAGGGGTTCCGGAGAGGGAAGGGG TAGTCTGCTCACCTGCGGCGATGTTGAAGAAAATCCTGGTCCCATGTGTCCACAGAAGCTGACAATAAGTTGGTTTGCCATTGTCCTCCTGGTGAGCCCACTCATGGCAATGTGGGAACTCGAAAAGGATGTCTACGTGGTAGAAGTAGATTGGACTCCAGACGCGCCAGGGGAGACAGTGAATTTGACATGTGACACACCAGAAGAAGATGACATTACATGG ACATCTGACCAACGCCATGGCGTAATAGGGAGTGGGAAAACACTCACGATCACAGTTAAAGAGTTCTTGGATGCTGGTCAATATACTTGCCATAAAGGCGGCGAGACACTCAGCCACTCACATTTGCTTTTGCATAAAAAGAGAATGGCATTTGGAGCACTGAAATACTTAAGAACTTTAAGAACAAGACATTTCTCAAGTGTGAGGCCCCTAATTACAGCGGC AGGTTCACGTGCTCATGGCTGGTCCAGCGCAACATGGACCTCAAGTTTAACATAAAATTCTTCTTCCTCTTCACCTGACTCCAGAGCTGTTACTTGCGGCATGGCTTCTCTGAGCGCAGAAAAAGTAACGTTGGATCAAAGAGACTACGAAAAGTACTCTGTTTCTTGTCAAGAGGATGTTACGTGCCCGACGGCCGAAGAAACGCTTCCAATTGAACTCGCGTTG GAAGCTCGCCAACAAAAACAAGTATGAAAACTACAGTACAAGCTTCTTATACGGGATATAATTAAACCCGATCCCCCCAAGAACTTGCAAATGAAACCACTTAAGAACAGCCAGGTGGAAGTTTCCTGGGAGTATCCAGACTCATGGAGTACTCCTCACAGCTATTTTTCTCTGAAATTCTTTGTAAGGATACAACGGAAGAAAGAGAAGATGAAAGAGACCGAGGAGG GTTGTAATCAGAAGGGAGCGTTTCTCGTGGAGAAAACGTCTACCGAAGTCCAATGTAAAGGTGGCAATGTGTGCGTCCAAGTCAGGATAGATACTATAATTCAAGTTGCTCCAAGTGGGCCTGTGTTCCATGCCGCGTTCGGAGCGGGGGAGGTAGCGGAGGAGGTAGTGGGGGTGGGTCAGGAGGAGGGAGTCGAGTTATCCCGGTGT CAGGCCCCGCACGCTGCTTGAGCCAGAGTCGCAACCTCCTTAAGACAACAGATGACATGGTGAAAACAGCACGCGAAAAGCTTAAACACTACTCTTGTACGGCGGAGGATATTGATCACGAGGATATTACCCGAGACCAAACTAGCACTTTGAAAACCTGTCTGCCCCTTGAACTTCTAAAAATGAGAGCTGTCTGGCTACACGAGAGACGTCAAGTACGACTAG GGGCAGCTGTCTCCCGCCGCAAAAGACAAGCCTCATGATGACGCTCTGTTTGGGTTCCATTTACGAGGACTTGAAAATGTATCAAACGGAGTTCCAGGCTATAAATGCGGCGTTGCAGAACCATAACCATCAACAAATTATACTTGATAAAGGCATGTTGGTGGCGATTGATGAACTCATGCAGAGTCTCAATCACAACGGGGAAACGTTGAGACA GAAACCCCCAGTCGGTGAAGCGGACCCATATCGAGTAAAAATGAAGCTCTGCATTCTGCTTCACGCATTCAGCACTAGAGTTGTTACCATCAACCGGGTAATGGGATATCTCTCCAGTGCGtaG IL7 (mouse) (SEQ ID NO: 64) ATGTTTCATGTGTCCTTCAGGTACATATTGGTATCCACCACTTATATTGGTGCTCTTGCCTGTAACCAGCTCTGAATGTCATATAAAAGACAAGGAGGGCAAAGCATACGAGTCCGTATTGATGATCTCAATCGATGAACTTGACAAGATGACAGGGACCGATTCTAATTGTCCAAATAACGAGCCAAACTTCTTTCGGAAACACGTGTGTGATGATACAA AAGAAGCTGCTTTTCTTAACAGAGCTGCCAGAAAACTCAAGCAGTTCCTCAAAGATGAATATATCCGAGGAATTTAACGTGCATCTCCTCACAGTATCTCAGGGAACTCAAACCCTTGTAAACTGCACTTCTAAGGAGGAGAAGAATGTCAAAGAGCAGAAGAAAAATGATGCATGTTTTTTGAAACGGCTGTTGAGGGAGATCAAAACATGCTGGAATAAATCCT CAAGGGCTCAATTtaG IL-15 (human) (SEQ ID NO: 65) ATGGAAACAGACACATTGCTGCTTTGGGTATTGTTGCTCTGGGTGCCTGGATCAACAGGAAACTGGGTAAACGTAATTTCAGATCTGAAGAAGATCGAGGACCTTTATTCAATCCATGCACATCGATGCCACTTCTCTACACCGAAAGCGACGTTCACCCATCTTGCAAGGTGACCGCTATGAAATGTGAATTGTTGGAACTTCAGGTAATTTCT CTGGAGAGCGGCGATGCCTCAATACATGACACCGTTGAAAATCTTATCATCCTTGCTAATGATTCACTCTCTAGTAATGGGAACGTAACAGAGAGCGGGTGTAAGGAGTGTGAAGAACTGGAGGAGAAAAAACATTAAGGAATTTTTGCAGTCATTCGTCCATATAGTGCAAATGTTCATAAACACTTCCAGAAAGAAAGCGAGGCTCTGGGGAGGGGCGAGGCT CTCTGCTGACCTGTGGGGATGTAGAAGAGAATCCAGGTCCCATGGACCGGCTGACCAGCTCATTCCTGCTTCTGATTGTGCCAGCCTACGTGCTCTCCATCACATGTCCTCCCCCAATGAGCGTCGAGCATGCTGACATCTGGGTGAAGTCATACTCCTTGTACAGCAGAGAGATACATTTGTAATTCCGGATTCAAGCGCAAGGCCGGCACCTCCTCT CTGACAGAGTGCGTCCTTAACAAAGCAACCAACGTAGCACATTGGACCACACCATCCTGAAGTGCATACGAGAACCTAAATCTTGCGATAAGACTCATAACTTGTCCACCTTGTCCAGCCCCAGAACTGCTTGGCGGACCCCAGTTTTTGTTCCCCACCAAAGCCAAAAGACACACTCATGATATCCAGAACTCCTGAGGTGACCTGTGTCGTTGTAGACGTTTCCCACGA AGATCCTGAAGTAAAATTCAACTGGTACGTGGATGGGGTCGAAGTCCATAACGCCAAGACTAAACCAAGGGAGGAACAGTATAACTCTACTTACCGAGTAGTTTCTGTGTTGACCGTGCTGCACCAGGACTGGTTGAACGGGAAGGAGTACAAATGCAAGGTGAGCAATAAAGCTCTGCCCGCACCAATCGAAAGACAATATCTAAGGCCAAGGGGCAGCC ACGAGAGCCCCAGGTATACACACTGCCACCCTCACGCGATGAATTGACTAAGAACCAGGTTTCCCCTGACCTGTCTTGTAAAAGGTTTCTACCCCTTCCGACATAGCTGTTGAGTGGGAAAGTAACGGGCAGCCAGAGAACAATTACAAGAGAACTCCACCCGTTCTTGATAGCGATGGATCATTTTTTCGTATTCCAAACTCACTGTCGATAAAAGTCGCTGGC AGCAAGGCAATGTTTTTAGCTGCTCAGTCATGCACGAAGCACTGCATAATCACTACACACAAAAAAGTTTGTCCCTTAGCCCTGGTAAGtaG IL-15 (human) (SEQ ID NO: 66) ATGTACTCAATGCAGTTGGCCTCCTGTGTAACATTGACCTTGGTCCTCTTGGTCAACAGCAATTGGATCGATGTACGCTACGACTTGGAGAAGATTGAGTCCCTTATACAGAGTATACACATAGATACAACCTTGTATACTGACAGTGACTTCCATCCCAGCTGTAAAGTGACTGCAATGAACTGTTTTTTGTTGGAGTTGCAAGTAATTCTGCATGAATAC AGCAACATGACCCTCAATGAAACCGTTAGGAATGTCCTTTTATCTCGCAAATTCTACTCTGAGTAGCAATAAGAATGTTGCCGAAAGCGGCTGCAAGGAGTGCGAAGAACTGGAGGAAAAAACTTTTCACCCGAGTTTCTCAGAGTTTCATCAGAATTGTCCAAATGTTCATTAATACAAGTAGTGGTGGTGGGAGCGGGGGTGGAGGCAGTGGGGGAGGT GGGAGCGGAGGTGGAGGGTCCGGAGGGGGGAGCCTTCAAGGCACTACTTGTCCTCCACCCGTATCCATCGAGCACGCCGATATTCGAGTTAAAAATTATAGTGTTAATAGCAGAGAACGATACGTCTGCAACTCAGGGTTTAAGAGAAAGGCCGGAACTTCAACTCTCATAGAATGCGTGATTAATAAGAATACTAACGTCGCACATTGGACTAC TCCCAGTCTCAAGTGCATACGCGATCCATCTCTCGCTCATTACTCACCAGTACCTACAGTGGTTACTCCTAAGGTGACCTCTCCAGCCCGAATCACCATCTCCCAGCGCAAAAGAGCCTGAGGCCTTTTCTCCTAAATCAGACACTGCTATGACTACAGAAACAGCCATAATGCCAGGAAGCCGGCTGACACCATCTCAAAACTACCAGCGCAGGCACAACTGGGACTGGCTCCCAC AAAAGCTCACGCGCACCAAGTCTCGCCGCAACAATGACATTGGAGCCTACAGCCAGCACATCTCTTAGAATCACAGAAATTTCCCACAGTAGCAAGATGACCAAGGTGGCAATTAGTACCAGCGTCCTTCTTGTAGGAGCTGGAGTTGTGATGGCATTTTTGGCATGGTATATCAAAAGCAGGtaG IL-15 (mouse) (SEQ ID NO: 67) ATGAAGATCCTCAAGCCATACATGCGAAACACTAGTATTAGCTGTTACTTGTGTTTTCTGCTGAATAGTCATTTTTTGACTGAAGCAGGAATCCATGTATTTATACTCGGTTGTGTGTCTGTAGGTCTGCCAAAGACTGAGGCTAATTGGATTGACGTGCGCTATGATCTTGAAAAAATAGAGTCCTTGATTCAATCAATACACATCGATACCACTCTTCTACACCGA CAGTGATTTCCATCCTTCCTGCAAGGTAACAGCTATGAATTGCTTCCTCCTGGAGCTCCAAGTCATTCTCCATGAGTACTCCAACATGACTTTGAACGAAACTGTAAGAAACGTATTGTATCTGGCTAATAGCACCTTGTCTAGTAACAAAAATGTGGCAGAGAGCGCTGCAAAGAATGTGAAGAATTGGAAGAGAAAACATTTACAGAGTTCCTGCAATCCTTTATTCG CATCGTCCAAATGTTTTATCAATACCCTCTtaG IL-15 (mouse) (SEQ ID NO: 68) ATGTATTCCATGCAACTTGCCAGTTGTGTAACCCTTACTCTCGTCCTGCTCGTTAATTCCGCTGGTGCTAACTGGATAGATGTTCGATACGATCTGGAAAAGATTGAGTCCCCTTATCCAATCCATTCATATAGATACCACCCCTTTATACTGACAGCGACTTCCATCCTTCTTGCAAGGTGACCGCTATGAATTGTTTCCTGCTGGAACTCCAAGTTATT CTGCATGAATACTCTAATATGACACTTAACGAGACCGTAAGAAATGTTTCTCTATCTCGCTAATAGTACTTTGAGCTCAAATAAGAACGTGGCCGAGTCTGGGTGTAAGGAATGCGAAGAGCTGGAAGAAAAGACATTCACCGAGTTTCTCCAGTCTTTCATACGGATTGTGCAGATGTTTATCAACACATCAGATTACAAAGACGACGATGATAAGtaG IL-18 (mouse) (SEQ ID NO: 69) ATGGCAGCCATGTCTGAGGACTCTTGTGTGAACTTTAAAAGAAATGATGTTCATAGACAATACACTCTACTTTATACCTGAGGAGAATGGAGATTTGGAATCTGACAACTTTGGCAGGCTGCATTGCACTACCGCAGTTATCCGAAACATCAACGATCAGGTACTGTTTGTTGATAAAAGACAACCTGTATTCGAGGACATGACCGACATAGATCAGTCTGC CTCAGAGCCCCAGACTAGGCTTATCATCTATATGTACAAGGACAGCGAAGTACGAGGCCTGGCTGTTACACTCTCAGTCAAAGACTCTAAGATGAGCACCCTGTCATGCAAAGAACAAAATTATCAGTTTTGAGGAGATGGACCACCTGAAAACATAGATGACATTCAGTCAGACCTCATTTTTTTTCAAAAGCGGGTACCAGGACACAACAAAAATGGAATTTGAAT CATCACTCTACGAAGGACATTTCCTTGCATGCCAGAAAGAGGATGACGCATTCAAATTGATCCTGAAAAAAAAGGACGAAAATGGTGATAAATCAGTCATGTTTACATTGACCAAATCTTCACCAAAGTtaG IL-18 (mouse) (SEQ ID NO: 70) ATGGCTGCAATGTCTGAAGATAGCTGTGTCAACTTTAAGGAGATGATGTTCATTGATAATACTTTGTACTTTATACCTGAAGAAAATGGAGACCTTGAGTCAGACAACTTCGGGAGACTGCACTGCACAACTGCCGTTATCCGAAACATAAATGATCAAGTATTGTTCGTGGACAAAAAGACAACCAGTCTTTGAGGATATGACAGACATCGACCAATCC GCATCTGAACCTCAGACTAGGCTGATCATCTATATGTACGCCGACTCCGAAGTAAGAGGCCTTGCTGTGACACTTAGTGTTAAGGATAGTAAGATGAGCACACTGTCCTGTAAGAATAAGATTATATCTTTTGAAGAGATGGACCCTCCCGAGAACATAGATGACATCCAGAGCGACTTGATCTTCTTTCAGAAGCGAGTGCCAGGCCATAACAAGATGGAATTTGAATC ATCTCTTTATGAAGGCCATTTCCTCGCATGTCAAAAAGGAGGACGATGCCTTCAAAGCTCATTCTGAAAAAAAAAAGACGAGAACGGTGATAAGAGCGTGATGTTCACTCTGACAAATCTGCACCAGTCAtaG IL-18 (human) (SEQ ID NO: 71) ATGTATCGCATGCAACTCCTGTCCTGCATTGCTCTGAGCTTGGCTTTGGTAACCAACTCATACTTCGGGAAACTGGAGAGTAAACTCTCCGTAATCAGGAATCTTAATGACCAAGTATTGTTTATTGACCAGGGCAACCGCCCGTTGTTCGAGGATATGACTGATTCTGACTGTCGGGATAACGCTCCGAGAACTATCTTTATCATTTCAATGTACAAG GACAGCCAACCGCGGGGTATGGCTGTGACAATCAGTGTCAAATGTGAGAAGATTTCCACGCTGTCCTGCGAAAACAAGATAATTTCTTTCAAAGAAATGAACCCCCCTGACAATAAAGGATACAAAGAGTGATATCATCTTCTTTCAGAGGTCCGTGCCCGGCCACGATAATAAGATGCAATTTGAAAGTTCATCTTATGAGGGGTACTTTTTGGCATGCGAGA AAGAAAGGGATCTCTTCAAGTTGATCCTGAAGAAGGAGGACGAATTGGGCGACCGCTCCATCATGTTCACAGTCCAGAACGAGGACtaG IL-18 (human) (SEQ ID NO: 72) ATGTACCGCATGCAGCTCCTGAGTTGTATTGCCCTTTCCCTCGCTCTCGTTACCAATTCTTACTTCGGTAAGCTTGCCTTCTAAACTCTCTGTTATTAGGAACTTGAACGACCAAGTCCTTTTCATAGACCAAGGGAACAGACCACTGTTTGAAGATATGACGGATAGCGATTGCCGAGATAATGCCCCTAGGACGATTTTTATCATTAGTATGTATGCGGACTCTCAAC CGAGGGGGATGGCCGTTACTATAAGTGTGAAATGCGAGAAAATATCAACGCTCAGTTGTGAGAACAAAATCATAAGTTTCAAGGAGATGAATCCACCTGATAACATCAAAGACACTAAGTCTGATATTATATTTTTCCAACGAAGTGTTCCGGGACACGATAACAAAATGCAATTTGAGAGCTCCTCATACGAGGGCTACTTCCTCGCGTGTGAGAAAGAAAG GGATTTGTTTAAGCTTATCCTCAAGAAAGAGGACGAGTTGGGGGATCGGAGCATAATGTTTACCGTACAGAATGAGGACtaG IL-21 (mouse) (SEQ ID NO: 73) ATGGAGCGGACACTCGTGTGTCTTGTCGTAATTTTTCTCGGGACAGTCGCACACAAGTCCTCACCCCAGGGTCCTGATCGCCTTCTCATACGCCTCCGACATTTGATCGACATTGTAGAGCAGCTCAAAATTTACGAGAATGACCTCGATCCCGAGCTTTTGAGTGCTCCCCAAGACGTTAAGGGTCATTGCGAGCACGCAGCTTTTGCTTGCTTCCAGAAGGCCA AGTTGAAACCAAGCAACCCTGGTAATAATAAGACTTTTCATCATCGACTTGGTCGCCCAACTCCGAAGGAGGCTGCCTGCCCGGCGCGGAGGAAAAAAACAAAAGCATATTGCAAAGTGTCCTTCATGTGATTCATACGAAAAGCGGACTCCCAAAGAGTTCTTGGAAAGGTTGAAATGGCTTCTTCAGAAGATGATTCATCAACATTTGTCAtaG IFN-beta (human) (SEQ ID NO: 74) ATGACCAACAAATGCCTTTTGCAAATTGCCCTGCTTTTGTGTTTTAGCACTACCGCATTGAGCATGTCATATAACCTCCTCGGCTTCCTTCAGAGATCATCAAACTTTCAGTGTCAGAAACTGCTTTGGCAACTTAATGGCAGGCTCGAATATTGTCTGAAAGATCGGATGAATTTCGACATTCCAGAAGAAATAAAACAGCTTCAACAATTCCAGAAAGAGGACG CCGCCCTGACTATTTACGAGATGCTCCAGAATATCTTCGCCATTTTCCGGCAGGACAGCTCATCCACGGGGTGGAATGAGACTATTGTAGAAAATCCTTCTGGCTAATGTGTACCATCAATTAATCACCTCAAACGGTGCTTGAGGAAAAACTTGAAAAGGAAGATTTCACACGGGGCAAGTTGATGTCCTCCCTGCACCTTAAACGATACTACGGCAGGATT CTTCATTACTTGAAGGCTAAGGAGTATAGCCATTGCGCGTGGACAATTGTACGGGTAGAAATACTGCGAAACTTTTATTTCATCAACCGGCTCACTGGATACCTTAGAAATtaG IFN-β (mouse) (SEQ ID NO: 75) ATGAACAATCGGTGGATACTCCACGCCGCATTTCTCCTCTGCTTTAGCACGACGGCCCTGTCCATCAACTACAAACAGCTTCAGTTGCAGGAGCGGACTAACATAAGGAAGTGCCAGGAACTGCTGGAACAGCTTAATGGTAAAATTAATCTTACATACCGAGCTGACTTCAAAAATTCCTATGGAAATGACCGAGAAGATGCAGAAATCCTACACGGCAT TCGCCATCCAGGAAATGCTCCAGAACGTATTTCTCGTGTTCCGCAATAATTTTCTCTTACGGGTTGGAACGAAAACCATTGTTGTTAGACTGCTTGACGAACTGCATCAGCAAAACCGTGTTCCTTAAAACCGTGCTTGAGGAGAAGCAGGAGGAGCGCCTGACTTGGGAGATGTCTAGTACCGCACTTCACTTGAAATCCTACTACTGGCGCGTTCAGCG GTATCTGAAGCTGATGAAGTATAACTCATACGCCTGGATGGTAGTGCGCGCAGAGATCTTCAGAAACTTTCTTATCATCCGGCGACTGACCCGAAACTTTCAGAATtaG IFN-γ (human) (SEQ ID NO: 76) ATGAAGTACACTAGCTATATATTGGCCTTCCAGCTTTGCATCGTATTGGGTAGCCTCGGATGCTATTGCCAAGACCCGTATGTCAAAGAGCCGAAAATCTCAAAAGTATTTCAATGCCGGACACTCAGACGTCGCGGATAACGGTACACTGTTTCTTGGCATCCTGAAAAATTGGAAGGAAGAGAGTGACAGAAAAATAATGCAGTCACAAATAGTGT CCTTTTACTTTTAAGCTGTTCAAAAATTTCAAGGATGACCAAAGTATCCAGAAGAGTGTTGAAACTATCAAAGAGGACATGAATGTGAAATTCTTTAACAGTAATAAGAAGAAGCGCGATGACTTCGAGAAACTCACTAATTACAGCGTAACGGATCTTAACGTCCAACGCAAGGCAATCCACGAGCTTATACAGGTAATGGCTGAGCTTAGTCCCGCAGCCAAGACAG GGAAGAGAAAAAGGTCTCAAATGCTTTTTCGGGGCCGGCGAGCTTCACAAtaG IFN-γ (mouse) (SEQ ID NO: 77) ATGAACGCTACGCATTGCATCCTCGCACTCCAATTGTTCCTCATGGCTGTGTCAGGGTGTTACTGTCACGGTACTGTCATAGAAAGCCTCGAATCCCTGAATAACTATTTTAACAGTAGCGGTATAGATGTAGAAGAAAAGTCTCTCTTTCTTGACATCTGGAGGAATTGGCAAAAGGATGGAGACATGAAGATTCTCCAATCTCAGATTATATCATTTTT ACTTGAGGCTTTTTGAGGTTCTGAAGGATAACCAGGCGATCAGCAATAATATCAGCGTAATTGAATCTCACCTTATTACAACATTTTCTCAAATTCCAAGGCAAAGAAAGATGCTTTCATGTCTATCGCGAAATTTGAGGTGAACAATCCTCAGGTACAAAGGCAAGCCTTTAACGAGCTGATTAGAGTTGTACATCAGTTGTTGCCCGAAAGTAGTCTTAGAAAACGC AAACGGAGCCGATGCtaG IFN-α (mouse) (SEQ ID NO: 78) ATGGCAAGGTTGTGCGCTTTTCTCATGGTACTGGCTGTGCTCTCCTATTGGCCTACTTGTTCTCTGGGATGCGACTTGCCACAGACCCACAATCTGCGGAATAAGAGGGCTCTGACTCTGCTGGTGCAAATGAGACGGCTCTCTCCACTTAGCTGTTTGAAAGATAGAAAGGATTTCGGGTTCCCCCAGGAGAAGGTGGATGCCCAGCAGATC AAGAAGGCACAGGCTATCCCCGTCCTTTCCGAGCTGACCCAGCAAATTTTGAACATCTTTACCAAGTAAGGATAGTTCAGCTGCATGGAATACCACACTTTTGGATTCTTTTTGTAACGATCTGCATCAGCAGCTGAACGATCTCCAGGGATGCCTGATGCAGCAAGTCGGCGTGCAAGAATTTCCACTCACCCAGGAGGACGCTCTGCTCGCAGTGCGAAAGTATTT TCACCGAATTACCGTGTACCTCCGGGAGAAAAAGCATTCACCCTGCGCTTGGGAAGTAGTCAGGGCCGAAGTATGGAGAGCCCTAGTAGCTCCGCTAATGTACTGGGCCGGTTGCGGGAAGAGAAAtaG CCL21 (human) (SEQ ID NO: 79) ATGGCGCAAAGTCTGGCTCTTTCACTCCTGATCCTGGTCTTGGCCTTCGGGATTCCGAGGACCCAAGGAAGTGATGGTGGCGCCCAAGATTGTTGCCTTAAATACAGCCAGCGGAAAATACCCGCGAAAGTGGTCAGGAGTTATAGAAAACAGGAGCCTTCCCTGGGTTGTAGTATCCCCGCCATACTTTCCTCCCGAGAAAACGGAGCCAGGCC GAACTGTGCGCTGACCCTAAGGAACTTTGGGTGCAACAACTTATGCAACACCTGGATAAGACACCTTCTCCTCAAAAGCCAGCTCAGGGCTGCCGAAAAGATAGAGGCGCCTCAAAAACCGGAAAAAAGGGCAAAGGTTCTAAAGGATGTAAGCGGACTGAACGCTCTCAAACGCCTAAAGGGCCGtaG CCL21a (mouse) (SEQ ID NO: 80) ATGGCGCAAATGATGACCCTTTCCCTGCTGAGTCTTGTCCTCGCGCTCTGCATCCCGTGGACGCAGGGGTCTGATGGGGGGGCCAAGACTGTTGCCTGAAGTATTCACAAAAAAGATACCGTACTCTATTGTCAGAGGGTACAGGAAGCAAGAACCCTCCTTGGGTTGCCCTATACCAGCAATTCTTTTTCCCCCACGCAAGCATTCCAAACCAG AACTGTGTGCGAACCCCGAGGAGGGTTGGGTACAGAACTTGATGCGAAGGCTTGACCAGCCCCCAGCCCCTGGCAAGCAGTCACCTGGGTGCAGAAAAAACAGAGGTACTTCAAAAGAGCGGCAAGAAAGGCAAAGGGAGTAAAGGATGTAAAAGAACGGAGCAGACCCAGCCTTCACGAGGCtaG Tailless CCL21 (human) (SEQ ID NO: 81) ATGGCGCAAAGTCTGGCTCTTTCACTCCTGATCCTGGTCTTGGCCTTCGGGATTCCGAGGACCCAAGGAAGTGATGGTGGCGCCCAAGATTGTTGCCTTAAATACAGCCAGCGGAAAATACCCGCGAAAGTGGTCAGGAGTTATAGAAAACAGGAGCCTTCCCTGGGTTGTAGTATCCCCGCCATACTTTCCTCCCGAGAAAACGGAGCCAGGCC GAACTGTGCGCTGACCCTAAGGAACTTTGGGTGCAACAACTTATGCAACACCTGGATAAGACACCTTCTCCTCAAAAGCCAGCTCAGGGCtaG Tailless CCL21 (mouse) (SEQ ID NO: 82) ATGGCGCAAATGATGACCCTTTCCCTGCTGAGTCTTGTCCTCGCGCTCTGCATCCCGTGGACGCAGGGGTCTGATGGGGGGGCCAAGACTGTTGCCTGAAGTATTCACAAAAAAGATACCGTACTCTATTGTCAGAGGGTACAGGAAGCAAGAACCCTCCTTGGGTTGCCCTATACCAGCAATTCTTTTTCCCCCACGCAAGCATTCCAAACCAG AACTGTGTGCGAACCCCGAGGAGGGTTGGGTACAGAACTTGATGCGAAGGCTTGACCAGCCCCCAGCCCCTGGCAAGCAGTCACCTGGGtaG CCL19 (mouse) (SEQ ID NO: 83) ATGGCACCCCGCGTCACACCCTTGCTTGCTTTTTCTCTGCTTGTCCTCTGGACCTTCCCCGCTCCTACCCTTGGAGGAGCCAATGATGCCGAGGATTGCTGCCTGAGTGTTACACAAAGGCCAATACCAGGGAATATAGTGAAGGCATTCCGGTATCTGCTCAATGAAGATGGGTGCAGAGTCCCCGCAGTTGTCTTTACAACATTGCGAGGTTACCAG CTTTGTGCTCCCCCAGACCAGCCTTGGGTAGATCGCATTATTCGCCGGTTGAAGAAGAGCTCAGCAAAGAATAAGGGCAATTCCACACGGAGAAGCCCCGTCTCCtaG CCL19 (mouse) (SEQ ID NO: 84) ATGAAAATCAGCAGTCCTTTTCTTGCTCGGGATTATTTTTCTGGAACAATGTGGAGTGAGGGGAACACTCGTAATAAGAAACGCTCGGTGCTCATGCATATCAACATCACGGGGCACTATCCACTACAAATCCCTGAAGGATCTGAAGCAGTTCGCCCCAAGCCTAACTGTAACAAGACCGAAATTATCGCAACTTCCAAAAAATGGAGATCAGACTTGTCTT GACCCAGATTCAGCAAATGTCAAGAAGCTGATGAAAGAGTGGGAAAAGAAGATTTCACAAAAAAAAAAGCAAAAACGCGGCAAGAAACATCAAAAGAACATGAAAACAGGAAACCTAAGACTCCCAGTCAAGGAGAAGATCCCCGCAAGACAACCtaG CXCL11 (mouse) (SEQ ID NO: 85) ATGAACAGAAAAAGTTACCGCTATAGCACTTGCTGCCATAATATGGGCCACCGCAGCTCAAGGGTTCCTGATGTTCAAGCAGGGCCGATGCCTCTGCATTGGCCCTGGAATGAAGGCCGTGAAAATGGCCGAAATAGAAAAAGCTAGTGTCATATACCCCTCTAACGGTTGCGATAAAGTCGAGGTTATAGTCACAATGAAAGCTCATAAACGCCAACGCTG CCTCGACCCCCCGGTCTAAGCAGGCTAGGCTCATAATGCAAGCAATCGAGAAGAAAAACTTTTCTTAGACGGCAAAACATGtaG CXCL10 (mouse) (SEQ ID NO: 86) ATGAACCCATCTGCCGCCGTTATTTTTCTGTCTGATACTCCTTGGGCTGAGTGGCACACAAGGCATACCCCTCGCCCGCACAGTCCGGTGTAATTGTATACATATTGACGACGGCCCTGTTAGAATGCGGGCCATCGGTAAGCTGGAGATTATACCAGCAAGCCTTAGTTGTCCCAGGGTTGAAATCATAGCAACTATGAAAAAAAACGACGAACAA AGATGTTTGAATCCCGAGAGCAAGACAATCAAAAACCTTATGAAAGCATTTAGTCAAAAACGCTCTAAACGCGCTCCAtaG CXCL10 (human) (SEQ ID NO: 87) ATGAATCAGACGGCAATCCTTATATGCTGCCTTATATTCCTTACTCTCTCAGGGATACAAGGGGTACCACTTTCTCGGACTGTTCGCTGCACTTGCATTTCAATATCTAACCAACCTGTAAATCCGCGGAGCCTGGAAAAATTGGAGATTATACCTGCTTCTCAATTCTGCCCTCGGGTGGAAATCATCGCCACTATGAAGAAGAAGGGCGAGAAAAGGTGT CTGAATCCAGAGTCAAAGGCAATCAAAAACCTGCTGAAAGCGGTGTCAAAGGAACGGTCCAAGAGATCACCCtaG CXCL11-CXCL10 (mouse) (SEQ ID NO: 88) ATGAACAGGAAAGTAACAGCATTGCATTGGCTGCCATCATCTGGGCCACCGCAGCACAGGGTTTTCTGATGTTTAAGCAAGGGCGCTGTCTCTGTATAGGCCCAGGCATGAAGGCCGTGAAGATGGCAGAGATTGAGAAGGCATCTGTGATTTATCCTTCTAACGGGTGCGATAAAGTCGAAGTTATTGTGACAATGAAGGCACACAAACGCCAACGGTGT TTGGACCCACGATCTAAACAGGCAAGATTGATTATGCAAGCCATCGAGAAAAAGAACTTTCTCCGAAGGCAAAATATGATCCCTTTGGCTCGGACAGTGCGGTGTAACTGTATTCACATCGACGATGGGCCAGTACGGATGAGAGCAATAGGAAAGCTCGAAATCATACCCGCCTCATTGTCTTGTCCCAGGGTGGAAATAATCGCCACTATGAAAAAGAACG ATGAACAGAGGTGTCTCAACCCAGAGAGTAAGACTATCAAGAACCTTATGAAGGCATTCAGTCAGAAGAGGTCAAAGCGAGCACCAtaG XCL1 (human) (SEQ ID NO: 89) ATGAGACTTCTCATATTGGCGCTTCTCGGGATATGTTCTCTTACGGCATACATAGTTGAGGGGGTGGGATCTGAGGTTAGCGATAAACGAACTTGTGTTAGTCTTACAACACAGAGGCTTCCAGTCTCAGGATAAAAACATACGATAACTGAGGGATCTCTCAGAGCGGTCATTCTAACGAAGAGGGGCCTGAAGGTCTGTGCTGACCCA CAAGCGACTTGGGTAAGGGACGTTGTGCGGAGCATGGACAGGAAGAGCAATACTCGCAACAACATGATCCAAACCAAACCTACGGGCACCCAACAGTCAACCAATACTGCGGTAACATTGACGGGGtaG XCL1 (mouse) (SEQ ID NO: 90) ATGCGCCTCCTTCTGCTGACTTTTCTGGGTGTATGTTGCCTGACACCCTGGGTCGTAGAAGGAGTAGGAACCGAGGTTCTGGAAGAGTCCTCATGTGTAAACTTGCAGACACAACGACTCCCCGTCCAAAAAATCAAGACCTATAATCTGGGAGGGGGCAATGCGGGCCGTCATTTTCGTGACTAAACGAGGTCTCAAAAATCTGCGCCGACC CCGAGGCTAAGTGGGTGAAGGCAGCCATTAAGACCGTGGATGGGAGAGCCAGCACCAGAAAGAACATGGCCGAAACAGTACCTACTGGCGCACAGCGGTCAACCTCAACTGCTATAACCTTGACAGGAtaG m_sCD40L No. 1 (SEQ ID NO: 91) ATGGAGACTGACACTCTGCTTCTGTGGGTGTTGCTGCTGTGGGTGCCTGGCAGTACAGGCGATATGCAACGAGGTGACGAGGACCCTCAAATCGCCGCCCATGTAGTCTCTGAAGCTAATAGCAACGCTGCATCCGTCTTGCAGTGGGCAAAGAAAGGCTACTATACTATGAAGTCCAACTTGGTAATGCTTGAAAACGGCAAGCAGTTGACTGT CAAGAGAGAGGGACTTTATTACGTCTACCCAAGTCACATTCTGTAGCAATCGAGAACCCTCCTCACAGAGGCCTTTTATAGTGGGACTCTGGCTTAAACCAAGTAGCGGCTCTGAGCGCATACTGTTGAAAGCCGCAAACACACAGCTCTTCCCAACTCTGCGAGCAGCAATCCGTGCATCTCGGTGGAGTATTTGAGCTTCAAGCCGGTGCCTCAGTGTTTGTGA ACGTCACTGAGGCCTCCCAGGTCATACATCGAGTTGGGTTCAGCTCCTTCGGCTTGCTCAAGCTCtaG m_sCD40L No. 2 (SEQ ID NO: 92) ATGGAAACTGATACATTGCTGCTCTGGGTTTTGCTGCTCTGGGTGCCTGGGAGTACAGGCGACATGAGGAGGCAGTTCGAGGATCTCGTTAAGGATATTACCCTTAATAAGGAGGAGAAGAAAGAAAACTCTTTTGAGATGCAACGAGGGGACGAAGATCCTCAGATCGCTGCTCACGTGGTCTCTGAAGCTAACAGCAACGCCGCTTCTGT CCTCCAGTGGGCCAAGAAAGGTTATTACACCATGAAATCAAACCTTGTAATGCTTGAAACCTTGTAATGCTTGAAAACGGGAAACAGCTTACAGTGAAGAGGGAAGGTCTTTACTACGTCTATACCCAGGTAACCTTCTGCTCAAACAGAGAACCATCAAGCCAGAGGCCATTCATAGTGGGGCTCTGGCTCAAACCTTCCAGTGGCAGCGAGAGAATCTTGTTGAAAGCTGCTAATACACAT AGTAGTAGCCAGCTTTGCGAGCAACAGTCAGTCCACCTCGGGGGGGTGTTTGAGTTGCAAGCAGGGGCCTCAGTATTCGTGAATGTCACTGAGGCTTCCCAGGTAATTCACAGGGTAGGCTTTAGTTCATTCGGTTTGCTGAAGCTTtaG m_sCD40L No. 3 (SEQ ID NO: 93) ATGCGAAGAATGCAGCTTCTGCTCCTTATTGCTCTGAGTCTCGCCCTTGTCACCAACTCCGGGGACAGAATGAAACAAATCGAGGACAAAATTGAAGAAATACTGAGTAAAATATCACATCGAAAACGAAATTGCACGCATTAAGAAATTGATTGGCGAACGCACCAGTGGCGGCTCTGGTGGCACCGGAGGTTCAGGCGGGACCGGGGGCT CTGACAAAGTCGAAGAGGAGGTTAACCTTCATGAGGACTTTGTGTTCAATCAAGAAGCTGAAACGGTGCAATAAAGGAGAAGGTTCTTTGAGCCTCCTTAATTGCGAAGAGATGCGACGACAGTTCGAGGATCTGGTTAAGGACATTACACTTAATAAGGAAGAGAAAAAGGAGAACTCTTTCGAAATGCAGCGCGGCGATGAAGATCCCCAGATAGCCGCCCATGTCGT CTCTGAGGCCAACTCTAACGCAGCATCCGTCCTCCAGTGGGCTAAGAAAGGATATTATACTATGAAAAGCAATTTGGTCATGCTCGAAAACGGTAAACAGCTCACTGTTAAGAGAGAAGGCCTCTATTACGTATATACTCAAGTAACTTTCTGTTCTAATAGGGAACCCTCTCTCAAAGACCTTTTATCGTAGGACTCTGGTTGAAACCCAAGTAGCGGTAGTGAAAG GATTCTGCTCAAAGCAGCTAATACTCACTCCAGCAGTCAACTGTGCGAACAACAAAGCGTTCACCTCGGGGGCGTCTTTGAACTTCAGGCAGGTGCCAGTGTTTTCGTCAACGTAACAGAAGCATCCCAGGTAATTCATCGAGTAGGGTTTTTCTAGCTTTGGTTTGCTGAAGCTGtaG Anti-CD40_FGK4.5 (SEQ ID NO: 94) ATGGAAACTGATCGCCTGTTGCTCTGGGTACTTCTTCTGTGGGTGCCTGGGTCCACTGGTGACACTGTACTTACACAATCACCCGCTTTGGCCGTTTCTCCTGGTGAACGGGTCACAATTAGTTGCCGAGCTTCCGATTCTGTATCTACTCTTATGCATTGGTATCAAAAAACCTGGTCAGCAGCCAAAATTGCTCATTTATTCTTGCTAGTCAC TTGGAGTCCGGCGTACCTGCTCGATTCAGCGGTAGTGGGTCTGGCACAGATTTCACTTTGACCATAGATCCCGTGGAGGCCGATGACACTGCAACCTACTATTGCCAGCAATCCTGGAACGACCCTTGGACTTTCGGCGGCGGCACCAAGCTGGAACTCAAGCGAGCAGATGCTGCCCCAACCGTTAGTATTTCCCACCCTCAACCGAACAACTCGCCAC AGGAGGCGCTAGTGTCGTGTGTCTTATGAACAATTTCTATCCACGAGACATTAGCGTCAAGTGGAAAATTGATGGGACAGAAAGGCGAGATGGAGTTTTGGATTCAGTAACAGACCAGGATTCAAAGGATTCTACCTATAGCATGAGCTCCACCTTGAGCCTGACCAAAGCTGATTATGAATCTCATAACCTGTATACTTGTGAAGTGTGCATAAGACTTCTAGC TCACCAGTGGTTAAATCTTTTAACCGCAACGAATGTCGGCGCAAGAGGGGTTCCGGAGAGGGAAGGGGTAGTCTGCTCACCTGCGGCGATGTTGAAGAAAATCCTGGTCCCATGGACATTCGGCTCTCTTTGGTATTCCTGGTACTTTTTATAAAGGGGGTGCAATGTGAAGTCCAGCTCGTGGAAAGCGGTGGGGGCCTGGTTCAGCC CGGTCGCAGCCTTAAACTTAGTTGCGCAGCATCCGGATTTACATTTTCTGACTATAACATGGCCTGGGTTCGACAGGCACCCAAAAAAAGGGCTGGAGTGGGTCGCAACTATCATACGATGGTTCCCGGACATACTATAGAGATTCAGTGAAGGGGCGCTTTACAATAAGCAGGGACAATGCTAAGTCTACCTTGTATCTTCAGATGGACTCCCTGAG GAGCGAAGATACAGCAACATATTTTGTGCTACAAACCGCTGGTTGCTGCTTCATTATTTCGACTACTGGGGTCAGGGCGTCATGGTAACTGTATCAAGCGCCGAGACCACAGCCCCTTCTGTATATCCATTGGCACCAGGTACTGCTCTGAAATCCAACTCAATGGTAACCCTTGGATGTCTGGTTAAGGGTTATTTTCCCGAGCCCGTCACAGTTA CTTGGAACTCTGGGGCCCTTTCTAGCGGAGTCCATACCTTTCCCGCCGTTTTGCAGAGTGGTCTGTACACCTTACTCTAGCACATGGAGCTCCCAGGCAGTAACTTGTAATGTGGCCCATCCAGCCTCCTCAACTAAGGTAGATAAAAAGATCGTTCCCAGAGAATGCAATCCATGTGGATGCACCGGGTCTGAGGTCAG CAGTGTGTTCATTTTCCCACCCAAGACTAAAGATGTATTGACTATTACTCTTACACCAAAGTAACCTGCGTGGTGGTTGATATTAGTCAAAATGATCCCGAGGTACGGTTCTCTTGGTTTATCGACGTCGAAGTACATACAGCTCAGACACACGCTCCCGAGAAACAAAAGCAATTCCACTCTTAGGAGCGTGTCCGAGTTGCCAATCGTACATAGGGATTGG CTTAATGGCAAGACCTTTAAGTGTAAGGTCAATTCAGGGGCATTCCCGCACCAATAGAGAAGGTATAAGCAAACCCGAGGGGACACCCAGAGGTCCACAGGTCTATACAATGGCTCCCCCCAAGGAAGAGATGACCCAAAGTCAAGTCTCAATTACATGTATGGTGAAGGGCTTTTATCCACCCGACATATACACTGAGTGGAAGATGAATGGACAGCCCCAAGAGAA TTATAAAAAACACTCCCCTACCATGGACACCGACGGGTCCTATTTTCTTATAGTAAATTGAACGTGAAAAAGGAGACCTGGCAACAAGGCAACACTTTTCACCTGCTCCGTTCTTCACGAGGGCCTGCATAATCATCATACCGAAAAGTCTCTCAGTCATTCTCCAGGTAAGtaG CD40L_2 (human) (SEQ ID NO: 95) ATGGAAACAGATACGTTGCTGTTGTGGGTACTTCTCCTTTGGGTCCCTGGCAGCACAGGGGACGAGAATAGTTTCGAAATGCAGAAGGGCGACCAGAACCCAGATCGCGGCTCACGTTATATCAGAAGCAAGTAGTAAGACCACTTCCGTACTTCAGTGGGCTGAAAAAAGGATATTACACCATGTCCAACAATCTCGTGACACTGGAGAACG GTAAACAACTTACGGTGAAACGACAGGGCCTCTATTACATCTACGCTCAGGTGACATTCTGCTCAAATAGGGAGGCTTCTTAGTCAAGCGCCCTTCATCGCCAGCCTGTGCCTCAAATCTCCCGGCCGGTTCGAACGAATCCTGTTGCGAGCGGCCAATACCCCATAGCTCAGCTAAACCTTGCGGCCAGCAGAGTATTCATCTTGGTGGTGTGTTTGAACTTCAGCCG GGAGCATCTGTGTTCGTCAACGTAACGGACCCTAGCCAAGTGTCTCATGGGACAGGTTTTCATCCTTCGGACTCCTCAAGTTGtaG Flt3L (human) (SEQ ID NO: 96) ATGACAGTTCTCGCGCCAGCTTGGAGTCCCACCCACATACTTGCTTTTGCTTCTGCTTCTGTCCTCTGGCCTGAGTGGGACCCAAGATTGTTCCTTTCAACATTCCCCAATTAGTTCTGATTTTGCAGTGAAGATTAGAGAGCTCTCAGACTATCTGCTGCAAGATTATCCTGTCACAGTCGCTTCAAACCTGCAAGACGAAGAGCTCTGCGGTGCCTTGTGGCG GTTGGTCTTGGCTCAAAGATGGATGGAGAGACTGAAAACCGTAGCAGGCAGCAAGATGCAGGGTCTCCTGGAAAGGGTGAACACGGAAATCCATTTTGTGACCAAGTGCGCGTTCCAGCCCCCACCGAGTTGTCTCCGGTTTGTTCAAACGAATATATCCCGGTTGCTCCAGGAAACCTCAAGAACAACTGGTGGCTTTGAAACCCTGGATCACAAGACA AAACTTTAGTCGGTGCCTCGAACTCCAGTGCCAAACCAGATTTCTTCTACACTTCCCCCCCCCGTGGTCCCCGCGCCCGTTGGAAGCAACGGCCCCAtaG TGFb TRAP (human) (SEQ ID NO: 97) ATGGCCTGGAGTCCTCTGTTTCTGACTCTTATAACTCACTGTGCCGGCAGTTGGGCTATACCCCTCATGTACAGAAGTCTGTAAACAACGACATGATTGTAACCGACAATAATGGCGCAGTGAAATTCCCACAACTGTGTAAGTTCTGTGATGTACGGTTTAGTACATGCGACAATCAAAAAAGCTGTATGTCTAACTGCTCTATTACATCCATATGTGAAA AACCTCAGGAGGTGTGTGTTGCCGTTTGGCGAAAAAATGATGAGAATATCACACTGGAGACAGTATGTCATGACCCTAAACTGCCATACCATGATTTCATACTGGAGGACGCCGCCGTCTCTAAGTGCATTATGAAAGAGAAAAAGAAACCCGGTGAAACATTCTTTATGTGCTCTTGTAGCTCTGACGAGTGTAACGACAACATTATATTCAGCGAGGAGTACAA TACAAGCAACCCCCGATATACCCACCTCACGTACAAAAAAGTGTCAACAACGATATGATTGTTACCGACAATAACGGAGCTGTTAAGTTCCTCAGTTGTGCAAGTTCTGCGATGTACGATTCTCTACCTGCGACAACCAAAAGTCATGTATGTCTAACTGTTCCATAACCTCCATCTGCGAGAAGCCCCAGGAAGTCTGCGTCGCCGTGTGGCGGAAAAACGACGAGAAT ATCACTCTTGAAACCGTTTGTCATGATCCTAAACTGCCCTATCACGACTTTATTCTGGAAGATGCTGCTTCCCCTAAGTGTATCATGAAAGAAAAGAAGAAACCTGGGGAGACATTCTTTATGTGTTCATGCTCCTCCGATGAGTGTAACGACAATATCATCTCTCTGAGGAATACAACACTTCTAACCCTGATtaG Fresolimumab (Human) (SEQ ID NO: 98) ATGGCCTGGTCCCCTCTTTTTCTGACCCTCATCACACACTGTGCAGGCTCATGGGCTGAGACCGTCTTGACCCAGTCCCAGGAACTTTGTCTCTGTCTCCTGGTGAAAGAGCTACCCTTAGTTGTCGAGCCTCTCCAGTCCCTTGGTTCTAGCTATCTCGCTTGGTACCAGCAAAAGCCAGGCCAGGCCCCACGACTGCTGATCTACGGAGCATCTTCAC GGGCTCCCGGCATTCCCGATCGATTTTCCGGATCTGGTAGTGGTACAGATTTCACACTGACCATATCTCGCCTGGAGCCCGAGGACTTTGCTGTTTTATTATTGTCAGCAGTACGCCGATTCTCCTATCACTTTTGGACAGGGAACCCGCCTGGAGATTAAGCGCACAGTAGCAGCTCCATCCGTCTTATCTTTCCACCATCAGATGAACAGCTCAAGAGTGGGA CCGCAAGTGTAGTATGCCTGCTGAACAATTTTTACCCTAGAGAGGCCAAAGTGCAGTGGAAGGTGGATAACGCCCTCCAGAGTGGCAATAGTCAAGAAAGTGTTACTGAGCAAGATAGTAAGGACTCTACATACTCTTTGAGTTCTACTTTGACCCTGTCAAAAAGCAGATTATGAAAAACATAAGGTGTATGCATGTGAAGTTACACACCAAGGGTTGTCCTCCATCAAA ATCTTTTAATAGAGGAGAGTGCCGCCGCAAACGCGGTAGTGGAGAAGGTCGAGGCTCACTCTTGACCTGTGGCGACGTGGAAGAAAATCCCGGTCCTATGGATTGGACTTGGAGGGTATTTTGTCTTTTGGCAGTAACACCTGGAGCTCACCCCAAGTACAGCTCGTCCAATCTGGTGCCGAGGTTAAAAGCCTGGAAGTTCAGTGAAGGTCCTCT TGCAAGGCATCTGGATACACCTTTTCATCTAACGTCATATCCTGGGTACGGCAAGCCCCAGGACAGGGACTTGAGTGGATGGGAGGGGTCATCCCCATCGTGGACATTGCTAATTACGCTCAGCGATTCAAAGGGCGGGTTACTATAACTGCCGACGAGTCTACCTCCAACTACCTACATGGAGTTGTCCTCTCTCCGCTCCGAGGACACTGCT GTATATTACTGTGCCAGCACTCTCGGGTTGGTGTTGGATGCCATGGACTATTGGGGACAAGGAACCCTGGTGACAGTTAGCTCCGCAAGCACTAAAGGCCCTTCTGTTTTTCCCTTGGCACCTTGTAGTAGGTCTACCCTCTGAGTCTACAGCAGCACTTGGATGCTTGGTTAAGGACTATTTTCCCGAGCCAGTTACAGTCTCTTGGAACAGTGGTGC CCTCACAAGTGGGGTTCATACCTTTCCCGCAGTCCTCCAGAGTAGTGGCCTTTACAGCCTCTCATCAGTTGTGACTGTTCCTAGTTCATCACTCGGTACTAAGACATACATGTAACGTAGACCACAAAGCCAAGCAACACAAAAGTAGACAAAACGAGTCGAATCTAAGTATGGACCCCTTGTCCCTCCTGTCCTGCTCCCGAGTTCCTTGGGGGCCCTTCCGTGT TCTTGTTTCCTCCCAAGCCCAAGGATACCCTCATGATCTCACGAACCCCAGAGGTAACATGTGTGGTTGTTGACGTAAGTCAGGAAGTTCCCGAAGTGCAATTTAATTGGTACGTGGATGGCGTCGAAGTCCATAACGCTAAAACAAAACCCCGAGAGGAACAATTCAATTCCACATATCGGGTGGTGAGTGTATTGACCGTTTCTCACCAAGATTGGCTGA ACGGCAAGGAGTATAAGTGTAAAGTAAGCAACAAAGGTCTGCCAAGTAGCATAGAAAAAACAATATCTAAAGCTAAGGGCCAACCAAGGGAACCACAAGTATACATTGCCCCCCTCTCAGGAAGAGATGACAAAGAATCAAGTTAGCCTGACCTGTTTGGTAAAGGGGTTCTATCCCTCAGATATAGCAGTCGAGTGGGAATCTAACGGCCAGCCCGAGAATAATTA TAAAACAACCCCCCCTGTGTTGGACTCAGACGGCAGCTTCTTTCCTATTCACGGCTCACTGTTGATAAGTCCCGATGGCAGGAGGGGAATGTTTTTCAGCTGTAGCGTGATGCACGAAGCTCTCCACAACCACTATACACAGAAAAGTTTGTCTTTGTCCCTTGGAAAataG TGFb neutralizing peptide (human) (SEQ ID NO: 99) ATGAGTACATCCTTTCCAGAGCTGGATCTGGAGAATTTTGAGTATGACGACAGTGCCGAAGCCTGCTACCTCGGGGACATAGTCGCATTCGGGACAATCTTTTTGTCTGTATTTACGCCCTGGTGTTTACATTTGGCCTGGTTGGAAATCTGTTGGTCGTACTCGCTCTCACCAATTCCCGAAAACCCAAAAAGTATAACAGACATATACCTGTT GAATCTGGCACTGAGTGACCTTTTGTTCGTCGCCACCCTTCCTTTTTGGACACACTACCTTATCAGTCACGAGGGGCTTCATAATGCTATGTGCAAGCTCACTACTGCCTTCTTCTTTATCGGATTCTTCGGGGGTATCTTTTTTATCACAGTTATTAGCATTGACCGATACCTTGCCATAGTGCTCGCAGCCAACTCAATGAACAACCGCACCGTGCAGCATGGAGT GACTATTTCCTTGGGTGTGTGGGCCGCTGCTATACTTGTCGCCAGCCCTCAATTCATGTTTACCAAAAAGGAAAGACAATGAGTGCCTCGGAGATTACCCTGAGGTGTTGCAAGAAATGTGGCCTGTACTTCGAAATAGCGAAGTGAATATACTCTGCTCATCATGTCATTCTGTTATTTTCGAATAATCCAAACATTGTTCAGCTG TAAGAACCGAAAGAAAGCCCGCGCCGTACGCCTGATTCTGCTCGTTGTGTTCGCCTTTTTTCTGTTTTGGACTCCTTACAACATAATGATATTCCTGGAGACTCTCAAATTCTATAACTTTTTTCCCTCCTGTGATATGAAAAGGGACCTTAGATTGGCTCTCAGTGTCACTGAAACAGTAGCCTTTAGCCATTGTTGTCTCCAACCCTTTCATATGCATTTGCAGGGGA AAAGTTCCGGCGGTATCTCGGACATTTGTATCGGAAGTGCTTGGCCGTGTTGTGTGGTCATCCTGTCCATACCGGATTCTCTCCTGAGAGTCAACGGAGCCGCCAAGATTCAATCCTGTCCAGTTTCACTCACTATACTTCAGAGGGGGATGGCAGCCTTCTGCTC Kynurenase No. 1 (SEQ ID NO: 100) ATGGAGACCGACACTTTGTTGCTGTGGGTACTTTTGTTGTGGGTCCCAGGATCTACCGGGGATATGGAACCCTCTCTCTTGAACTGCCAGTAGACGCCGTGCGCCGCATTGCAGCCGAGTTGAATTGCGATCCAACAGATGAACGCGTTGCCCTGAGGCTCGACGAAGAGGATAAATTGTCACATTTCAGGAACTGCTTTTACATTCAAAGAT GAGGGATCTTCCATCCATAGATCTTAGCCTCGTGTCCGAGGATGACGATGCCATATTTTTCTTGGGAACAGTCTTGGGTTGCAGCCAAAAATGGTACGGACATATCTCGAAGAGGAGCTGGACAAATGGGCTAAAATGGGTGCTTACGGCCACGACGTGGGAAAACGCCCCTGGATAGTTGGCGACGAATCTATCGTGAGTCTTATGAAAG ATATAGTTGGAGCACATGAGAAAGAAATTGCACTGATGAATGCCCTTACTATCAATCTGCATCTCCTCTTGCTTTCATTCTTTAAGCCCACTCCTAAACGCCACAAAATACTTTTGGAAGCAAAAGCCTTTCCAAGCGACCACTACGCTATTGAGTCACAATACAACTCCATGGACTTGATGTGGAAAAGTCTATGCGGATGGTAAAACCACGCGAAGGCGAGGAGA CCCTTCGAATGGAGGACATACTTGAGGTCATCGAAGAAGAAGGAGATAGTATAGCAGTTATTCCTTTTCAGCGGGCTGCACTTCTACACACAGGTCAACTCTTTAACATTCCAGCTATTACTAAGGCAGGCCACGCTAAAGGATGCTTCGTGGGCTTTGACCTTGCACACGCAGTAGGAAACGTAGAGCTCCGCTTGCACGATTGGGGCGTTGATTTCGCCTGCTG GTGTTCATATAAGTATCTTAACTCAGGAGCTGGTGGGTTGGCAGGCGCATTCGTACACGAGAAACACGCTCATACCGTAAAGCCTGCACTGGTAGGGTGGTTCGGACACGATCCTCTCTACCCGCTTCAATATGGATAATAAACTCCAGCTATACCTGGCGCCAATGGATTCAGGATCTCAAATCCTCCTATTTTGCTCGTTTGCAGTTTGCACGCATCTCTT GAGGTGTTCCAGCAGGCTACCATGACTGCACTCCGCCGGAAGTCAATCCTTTTGACCGGATACTTGGAGTATATGCTGAAACATTATCACTCAAAAGATAACACTGAGAATAAGGGCCCCATAGTAAACATTATCACTCCATCTCGGGCTGAAGAGCGCGGCTGCCAACTCATTGACTTTTTCCATTCCCAAGAAGTCAGTGTTCAAAAGAGTTGGAGAAACG GGGGGTTGTATGTGATAAGCGGGAGCCAGATGGAATCCGCGTTGCCCCAGTCCCCCCTATAATTCTTTTCACGATGTATACAAGTTTATTAGACTGCTGACAAGTATCTTGGACTCATCTGAGCGATCTtaG Kynurenase No. 2 (SEQ ID NO: 101) ATGGAACCCTCTCCTCTTGAACTGCCAGTAGACGCCGTGCGCCGCATTGCAGCCGAGTTGAATTGCGATCCAACAGATGAACGCGTTGCCCTGAGGCTCGACGAAGAGGATAAATTGTCACATTTCAGGAACTGCTTTTACATTCCAAAGATGAGGGATCTTCCATCCATAGATCTTAGCCTCGTGTCCGAGGATGACGATGCCATATATTTTCTTGG GAACAGTCTTGGGTTGCAGCCAAAAATGGTACGGACATATCTCGAAGAGGAGCTGGACAAATGGGCTAAAATGGGTGCTTACGGCCACGACGTGGGAAAACGCCCCTGGATAGTTGGCGACGAATCTATCGTGAGTCTTATGAAAGATATAGTTGGAGCACATGAGAAAGAAATTGCACTGATGAATGCCCTTACTATCAATCTGCATCTCCTCTTGC TTTCATTCTTTAAGCCCACTCCTAAACGCCACAAAATACTTTTGGAAGCAAAAGCCTTTCCAAGCGACCACTACGCTATTGAGTCACAAATACAACTCCATGGACTTGATGTGGAAAAGTCTATGCGGATGGTAAAACCGCGAAGGCGAGGAGACCCTTCGAATGGAGGACATAACTTGAGGTCATCGAAGAAGAAGGAGATAGTATAGCAGTTATTCCTTTTCAGC GGGCTGCACTTCTACACAGGTCAACTCTTTAACATTCCAGCTATTACTAAGGCAGGCCACGCTAAAGGATGCTTCGTGGGCTTTGACCTTGCACACGCAGTAGGAAACGTAGAGCTCCGCTTGCACGATTGGGGCGTTGATTTCGCCTGCTGGTGTTCATATAAGTATCTTAACTCAGGAGCTGGTGGGTTGGCAGGCGCATTCGTACACGAGAAACACG CTCATACCGTAAAGCCTGCACTGGTAGGGTGGTTCGGACACGATCTCCTTACCCGCTTCAATATGGATAATAAACTCCAGCTTATACCTGGCGCCAATGGATTCAGGATCTCAAATCCTCCTATTTTGCTCGTTTGCAGTTTGCACGCATCTCTTGAGGTGTTCCAGCAGGCTACCATGACTGCACTCCGCCGGAAGTCAATCCTTTTGACCGGATACTTGGA GTATATGCTGAAACATTATCACTCAAAAGATAACACTGAGAATAAGGGCCCCATAGTAAACATTATCACTCCATCTCGGGCTGAAGAGCGCGGCTGCCAACTCACATTGACTTTTTCCATTCCCAAGAAGTCAGTGTTCAAAGAGTTGGAGAAACGGGGGGTTGTATGTGATAAGCGGGAGCCAGATGGAATCCGCGTTGCCCCAGTCCCCCCTATAATT CTTTTCACGATGTATACAAGTTTATTAGACTGCTGACAAGTATCTTGGACTCATCTGAGCGATCTtaG VEGF (SEQ ID NO: 102) ATGAATTTCTTGCTGAGCTGGGTGCATTGGACACTCGCATTGTTGCTGTACTTGCACCATGCCAAGTGGTCCCAGGCTGCACCCACTACTGAGGGCGAGCAAAAGTCTCATGAGGTGATTAAATTTATGGACGTTTACCAACGATCATACTGTCGGCCAATCGAAACCCTCGTAGATATATTCCAGGAGTACCCAGACGAGATCGAATACATTT TCAAGCCCTCATGTGTCCCATTGATGCGATGTGCTGGGTGCTGTAACGACGAAGCACTTGAATGTGTCCCACCTCCGAGAGTAACATCACAATGCAAATAATGAGAATCAAGCCCCACCAATCCCAACATATCGGTGAAATGTCATTCTTCAGCATTCCCGCTGCGAGTGCCGGCCTAAGAAGGACCGCACCAAACCGAGAACCATTGTGAACCCTGTTCTGA GAGACGGAAGCACTTGTTCGTACAGGACCTCAAAACATGCAAGTGCAGCTGTAAGAATACCGACTCACGGTGTAAAGCTAGGCAACTGGAGCTTAATGAAAGGACCTGCCGATGCGATAAACCCAGGAGGtaa GM-CSF (SEQ ID NO: 103) ATGTGGTTGCAGAATTTGCTCTTCCTGGGGATTGTGGTCTACAGCCTCTCCGCACCTACCCGCTCTCCTATCACAGTTACAAGACCCTGGAAACATGTGGAGGCCATTAAAGAAGCATTGAATTTGTTGGACGATATGCCCGTCACCCTGAATGAAGAAGTAGAAGTTGTTTCTAATGAGTTCAGCTTTAAAAATTGACCTGTGTGCAGACACGGCT TAAAATTTTTGAACAGGGACTTAGAGGAAACTTTACTAAGCTGAAGGGGGCACTTAACATGACAGCTTCTTATTATCAGACCTATTGTCCTCCAACACCTGAAACCGACTGTGAAACACAGGTAACCACTTACGCCGATTTTATTGAAAACATTCCTCACCGATATACCATTTGAGTGTAAGAAGCCAGGCCAAAAGtaG Anti-PD1 (SEQ ID NO: 104) ATGGAAACTGACACACTTCTTCTGTGGGTCTTGCTCCTGTGGGTCCCAGGCTCTACTGGTGACAGTCCTGATAGGCCATGGAACCCACCTACCTTTAGTCCAGCCTTGCTCGTCGTAACCGAAGGGGACAACGCTACATTCACCTGCTCTTTTAGCAATACTTCTGAGAGTTTCTGTAGTCTGGCATCGGGAGAGTCCATCCGGACAAACAGATACT TTGGCCGCTTTTCCAGAGGATAGGTCTCAACCTGGGCAAGACGCAAGGTTTCGAGTCACACAGCTCCTAACGGGAGAGATTTTCACATGTCTGTAGTTCGGGCACGCCGAAATGATTCTGGCACATATGTTTGCGGTGTGATCTCACTTGCTCCAAAGATTCAAATAAAGGAGAGCCTTCGCGCCGAGTTGCGGGTGACTGAGCGGGAGCCCAAGTCCT GCGACAAAACCCATACTTGTCCACCCTGTGGCGGCGGGTCATCCGGTGGCGGGTCTGGGGGGCAACCAAGAGAGCCACAGGTATATACTTCTTCCCCCCAGCAGAGAAGAAATGACAAAAAACCAAGTGTCCCTGACATGTCTGGTTAAAGGATTTTATCCCAGTGACATTGCTGTAGAATGGGAATCCAATGGTCAACCCGAGAATAACTACAAAACCAC TCCTCCAGTATTGGACAGTGACGGTTCCTTCTTCCTCTATTCCAAACTTACAGTGGATAAATCCCGCTGGCAGCAAGGGAATGTATTCAGCTGTAGTGTCATGCACGAAGCTCTTCATAACCATTATACACAGAAATCTCTTTCCCCTGAGCCCAGGTAAAtaG Adenosine deaminase (ADA) No. 1 (mouse) (SEQ ID NO: 105) ATGGAGACTGATACACTTTTGCTCTGGGTTTTGCTCTTGTGGGTACCAGGGTCTACTGGAGATGCACAAACTCCTGCATTCAACAAGCCTAAGGTAGAGCTTCATGTCCATTTGGACGGAGCCATAAAACCTGAAACCATACTCTATTTCGGCAAGAAACGGGGTATAGCACTTCCCGCTGATACCGTGGAAGAGTTGAGAAATATCATTGGCATGGACAAACCTCTTAGCCTGCCTGGCTTTCTTGCAAAGTTCGACTACTATATGCCAGTTATAGCAGGGTGTAGAGAAGCAATAAAGCGAATCGCCTATGAGTTCGTTGAGATGAAGGCTAAAGAAGGAGTTGTTTACGTGGAAGTCCGGTACTCACCTCATCTGCTTGCTAATAGCAAGGTGGACCCAATGCCATGGAATCAAACTGAAGGTGATGTAACCCCTGACGATGTGGTCGATTTGGTCAATCAAGGTCTCCAAGAAGGCGAGCAGGCTTTCGGCATTAAGGTAAGAAGTATATTGTGCTGTATGCGACATCAACCTTCATGGTCCCTGGAGGTCCTCGAATTGTGCAAAAAGTACAATCAAAAAACAGTGGTCGCAATGGATCTCGCTGGAGATGAGACCATAGAAGGTTCCTCTCTTTTCCCCGGTCATGTCGAAGCATATGAAGGGGCTGTCAAAAATGGTATCCACCGCACCGTCCACGCAGGGGAAGTAGGGTCCCCAGAAGTAGTCAGGGAAGCCGTTGACATTTTGAAAACAGAAAGAGTCGGGCATGGCTACCATACAATAGAGGACGAAGCCTTGTACAATCGACTTTTGAAAGAAAATATGCACTTCGAGGTCTGTCCCTGGAGTTCATATCTCACCGGAGCATGGGACCCCAAAACAACCCACGCCGTCGTACGCTTCAAGAATGATAAGGCAAACTACAGTTTGAATACAGATGATCCACTGATATTCAAGTCAACACTTGACACTGACTACCAGATGACAAAAAAAGATATGGGTTTCACCGAAGAAGAGTTCAAGAGATTGAACATTAACGCAGCAAAAAGCTCCTTCCTGCCAGAGGAAGAGAAAAAAGAATTGCTTGAAAGGTTGTATCGAGAATACCAA Adenosine deaminase (ADA) No. 2 (mouse) (SEQ ID NO: 106) ATGGCACAAACTCCAGCTTTATAATAAGCCCAAAGTGGAACTTCATGTTCATCTGGATGGGGCAATTAAGCCCGAAACTATATTGTACTTTGGCAAAAAGAGGGGTATTGCCCTGCCAGCAGATACCGTTGAGGAGCTTCGCAACATCATTGGGATGGACAAGCCCCCTCTCTCTGCCAGGTTTTCTCGCTAAATTCGATTATTATATGCCTGTTA TTGCTGGTTGCCGGGAGGCCATCAAGAGGATAGCCTACGAGTTTGTTGAGATGAAGGCCAAAGAGGGCGTGGTGTACGTAGAGGTCAGATACAGCCCTCACCTGCTTGCCAACAGCAAGGTGGACCCAATGCCCTGGAACCAAACCGAGGGGGATGTCACTCCCGACGACGTTGTAGACCTCGTAAATCAGGGCCTTCAAGAGGGCGAGCAGGCAT TTGGCATAAAAGTCCGGTCTATACTCTGCTGTATGAGGCACCAACCCTCCTGGTCTTTGGAGGTACTTGAGTTGTGTAAGAAATACAATCAAAAGACTGTAGTCGCCATGGATCTTGCAGGCGATGAAAACCATCGAGGGTAGCTCCTTGTTCCCTGGACATGTTGAAGCCTACGAGGGGGCCGTAAAAAATGGGATACACAGGACTGTCCACGCT GGTGAAGTCGGAAGCCCAGAGGTGGTAAGGGAGGCAGTTGACATACTCAAGACAGAGCGGGTTGGACACGGATACCACACAATTGAGGACGAGGCCCTGTATAACCGCCTCCTCAAAGAGAACATGCATTTTGAGGTGTGTCCTTGGTCCAGCTACCTGACTGGTGCTTGGGACCCTAAAACAACTCACGCCGTGGTCCGGTTCAAGAACGATAAAGC CAATTACTCTTTGAATACCGACGACCCCTCATATTCAAATCAACATTGGATACCGACTACCAAATGACCAAAAAGGATATGGGGTTTACTGAAGAGGAGTTCAAGAGGCTCAACATAAATGCCGCTAAATCCTCCTTTCTCCCCGAGGAAGAAAAAAAAGAACTCCTTGAGCGGCTGTATAGGGAGTATCAA 4-1BBL No. 1 (mouse) (SEQ ID NO: 107) ATGGAAACAGATACACTCTTGCTCTGGGTACTGCTTCTGTGGGTCCCCGGCTCTACTGGGGATGAAGATGATGTAACTACTACAGAAGAACTCGCTCCCGCTCTTGTCCCCCCACCCAAGGGTACCTGCGCCGGTTGGATGGCTGGCATCCCAGGACATCCAGGTCACAACGGTACCCCGGAAGAGATGGTCGGGATGGAACTCCC GGCGAGAAGGGCGAAAAAGGGGATGCAGGGCTTCTGGGACCTAAAGGTGAAACAGGGGACGTTGGAATGACTGGTGCAGAAGGGCCTCGCGGCTTTCCTGGCACCCCTGGGAGGAAAGGAGAGCCCGGAGAGCTCCAGAGAACTGAACCTCGGCCTGCACTCACTATAACTACTTCCCCTAATCTTGGGACCGCGAGAACAACGCCGATC AGGTTACACCTGTAAGCCATATCGGGTGCCCCAATACTACCCAGCAAGGGAGTCCCGTGTTCGCAAAGCTTTTGGCTAAAAACCAAGCATCCCTGTGTAACACTACTCTTAATTGGCATTCACAAGACGGTGCTGGTAGCTCTATCTTTCTCAGGGGCTGCGGTACGAAGAAGATAAGAAGGAATTGGTTGTGGATTCTCCAGGACTCTATTATGTCTT TCTCGAATTGAAGCTCAGTCCACCTTCAAAACACTGGACACAAAGTCCAGGGCTGGGTAAGTCTGGTACTCCAAAGCAAAGCCCCAGGTTGACGATTTCGACAATTTGGCACTCACCGTAGAGCTTTTCCCATGCTCCATGGAAAATAAACTTGTTGATCGGTCATGGTCACAGCTCTTGCTGCTTAAGGCAGGGCATCGCCTCTCAGTGGGTCTGAGAGC TTATTTGCATGGTGCACAAGATGCTTACAGGGATTGGGAATTGTCCTACCCAAACACTACAAGTTTCGGGTTGTTCCTTGTCAAACCTGATAACCCATGGGAGtaG 4-1BBL No. 2 (mouse) (SEQ ID NO: 108) ATGGAAACTGATACACTCCTCCTGTGGGTCCTTCTTTTGTGGGTGCCCGGATCAACCGGCGATGGCTGGATGGCAGGCATCCCAGGACACCCAGGACACAACGGTACTCCAGGTCGAGACGGTCGGGATGGGACTCCTGGGGAGAAAGGCGAGAAAGGGGACGCTGGTTTGCTCGGTCCTAAGGGGGAAACCGGGG ATGTAGGAATGACAGGGGCTGAAGGGCCTCGGGGATTTCCTGGGACACCAGGCAGGAAGGGTGAACCAGGGGAGGCCCTCCAGCGCACCGAGCCACGGCCAGCTCTGACCATAACAACAAGTCCAAACCTGGGCACACGCGAAAACAATGCTGACCAGGTGACTCCTGTAAGTCACATCGGATGCCCTAACACTACACAACAGGGCTCTCCTGTATTT GCAAAGCTTCTCGCAAAAAATCAAGCATCACTTTGTAATACAACCCCTGAACTGGCATTCTCAGGACGGAGCAGGGTCCTCTTATTTGTCTCAAGGGCTCCGCTACGAAGAAGATAAAAAGGAATTGGTTGTTGACAGTCCAGGTTTGTATTATGTGTTTTTGGAACTTAAGCTGTCACCAACCTTCACTAACACCGGCCACAAGGTCCAAGGCTGGGTTAGCT TGTTTTGCAAGCCAAACCTCAAGTGGATGATTTTGACAATCTGGCTTTGACTGTTGAGCTTTTTCCATGCAGTATGGAGAATAAACTGGTTGATCGGTCATGGTCACAGCTCCTTCTGCTCAAGGCCGGACATAGGCTGAGTGTGGGACTTCGGGCCTACTTGCACGGCGCCCAGGACGCATACCGAGACTGGGAACTCAGCTACCCTAACACAA CTTCTTTTGGGTTGTTCCTTGTCAAACCCGATAATCCTTGGGAAtaG HPGE2 No. 1 (mouse) (SEQ ID NO: 109) ATGGAGACTGATACTTTGCTCCTGTGGGTTCTTCTCCTGTGGGTTCCTGGTTCCACAGGGGATATGCATGTCAATGGCAAGGTAGCACTCGTGACTGGGGCTGCACAGGGTATCGGGAAAGCTTTTGCCGAGGCCCTGTTGCTGCATGGCGCCAAGGTCGCTTTGGTAGATTGGAACTTGGAGGCTGGAGTTAAATGCAAAGCT GCACTCGACGAACAATTTGAGCCTCAAAAAACCCCTTTGTGCAGTGTGACGTTGCTGACCAAAGCAACTCAGGGACACATTCAGGAAGGTCGTAGACCATTTCGGACGCCTCGATATACTCGTTAATAATGCCGGGGTAAACAACGAAAAGAACTGGGAACAAACATTGCAAATCAACCTGGTAAGTGTCATTAGCGGAACTTATCTGGGTCTT GATTATATGAGCAAGCAGAACGGGGGCGAGGGCGGGATCATTATCAACATGTCAAGTCTTGCCGGATTGATGCCAGTTGCTCAGCAGCCTGTTACTGTGCCAGCAAGCACGGTATTATTGGGTTTACCGGAGTGCCGCCATGGCCGCAAATCTTATGAAGAGTGGGGTAAGACTGAATGTTATCTGCCCAGGTTTCGTAGATACCCCAATCCT GGAGAGCATCGAGAAGGAGGAAAATATGGGACAATACATTGAATATAAAGATCAAATCAAGGCTATGATGAAGTTCTACGGGGTTCTGCATCCATCCACAATTGCCAACGGGCTCATTAATCTGATTGAGGACGCCTTGAACGGAGCTATAATGAAAATCAGCTTCCAAAGGCATTCACTTCCAAAGATTATGATATATCACCCTTGCTTGTCAAGGCTCCT CTGACAAGT HPGE2 No. 2 (mouse) (SEQ ID NO: 110) ATGCATGTCAATGGCAAGGTAGCACTCGTGACTGGGGCTGCACAGGGTATCGGGAAAGCTTTTGCCGAGGCCCTGTTGCTGCATGGCGCCAAGGTCGCTTTGGTAGATTGGAACTTGGAGGCTGGAGTTAAATGCAAAGCTGCACTCGACGAACAATTTGAGCCTCAAAAAACCCCTTTGTGCAGTGTGACGTTGCTGACCAAAG CAACTCAGGGACACATTCAGGAAGGTCGTAGACCATTTCGGACGCCTCGATATACTCGTTAATAATGCCGGGGTAAACAACGAAAGAACTGGGAACAAACATTGCAAATCAACCTGGTAAGTGTCATTAGCGGAACTTATCTGGGTCTTGATTATATGAGCAAGCAGAACGGGGGCGAGGGCGGGATCATTATCAACATGTCAAGTCTTGCC GGATTGATGCCAGTTGCTCAGCAGCCTGTTACTGTGCCAGCAAGCACGGTATTATTGGGTTTACCGGAGTGCCGCCATGGCCGCAAATCTTATGAAGAGTGGGGTAAGACTGAATGTTATCTGCCCAGGTTTCGTAGATACCCCAATCCTGGAGAGCATCGAGAAGGAGGAAAATATGGGACAATACATTGAATAAAGATCAAATCAAGGCTAT GATGAAGTTCTACGGGGTTCTGCATCCATCCACAATTGCCAACGGGCTCATTAATCTGATTGAGGACGACGCCTTGAACGGAGCTATAATGAAAATCACAGCTTCCAAAGGCATTCACTTCCAAAGATTATGATATATCACCCTTGCTTGTCAAGGCTCCTCTGACAAGT Human IL-15 polypeptide sequence (SEQ ID NO: 199) MVLGTIDLCSCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS Human IL-15Rα polypeptide sequence (SEQ ID NO: 200) MAPRRARGCRTLGLPALLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVA ISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL Human IL-15Rα sushi domain polypeptide sequence (SEQ ID NO: 201) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR Human IL-15 / IL-15Rα sushi domain polypeptide sequence (SEQ ID NO: 202) MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGSGGGGSGGGSGGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFK RKAGTSSLETCVLNKATNVAHWTTPSLKCIR IL-12 (IL-12p70) polypeptide sequence (SEQ ID NO: 203) MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEY EYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGSGGGSGGGSGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQ KARQTLEFYPCTSEEIDHEDITKDKTSTEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS Secretion signals and signaling anchors

[0069] One or more effector molecules of the chimeric proteins provided herein may have a secretion signal peptide (also referred to as a signal Secretory effector molecules that direct newly synthesized proteins destined for secretion or membrane localization (also known as membrane insertion) to the appropriate protein processing pathway. For chimeric proteins having the formula MT-C-S, the membrane tether domain typically has a signal anchor sequence that directs the newly synthesized protein intended for membrane localization to the appropriate protein processing pathway (e.g., type II transmembrane protein signal anchor sequence). For chimeric proteins of the formula S-C-MT, a membrane tether domain with a reverse signal anchor sequence (e.g., that of certain type III transmembrane proteins) can be used, which generally does not incorporate predetermined A separate secretory signal peptide for membrane localization of newly synthesized proteins that directs them to the appropriate protein processing pathway.

[0070] In general, for all membrane cleavable chimeric proteins described herein, the one or more effector molecules are secretable effector molecules (referred to in formula S-C-MT or MT-C-S "S"). In embodiments with two or more chimeric proteins, each chimeric protein can include a secretion signal. In embodiments with two or more chimeric proteins, each chimeric protein can comprise a secretion signal enabling secretion of each effector molecule from the engineered cell upon cleavage at a protease cleavage site.

[0071] A secretion signal peptide that is operably associated with an effector molecule can be a natural secretion signal peptide (eg, a secretion signal peptide that is typically endogenously associated with a given effector molecule). The secretion signal peptide operably associated with the effector molecule can be a non-native secretion signal peptide natural secretion signal peptide. Non-natural secretion signal peptides can promote improved expression and function, such as maintenance of secretion, in specific environments such as the tumor microenvironment. Non-limiting examples of non-natural secretion signal peptides are shown in Table 3. Table 3. Exemplary signal secretion peptides name protein sequence Source (Uniprot) dna sequence IL-12 MCHQQLVISWFSLVFLASPLVA (SEQ ID NO: 112) P29460 ATGTGTCACCAGCAGCTCGTTATATCCTGGTTTAGTTTGGTGTTTCTCGCTTCACCCCTGGTGGCA (SEQ ID NO: 31) IL-12 (codon optimized) MCHQQLVISWFSLVFLASPLVA (SEQ ID NO: 112) - ATGTGCCATCAGCAACTCGTCATCTCCTGGTTCTCCCTTGTGTTCCTCGCTTCCCCTCTGGTCGCC (SEQ ID NO: 32) IL-2 (optimized) MQLLSCIALILALV (SEQ ID NO: 113) - ATGCAACTGCTGTCATGTATCGCACTCATCCTGGCGCTGGTA (SEQ ID NO: 33) IL-2 (natural) MYRMQLLSCIALSL LVTNS (SEQ ID NO: 114) P60568 ATGTATCGGATGCAACTTTTGAGCTGCATCGCATTGTCTCTGGCGCTGGTGACAAATTCC (SEQ ID NO: 34) Trypsinogen-2 MNLLLILTFVAAAVA (SEQ ID NO: 115) P07478 ATGAATCTCTTGCTCATACTTACGTTTGTCGCTGCTGCCGTTGCG (SEQ ID NO: 35) Gaussian luciferase MGVKVLFALICIAVAEA (SEQ ID NO: 116) - ATGGGCGTGAAGGTCTTGTTTGCCCTTATCTGCATAGCTGTTGCGGAGGCG (SEQ ID NO: 36) CD5 MPMGSLQPLATLYLLGMLVASCLG (SEQ ID NO: 117) P06127 ATGCCGATGGGGAGCCTTCAACCTTTGGCAACGCTTTATCTTCTGGGGATGTTGGTTGCTAGTTGCCTTGGG (SEQ ID NO: 37) IgKVII (mouse) METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 118) ATGGAAACTGACACGTTGTTGCTGTGGGTATTGCTCTTGTGGGTCCCAGGATCTACGGGCGAC (SEQ ID NO: 38) IgKVII (human) MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 119) P01597 ATGGATATGAGGGTTCCCGCCCAGCTTTTGGGGCTGCTTTTGTTGTGGCTTCGAGGGGCTCGGTGT (SEQ ID NO: 39) VSV-G MKCLLYLAFLFIGVNC (SEQ ID NO: 120) - ATGAAGTGTCTGTTGTACCTGGCGTTTCTGTTCATTGGTGTAAACTGT (SEQ ID NO: 40) Prolactin MNIKGSPWKGSLLLLLVSNLLLCQSVAP (SEQ ID NO: 121) P01236 ATGAATATCAAAGGAAGTCCGTGGAAGGGTAGTCTCCTGCTGCTCCTCGTATCTAACCTTCTCCTTTGTCAATCCGTGGCACCC (SEQ ID NO: 41) serum albumin preproprotein MKWVTFISLLFLFSSAYS (SEQ ID NO: 122) P02768 ATGAAATGGGTAACATTCATATCACTTCTCTTTTCTGTTCAGCTCTGCGTATTCT (SEQ ID NO: 42) azucidin preproprotein MTRLTVLALLAGLLASSRA (SEQ ID NO: 123) 20160 ATGACAAGGCTTACTGTTTTGGCCTCTCCTCGCTGGACTCTTGGCTTCCTCCCGAGCA (SEQ ID NO: 43) osteonectin (BM40) MRAWIFFLLCLAGRALA (SEQ ID NO: 124) P09486 ATGAGGGCTTGGATTTTTTTTCTGCTCTGCCTTGCCGGTCGAGCCCTGGCG (SEQ ID NO: 44) CD33 MPLLLLLPLLWAGALA (SEQ ID NO: 125) P20138 ATGCCTCTTCTGCTTTTGCTTCCTCTTTTGTGGGCAGGTGCCCTCGCA (SEQ ID NO: 45) IL-6 MNSFSTSAFGPVAFSLGLLLVLPAAFPAP (SEQ ID NO: 126) P05231 ATGAACTCTTTCTCAACCTCTGCGTTTGGTCCGGTCGCTTTCTCCCTTGGGCTCCTGCTTGTCTTGCCAGCAGCGTTTCCTGCGCCA (SEQ ID NO: 46) IL-8 MTSKLAVALLAAFLISAALC (SEQ ID NO: 127) P10145 ATGACAAGTAAACTGGCGGTAGCCTTGCTCGCGGCCTTTTTGATTTCCGCAGCCCTTTGT (SEQ ID NO: 47) CCL2 MKVSAALLCLLLIAATFIPQGLA (SEQ ID NO: 128) P13500 ATGAAGGTAAGTGCAGCGTTGCTTTGCCTTCTCCTCATTGCAGCGACCTTTTATTCCTCAAGGGCTGGCC (SEQ ID NO: 48) TIMP2 MGAAARTLRLAGLLLLATLLRPADA (SEQ ID NO: 129) P16035 ATGGGAGCGGCAGCTAGAACACTTCGACTTGCCCTTGGGCTCTTGCTCCTTGCAACCCTCCTTAGACCTGCCGACGCA (SEQ ID NO: 49) VEGFB MSPLLRRLLLAALLQLAPAQA (SEQ ID NO: 130) P49765 ATGTCACCGTTGTTGCGGAGATTGCTGTTGGCCGCACTTTTGCAACTGGCTCCTGCTCAAGCC (SEQ ID NO: 50) osteoprotegerin MNNLLCCALVFLDISIKWTTQ (SEQ ID NO: 131) O00300 ATGAATAACCTGCTCTGTTGTGCGCTCGTGTTCCTGGACATTTCTATAAAATGGACAACGCAA (SEQ ID NO: 51) serine protease inhibitor E1 MQMSPALTCLVLGLALVFGEGSA (SEQ ID NO: 132) P05121 ATGCAAATGTCTCCTGCCCTTACCTGTCTCGTACTTGGTCTTGCGCTCGTATTTGGAGAGGGATCAGCC (SEQ ID NO: 52) GROα MARAALSAAPSNPRLLLRVALLLLLLVAAGRRAAG (SEQ ID NO: 133) P09341 ATGGCAAGGGCTGCACTCAGTGCTGCCCCGTCTAATCCCAGATTGCTTCGAGTTGCATTGCTTCTTCTGTTGCTGGTTGCAGCTGGTAGGAGAGCAGCGGGT (SEQ ID NO: 53) CXCL12 MNAKVVVVLVLVLTALCLSDG (SEQ ID NO: 134) P48061 ATGAATGCAAAAGTCGTGGTCGTGCTGGTTTTGGTTCTGACGGCGTTGTGTCTTAGTGATGGG (SEQ ID NO: 54) IL-21 (codon optimized) MERIVICLMVIFLGTLVHKSSS (SEQ ID NO: 135) Q9HBE4 ATGGAACGCATTGTGATCTGCCTGATGGTCATCTTCCTGGGCACCTTAGTGCACAAGTCGAGCAGC (SEQ ID NO: 55) CD8 MALPVTALLLPLALLLLHAARP (SEQ ID NO: 136) - ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (SEQ ID NO: 139) CD8 (codon optimized) MALPVTALLLPLALLLLHAARP (SEQ ID NO: 137) - ATGGCGCTCCCGGTGACAGCACTTCTCTTGCCTCTTGCCCTGCTGTTGCATGCCGCGCGCCCA (SEQ ID NO: 140) GMCSF MLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 138) - ATGTTGCTCGTGACATCCCTCTTGCTTTGTGAGTTGCCTCATCCCGCATTCCTGCTCATCCCA (SEQ ID NO: 141) NK2D PFFFCCFIAVAMGIRFIIMVA (SEQ ID NO: 192) CCCTTCTTTCTTCTGTTGCTTTATCGCCGTGGCCATGGGCATCCGCTTCATCATTATGGTGGCC (SEQ ID NO: 193) protease cleavage site

[0072] In certain embodiments, the chimeric proteins provided herein (e.g., generally, for all membrane cleavable chimeric proteins described herein) contain a protease cleavage site (e.g., at the The membrane cleavable chimeric proteins described herein are referred to as "C" in the formula S-C-MT or MT-C-S). In general, a protease cleavage site can be any amino acid sequence motif capable of being cleaved by a protease. Examples of protease cleavage sites include, but are not limited to, type 1 transmembrane protease cleavage site, type II 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, ADAM28 protease cleavage site, ADAM30 protease cleavage site point, ADAM33 protease cleavage site, BACE1 protease cleavage site, BACE2 protease cleavage site, SIP protease cleavage site, MT1-MMP protease cleavage site, MT3-MMP protease cleavage site, MT5-MMP protease cleavage site, Furin cleavage site, PCSK7 protease cleavage site, Protease protease cleavage site, Protease-2 protease cleavage site, MMP9 protease cleavage site or NS3 protease cleavage site.

[0073] An example of a protease cleavage site is the hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease cleavage site, including but not limited to NS3 / NS4A, NS4A / NS4B, NS4B / NS5A or NS5A / NS5B cleavage site. For a description of representative sequences of NS3 proteases of various HCV strains and their cleavage sites, see, for example, Hepatitis C Viruses: Genomes and Molecular Biology (S.L. Tan editors, Taylor and Francis, 2006), Chapter 6, Chapters 163- 206 pages; which is incorporated herein by reference in its entirety. For example, HCV NS4A / 4B protease cleavage site, HCV NS5A / 5B protease cleavage site, C-terminal degron with NS4A / 4B protease cleavage site, N-terminal with HCV NS5A / 5B protease cleavage site Sequence of degron. Representative NS3 sequences are listed in the National Center for Biotechnology Information (NCBI) database. See eg NCBI entries: accession numbers YP_001491553, YP_001469631, YP_001469632, NP_803144, NP_671491, YP_001469634, YP_001469630, YP_001469633, ADA68311, ADA68307, A FP99000, AFP98987, ADA68322, AFP99033, ADA68330, AFP99056, AFP99041, CBF60982, CBF60817, AHH29575, AIZ00747, AIZ00744 , ABI36969, ABN05226, KF516075, KF516074, KF516056, AB826684, AB826683, JX171009, JX171008, JX171000, EU847455, EF154714, GU085487, JX171065 , JX171063; all such sequences (if entered on the filing date of this application) are incorporated by reference incorporated into this article.

[0074] Another example of a protease cleavage site is an ADAM17-specific protease (also known as tumor necrosis factor-alpha converting enzyme [TACE]) cleavage site. The ADAM17-specific protease cleavage site may be an endogenous sequence of the substrate naturally cleaved by ADAM17. An ADAM17-specific protease cleavage site can be an engineered sequence capable of being cleaved by ADAM17. Engineered ADAM17-specific protease cleavage sites can be engineered for specific desired properties including, but not limited to, optimal performance of chimeric proteins, specificity for ADAM17, rate of cleavage by ADAM17, secreted Ratio of chimeric protein levels to membrane-bound chimeric protein levels, and cleavage in different cell states. Protease cleavage sites can be selected for specific cleavage by ADAM17. For example, certain protease cleavage sites that are cleaved by ADAM17 are also cleaved by additional ADAM family proteases such as ADAM10. Thus, ADAM17-specific protease cleavage sites can be selected and / or engineered such that cleavage by other proteases such as ADAM10 is reduced or eliminated. Protease cleavage sites can be selected for the rate of cleavage by ADAM17. For example, it may be desirable to select protease cleavage sites that exhibit a specific rate of cleavage by ADAM17, such as reduced cleavage kinetics relative to endogenous sequences of substrates naturally cleaved by ADAM17. In such cases, generally, a particular rate of cleavage can be selected to regulate the rate of processing of the chimeric protein, which in turn regulates the rate of release / secretion of the payload effector molecule. Thus, ADAM17-specific protease cleavage sites can be selected and / or engineered such that the sequence exhibits the desired rate of cleavage by ADAM17. Protease cleavage sites can be selected for both specific cleavage by ADAM17 and the rate of cleavage by ADAM17. Exemplary ADAM17-specific protease cleavage sites (including those exhibiting specific specificity and cleavage rate kinetics) reference cleavage sites are shown in Table 4A (P5-P1: N-terminus; P1'-P5': C-terminus) . Additional details of ADAM17 and ADAM10, including expression and protease cleavage sites, are described in: Sharma et al. (J Immunol 15 Oct 2017, 199 (8) 2865-2872); Pham et al. (Anticancer Res. 2017 Oct;37(10):5507-5513); Caescu et al (Biochem J. 2009 Oct 23;424(1):79-88) and Tucher et al (J. Proteome Res. 2014, 13, 4, 2205-2214), each of which is incorporated herein by reference for this purpose. Table 4A - Various ADAM17 protease cleavage site sequences P5 P4 P3 P2 P1 P1' P2' P3' P4' P5' full sequence SEQ ID NO P R A E. A V K G G PRAEA VKGG 179 P R A E. A L K G G PRAEALKGG 180 P R A E. Y S K G G PRAEYSKGG 181 P R A E. P I K G G PRAEPIKGG 182 P R A E. A Y K G G PRAEAYKGG 183 P R A E. S S K G G PRAESSKGG 184 P R A E. f T K G G PRAEFTKGG 185 D. E. P h Y S Q R R DEPHYSQRR 187 P P L G P I f N P G PPLGPIFNPG 188 P L A Q A Y R S S PLAQAYRSS 189 T P I D. S S f N P D. TPIDSSFNPD 190 V T P E. P I f S L I VTPEPIFSLI 191

[0075] In some embodiments, the protease cleavage site comprises a first region having the amino acid sequence of PRAE (SEQ ID NO: 176). In some embodiments, the protease cleavage site comprises a second region having the amino acid sequence of KGG (SEQ ID NO: 177). In some embodiments, the first region is located N-terminal to the second region. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 178), wherein X1 is A, Y, P, S or F, and wherein X2 is V, L, S, I, Y, T or A. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 178), wherein X1 is A, Y, P, S or F, and wherein X2 is V, L, S, I, Y or T. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAVKGG (SEQ ID NO: 179). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEALKGG (SEQ ID NO: 180). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEYSKGG (SEQ ID NO: 181). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEPIKGG (SEQ ID NO: 182). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAYKGG (SEQ ID NO: 183). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAESSKGG (SEQ ID NO: 184). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEFTKGG (SEQ ID NO: 185). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAAKGG (SEQ ID NO: 186).

[0076] In some embodiments, the protease cleavage site comprises the amino acid sequence of DEPHYSQRR (SEQ ID NO: 187). In some embodiments, the protease cleavage site comprises the amino acid sequence of PPLGPIFNPG (SEQ ID NO: 188). In some embodiments, the protease cleavage site comprises the amino acid sequence of PLAQAYRSS (SEQ ID NO: 189). In some embodiments, the protease cleavage site comprises the amino acid sequence of TPIDSSFNPD (SEQ ID NO: 190). In some embodiments, the protease cleavage site comprises the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191). The protease cleavage sites of SEQ ID NOs: 187, 189 and 191 can be cleaved by ADAM17.

[0077] In some embodiments, the protease cleavage site comprises the amino acid sequence of ITQGLAVSTISSFF (SEQ ID NO: 198), which is a natural cleavage site for CD16 and can be cleaved by ADAM17.

[0078] The protease cleavage site can be the C-terminus of the secretable effector molecule. The protease cleavage site can be the N-terminus of the secretable effector molecule. In general, for all membrane cleavable chimeric proteins described herein, the protease cleavage site is: (1) the C-terminus of the secretable effector molecule and the N-terminus of the cell membrane tether domain (in other words, the protease cleavage site point between the secretable effector molecule and the cell membrane tether domain); or (2) the N-terminus of the secretable effector molecule and the C-terminus of the cell membrane tether domain (also between the cell membrane where the secretable effector molecule and the reverse domain are oriented between tether domains). A protease cleavage site can be linked to a secretable effector molecule by a polypeptide linker (ie, a polypeptide sequence not normally considered part of the effector molecule or protease cleavage site). The protease cleavage site can be linked to the cell membrane tether domain by a polypeptide linker (ie, a polypeptide sequence not generally considered part of the cell membrane tether domain or protease cleavage site). The polypeptide linker can be any amino acid sequence linking the first polypeptide sequence and the second polypeptide sequence. A polypeptide linker can be a flexible linker (eg, a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS]4GG [SEQ ID NO: 182]), A(EAAAK)3A (SEQ ID NO: 183), and Whitlow linkers (e.g., "KEGS" Linkers, such as the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), eGK linkers, such as the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 185), and are described in more detail in issued U.S. Patent No. 5,990,275 linker, the U.S. patent is incorporated herein by reference). Additional exemplary polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, and SEQ ID NO: 197. Other polypeptide linkers can be selected based on desired properties (eg, length, flexibility, amino acid composition, etc.) and are known to those skilled in the art.

[0079] In a membrane cleavable system, after the chimeric protein is expressed and localized in the cell membrane, a protease cleavage site directs cleavage of the chimeric protein, allowing release ("secretion") of the effector molecule into the extracellular space of the cell.

[0080] Generally, a protease that cleaves a protease cleavage site is a protease that is specific for that specific protease cleavage site. For example, in the case of disintegrin and metalloprotease ("ADAM") family proteases, proteases that cleave a particular ADAM protease cleavage site are generally limited to one or more that specifically recognize a particular ADAM protease cleavage site motif. ADAM protease. Protease cleavage sites can be selected and / or engineered such that cleavage by undesired proteases is reduced or eliminated. Proteases can be membrane-bound or membrane-associated. Proteases can be secreted, eg, in specific cellular environments such as the tumor microenvironment ("TME").

[0081] A protease that cleaves the protease cleavage site of the chimeric protein can be expressed in the same cell that expresses the chimeric protein. The protease that cleaves the protease cleavage site of the chimeric protein may be endogenous to the cell expressing the chimeric protein. In other words, cells engineered to express a chimeric protein can endogenously express a protease specific for the protease cleavage site present in the chimeric protein. Endogenous expression of a protease refers to expression under generally homeostatic conditions (e.g., cells generally considered healthy), and also refers to differential expression under non-steady state conditions (e.g., upregulated expression in tumor cells) . Protease cleavage sites can be selected based on known proteases expressed endogenously by the desired cell population. In such cases, in general, cleavage of the protease cleavage site (and thus release / secretion of the payload) can be restricted to the cell of interest to them, since the cell-restricted protease needs to be associated with a chimera expressed in the same cell. The protease cleavage site of the synthetic protein is contacted. For example, and without wishing to be bound by theory, it is believed that the endogenous expression of ADAM17 is restricted to NK cells and T cells. Thus, selection of ADAM17-specific protease cleavage sites can limit cleavage of the protease cleavage sites to NK cells and T cells co-expressing the chimeric protein. In other examples, protease cleavage sites can be selected for specific tumor-associated proteases known to be expressed in a particular tumor population of interest (eg, in specific tumor cells engineered to express a chimeric protein). Protease and / or expression databases can be used to select appropriate protease cleavage sites, such as by consulting Oncomine (www.oncomine.org), European Bioinformatics Institute (www.ebi.ac.uk) (specifically Language (www.ebi.ac.uk / gxa)), PMAP (www.proteolysis.org), ExPASy Peptide Cutter (ca.expasy.org / tools / peptide cutter) and PMAP.Cut DB (cutdb.burnham.org) (each of which is incorporated by reference for all purposes) to select protease cleavage sites for cleavage by tumor-associated proteases.

[0082] The protease that cleaves the protease cleavage site of the chimeric protein may be heterologous to the cell expressing the chimeric protein. For example, a cell engineered to express a chimeric protein can also be engineered to express a protease specific for a protease cleavage site present in the chimeric protein that the cell does not normally express. Cells engineered to express both chimeric proteins and proteases can be engineered to generate from a single engineered nucleic acid or a polycistronic system (multicistronic and multi-promoter systems are described in more detail herein under the heading In the section "Multicistronic and Multiple Promoter Systems") represent each. Heterologous proteases and their corresponding protease cleavage sites can be selected with reference to endogenous proteases as described above.

[0083] The protease that cleaves the protease cleavage site of the chimeric protein may be expressed on a separate cell than the cell expressing the chimeric protein. For example, proteases can generally be expressed in specific cellular environments such as the tumor microenvironment. In such cases, in general, cleavage of protease cleavage sites can be restricted to their cellular environment of interest (eg, the tumor microenvironment), since environmentally restricted proteases need to make contact with the protease cleavage site. In embodiments with membrane-cleavable chimeric proteins, secretion of effector molecules can generally be restricted to their cellular environment of interest (e.g., tumor microenvironment) due to the requirement for environmentally-restricted proteases to interact with the protease cleavage site. touch. The protease that cleaves the protease cleavage site of the chimeric protein may be endogenous to individual different cells. The protease that cleaves the protease cleavage site of the chimeric protein may be heterologous to individual different cells. For example, a separate distinct cell can be engineered to express a protease that is not normally expressed by a separate distinct cell.

[0084] Proteases include but are not limited to Type 1 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, PCSK7 protease, protease protease, protease -2 protease and MMP9 protease. The protease can be NS3 protease. The protease can be ADAM17 protease.

[0085] The protease can be a tumor-associated protease, such as an autolysozyme, a cysteine ​​protease, an aspartic protease, a serine protease, or a metalloprotease. Specific examples of tumor-associated proteases include autolyse B, autolysozyme L, autolysozyme S, autolysozyme D, autolysozyme E, autolysozyme A, autolysozyme G, coagulation Enzymes, 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 may also include, but are not limited to, those listed in Table 4B below. Exemplary homologous protease cleavage sites for certain proteases are also listed in Table 4B. Table 4B: Exemplary proteases with cognate cleavage sites and inhibitors protease (UniProt accession number) homologous cleavage site Protease inhibitor HCV NS4A / 4B DEMEECSQHL (SEQ ID NO: 142) EDVVPCSMG (SEQ ID NO: 143) Simeprevir, Danoprevir, Asunaprevir, Siluprevir, Boceprevir, Sofaprevir, Paliferevir, Telaprevir, Glaprevir HCV NS5A / 5B DEMEECSQHL (SEQ ID NO: 142) EDVVPCSMG (SEQ ID NO: 143) Simeprevir, Danoprevir, Asunaprevir, Siluprevir, Boceprevir, Sofaprevir, Paliferevir, Telaprevir, Glaprevir HCV NS3 DEMEECSQHL (SEQ ID NO: 142) EDVVPCSMG (SEQ ID NO: 143) Simeprevir, Danoprevir, Asunaprevir, Siluprevir, Boceprevir, Sofaprevir, Paliferevir, Telaprevir, Glaprevir HCV NS2-3 DEMEECSQHL (SEQ ID NO: 142) EDVVPCSMG (SEQ ID NO: 143) Simeprevir, Danoprevir, Asunaprevir, Siluprevir, Boceprevir, Sofaprevir, Paliferevir, Telaprevir, Glaprevir HIV-1 protease (SEQ ID NO: 144) Amprenavir, Atazanavir, Darunavir, Fosamprenavir, Indinavir, Lopinavir, Nef Nelfinavir, Ritonavir, Saquinavir, Tipranavir Signal peptidases (P67812, P15367, P00804, P0803) eukaryotic signal peptidase at residue 20 of pre(∆pro)apoA-II (Xaa 20↓ ) post-cleavage is better: Ala, Cys > Gly > Ser, Thr > Pro > Asn, Val, Ile, Leu, Tyr, His, Arg, Asp. Proprotein convertases that cleave at hydrophobic residues (e.g., Leu, Phe, Val, or Met) (Q16549, Q8NBP7, Q92824, P29120, Q6UW60, P29122, Q9QXV0) (R / K)-X-(hydrophobic)-X↓, where X is any amino acid Proprotein convertases that cleave at small amino acid residues such as Ala or Thr (Q16549, Q8NBP7, Q92824, P29120, Q6UW60, P29122) (K / R)-(X)n-(K / R)↓, where n is 0, 2, 4 or 6 and X is any amino acid Proopiomelanocortin converting enzyme (PCE) (Q9UO77615, O776133) Cleavage at or between paired basic residues in certain prohormones or on the carboxyl side Chromophilic Granular Aspartic Acid Protease (CGAP) Prefers to cleave dipeptide bonds with hydrophobic residues and β-methylene groups Hormone prothiol protease (cytolysozyme L1) (P07154, P07711, P06797, P25975, Q28944) Carboxypeptidases (e.g., Carboxypeptidase E / H, Carboxypeptidase D, and Carboxypeptidase Z) (Q9M099, P15169, Q04609, P08819, P08818, O77564, P70627, O35409, P07519, Q8VZU3, P22792, P15087, P16870, Q9JHH6, Q96IY4, Q7L8A9) Cleavage of peptide bonds at the carboxyl terminus (C-terminus) of proteins or peptides Aminopeptidases (eg, arginine aminopeptidase, lysine aminopeptidase, aminopeptidase B) Cleavage of peptide bonds at the amino terminus (N-terminus) of proteins or peptides Prolyl endopeptidase (Q12884, P48147, P97321, Q4J6C6) Hydrolysis of Pro-|-Xaa in oligopeptide >> Ala-|-Xaa. When Yaa is Pro, the N-terminal dipeptide (Xaa-Yaa-|-Zaa-) is preferentially released from the polypeptide, provided that Zaa is neither Pro nor hydroxyproline Aminopeptidase N (P97449, P15144, P15145, P15684) The N-terminal amino acid (Xaa-|-Yaa-) is released from the peptide, amide or arylamide. Xaa is preferably Ala, but can be most amino acids, including Pro (slow acting). When the terminal hydrophobic residue is followed by a prolinyl residue, both are released as the complete Xaa-Pro dipeptide Insulin degrading enzymes (P14735, P35559, Q9JHR7, P22817, Q24K02) Degrade insulin, glucagon and other peptides. Has no effect on protein. Cleavage of multiple short peptides with considerable sequence variation Calpain (O08529, P17655, Q07009, Q27971, P20807, P07384, O35350, O14815, P04632, Q9Y6Q1, O15484, Q9HC96, A6NHC0, Q9UMQ6) No specific amino acid sequence is uniquely recognized by calpains. In protein substrates, tertiary structural elements rather than primary amino acid sequences appear to be responsible for directing cleavage to specific substrates. Among peptide and small molecule substrates, the most consistently reported specificities are for the small hydrophobic amino acids at the P2 position (e.g., leucine, valine, and isoleucine) and the large hydrophobic amino acids at the P1 position. Sexual amino acids (eg, phenylalanine and tyrosine). A calpain substrate system (EDANS)-Glu-Pro-Leu-Phe=Ala-Glu-Arg-Lys-(DABCYL), (EDANSEPLFAERKDABCYL (SEQ ID NO: 145)), at the Phe=Ala bond Cutting occurs. Caspase 1 (P29466, P29452) The P1 position is strictly required to be an Asp residue and has a preferred cleavage sequence of Tyr-Val-Ala-Asp-|- (YVAD; SEQ ID NO: 146). Caspase 2 (P42575, P29594) The P1 position is strictly required to be an Asp residue, 316-asp is essential for proteolytic activity, and has a preferred cleavage sequence of Val-Asp-Val-Ala-Asp-|- (VDVAD; SEQ ID NO: 147). Caspase 3 (P42574, P70677) The P1 and P4 positions are strictly required to be Asp residues. It has a preferred cleavage sequence of Asp-Xaa-Xaa-Asp-|- with a hydrophobic amino acid residue at P2 and a hydrophilic amino acid residue at P3, although Val is also accepted at this position or Ala. Caspase 4 (P70343, P49662) The P1 bit is strictly required to be Asp. It has a preferred cleavage sequence of Tyr-Val-Ala-Asp-|- (YVAD; SEQ ID NO: 146), but also at Asp-Glu-Val-Asp-|- (DEVD; SEQ ID NO: 148) cutting. Caspase 5 (P51878) The P1 bit is strictly required to be Asp. It has a preferred Tyr-Val-Ala-Asp-|-(YVAD; SEQ ID NO: 146) cleavage sequence, but also cuts Asp-Glu-Val-Asp-|--|-(DEVD; SEQ ID NO: 148 ). Caspase 6 (P55212) The P1 position is strictly required to be Asp and has a preferred cleavage sequence of Val-Glu-His-Asp-|-(VEHD; SEQ ID NO: 149). Caspase 7 (P97864, P55210) The P1 position is strictly required to be an Asp residue and has a preferred cleavage sequence of Asp-Glu-Val-Asp-|- (DEVD; SEQ ID NO: 148). Caspase 8 (Q8IRY7, O89110, Q14790) The P1 position is strictly required to be Asp and has a preferred cleavage sequence of (Leu / Asp / Val)-Glu-Thr-Asp-|-(Gly / Ser / Ala). Caspase 9 (P55211, Q8C3Q9, Q5IS54) Position P1 is strictly required to be an Asp residue and position P2 is clearly preferred to be His. It has a preferred cleavage sequence of Leu-Gly-His-Asp-|-Xaa (LGHD; SEQ ID NO: 150). Caspase 10 (Q92851) The P1 position is strictly required to be Asp and has a preferred cleavage sequence of Leu-Gln-Thr-Asp-|-Gly (LQTDG; SEQ ID NO: 151). Puromycin-sensitive aminopeptidase (P55786, Q11011) The N-terminal amino acid is released from a wide range of peptides, amides and arylamides, preferentially alanine. Angiotensin-converting enzyme (ACE) (P12821, P09470, Q9BYF1) SEQ ID NO: 156 When Xaa is not Pro, and Yaa is neither Asp nor Glu, the C-terminal dipeptide (oligopeptide-|-Xaa-Yaa) is released. Benazepril (Lotensin), Captopril, Enalapril (Vasotec), Fosinopril, Lisinopril (Prinivil, Zestril), Moexipril, Perindopril (Aceon), Quinapril Quinapril (Accupril), Ramipril (Altace), Trandolapril (Mavik), Zofenopril Pyroglutaminyl peptidase II (Q9NXJ5) Release of N-terminal pyroglutaminyl group from pGlu--His-Xaa tripeptide and pGlu--His-Xaa-Gly tetrapeptide Dipeptidyl peptidase IV (P27487, P14740, P28843) When Yaa is Pro, the N-terminal dipeptide (Xaa-Yaa-|-Zaa-) is preferentially released from the polypeptide, provided that Zaa is neither Pro nor hydroxyproline N-arginine binary convertase (O43847, Q8BHG1) Hydrolysis of polypeptides preferably at -Xaa-|-Arg-Lys- and less commonly at -Arg-|-Arg-Xaa-, where Xaa is not Arg or Lys Endopeptidase 24.15 (thimet oligopeptidase) (P52888, P24155) Preferentially cleaves bonds with hydrophobic residues at P1, P2 and P3' and small residues at P1' in substrates of 5 to 15 residues Endopeptidase 24.16 (neurolysin) (Q9BYT8, Q91YP2) Preferential cleavage of neurotensin: 10-Pro-|-Tyr-11 Amyloid precursor protein secretase alpha (P05067, P12023, Q9Y5Z0, P56817) Endopeptidase with broad specificity. Amyloid precursor protein secretase beta (P05067, P12023, Q9Y5Z0, P56817) Broad endopeptidase specificity. Cleavage of Glu-Val-Asn-Leu-|-Asp-Ala-Glu-Phe in the Swedish variant of Alzheimer's amyloid precursor protein (EVNLDAEF; SEQ ID NO: 152) Amyloid precursor protein secretase gamma (P05067, P12023, Q9Y5Z0, P56817) Intramembrane cleavage of integral membrane proteins MMP 1 (P03956, Q9EPL5uy) At 775-Gly-|-Ile-776 in the α-1(I) chain, starting from the N-terminus, the triple helix of collagen is cleaved at about three-quarters of the molecular length. Cleavage of synthetic substrate and α-macroglobulin at the bond of P1' is a hydrophobic residue. SB-3CT p-OH SB-3CT Phosphate SB-3CT ester RXP470.1 MMP2 (P08253, P33434) Cuts type I gelatin and type IV, V, VII, X collagen. Cleavage of the collagen-like sequence Pro-Gln-Gly-|-Ile-Ala-Gly-Gln (PQGIAGQ; SEQ ID NO: 153). SB-3CT p-OH SB-3CT Phosphate SB-3CT ester RXP470.1 MMP 3 (P08254, P28862) Preferential cleavage, wherein P1', P2' and P3' are hydrophobic residues. SB-3CT p-OH SB-3CT Phosphate SB-3CT ester RXP470.1 MMP 7 (P09237, Q10738) Cleavage of 14-Ala-|-Leu-15 and 16-Tyr-|-Leu-17 in insulin B chain. It has no effect on collagen types I, II, IV, and V. Cleavage of gelatin chains α-2(I) > α-1(I). SB-3CT p-OH SB-3CT Phosphate SB-3CT ester RXP470.1 MMP 8 (P22894, O70138) Degrades fibrous collagen types I, II and III. Cleavage of triple-helical domain interstitial collagen. Unlike EC 3.4.24.7, this enzyme cleaves type III collagen more slowly than type I. SB-3CT p-OH SB-3CT Phosphate SB-3CT ester RXP470.1 MMP9 (P14780, P41245) Cuts type I and V gelatin and type IV and V collagen. Cleaves KiSS1 at the Gly-|-Leu bond. Collagen types IV and V are cleaved into large C-terminal three-quarter fragments and shorter N-terminal quarter fragments. Degrades fibrin, but not laminin or Pz peptides. SB-3CT p-OH SB-3CT Phosphate SB-3CT ester RXP470.1 MMP 10 (P09238, O55123) Degrades fibrin, type I, type III, type IV and type V gelatin; weakly degrades collagen III, IV and V. SB-3CT p-OH SB-3CT Phosphate SB-3CT ester RXP470.1 MMP 11 (P24347, Q02853) A(A / Q)(N / A)↓(L / Y)(T / V / M / R)(R / K) G(G / A)E↓LR ↓ indicates the cleavage site SB-3CT p-OH SB-3CT Phosphate SB-3CT ester RXP470.1 MMP12 (P39900, P34960) Hydrolysis of soluble and insoluble elastin. Specific cleavage also occurs at 14-Ala-|-Leu-15 and 16-Tyr-|-Leu-17 in the insulin B chain Has significant elastolytic activity. Both large and small amino acids are acceptable at the P1' site, but leucine is preferred. Aromatic or hydrophobic residues are preferred at the P1 position, where a small hydrophobic residue (preferably alanine) occupies P3 SB-3CT p-OH SB-3CT Phosphate SB-3CT ester RXP470.1 MMP13 (P45452, P33435) Cleaves triple-helical collagen, including type I, type II and type III collagen, but has the highest activity on soluble type II collagen. Also degrades collagen types IV, XIV and X SB-3CT p-OH SB-3CT Phosphate SB-3CT ester RXP470.1 MMP14 (P50281, P53690) Progelatinase A is activated by cleavage of the propeptide at 37-Asn-|-Leu-38. Other hydrolytic linkages include 35-Gly-|-Ile-36 in the collagenase 3 pre-peptide, and 341-Asn-|-Phe-342, 441-Asp-|- in the interglobular domain of aggrecan Leu-442 and 354-Gln-|-Thr-355. SB-3CT p-OH SB-3CT Phosphate SB-3CT ester RXP470.1 Urokinase plasminogen activator (uPA) (P00749, P06869) Specifically cleaves the Arg-|-Val bond in plasminogen to form plasmin. Plasminogen Activator Inhibitor (PAI) Tissue plasminogen activator (tPA) (P00750, P11214) Specifically cleaves the Arg-|-Val bond in plasminogen to form plasmin. Plasminogen Activator Inhibitor (PAI) Tissue plasminogen activator (tPA) (P00750, P11214) Specifically cleaves the Arg-|-Val bond in plasminogen to form plasmin. Plasminogen Activator Inhibitor (PAI) Plasmin (P00747, P20918) Preferential cleavage: Lys-|-Xaa > Arg-|-Xaa, higher selectivity than trypsin. Converts fibrin to soluble products. Alpha-2-antiplasmin (AP) Thrombin (P00734, P19221) Cut bond after Arg and Lys Converts fibrinogen to fibrin and activates factors V, VII, VIII, XIII and complexes with thrombomodulin (protein C). BMP-1 (procollagen C-peptidase) (P13497, P98063) The C-terminal propeptide is cleaved at Ala-|-Asp in type I and II procollagens and at Arg-|-Asp in type III. ADAM (Q9P0K1, Q9UKQ2, Q9JLN6, O14672, Q13444, P78536, Q13443, O43184, P78325, Q9UKF5, Q9BZ11, Q9H2U9, Q99965, O75077, Q9H013, O43506) SB-3CT p-OH SB-3CT Phosphate SB-3CT ester RXP470.1 Granzyme A (P12544, P11032) Preferential cleavage: In small molecule substrates, -Arg-|-Xaa-, -Lys-|-Xaa- >> -Phe-|-Xaa-. Granzyme B (P10144, P04187) Bulk aromatic residues are preferred at the P1 position, and acidic residues at the P3' and P4' positions are preferred. Granzyme M (P51124, Q03238) Peptide substrates are cleaved after methionine, leucine, and norleucine. Tobacco etch virus (TEV) protease (P04517, P0CK09) E-Xaa-Xaa-Y-Xaa-Q-(G / S), where cleavage occurs between Q and G / S. The most common sequence is ENLYFQS (SEQ ID NO: 154) Chymotrypsin-like serine proteases (P08217, Q9UNI1, Q91X79, P08861, P09093, P08218) -Brown thermobifidobacterium (Thermobifida fusca) Thermopin -Pyrobaculum aerophilum Aeropin - Thermococcus kodakaraensis Tk-serine protease inhibitor - Alteromonas sp. Marinostatin - Streptomyces misionensis SMTI - Streptomyces sp. chymostatin Alphavirus proteases (P08411, P03317, P13886, Q8JUX6, Q86924, Q4QXJ8, Q8QL53, P27282, Q5XXP4) Chymotrypsin-like cysteine ​​proteases (Q86TL0, Q14790, Q99538, O15553) -Bifidobacterium thermopin - Aeropin Aeropin -Pyrococcus kakala Tk-serine protease inhibitor - Alteromonas sp. algalstatin - Streptomyces oryzae SMTI - Streptomyces sp. chymotrypsin inhibitor Papain-like cysteine ​​proteases (P25774, P53634, Q96K76) Picornavirus leader protease (P03305, P03311, P13899) HIV protease (P04585, P03367, P04584, P03369, P12497, P03366, P04587) Herpes virus protease (P10220, Q2HRB6, O40922, Q69527) Adenovirus protease (P03252, P24937, Q83906, P68985, P09569, P11825, P10381) Streptomyces griseus protease A (SGPA) (P00776) Streptomyces griseus protease B (SGPB) (P00777) Alpha-lytic protease (P85142, P00778) Serine proteases (P48740, P98064, Q9UL52, P05981, O60235) Cysteine ​​proteases (Q86TL0, Q14790, Q8WYN0, Q96DT6, P55211) Aspartic proteases (Q9Y5Z0, P56817, Q00663, Q53RT3, P0CY27) Threonine Protease (Q9UI38, Q16512, Q9H6P5, Q8IWU2) Mast cell (MC) chymosin (CMA1) (NM_001836) Abz-HPFHL(SEQ ID NO: 155)-Lys(Dnp)-NH2 BAY 1142524 SUN13834 Rat mast cell protease-1, -2, -3, -4, -5 (NM_017145, NM_172044, NM_001170466, NM_019321, NM_013092) Abz-HPFHL(SEQ ID NO: 155)-Lys(Dnp)-NH2 TY-51469 Rat Vascular Chymosin (RVCH) (O70500) Abz-HPFHL(SEQ ID NO: 155)-Lys(Dnp)-NH2 DENV NS3pro (NS2B / NS3) SEQ ID NO: 157, 158, 159, 160 It is observed that the P1 position has a strong preference for basic amino acid residues (Arg / Lys), while the P2-4 positions are preferably in the following order: P2 is Arg > Thr > Gln / Asn / Lys, and P3 is Lys > Arg > Asn, and P4 is Nle > Leu > Lys > Xaa. The major site substrate specificity is for small polar amino acids in P1 and P3. Anthraquinone BP13944 ZINC04321905 MB21 Polycresol SK-12 NSC135618 Biliverdin

[0086] The protease can be any of the following human proteases (MEROPS peptidase database numbers provided in parentheses; Rawlings N. D., Morton F. R., Kok, C. Y., Kong, J. and Barrett A. J. (2008) MEROPS: the peptidase database. Nucleic Acids Res. 36 Database issue, D320-325; which is incorporated herein by reference for all purposes): peptase A (MER000885), pepsin (MER000894), memapsin-2 (MER005870), Renin (MER000917), Autolysozyme D (MER000911), Autolysozyme E (MER000944), memapsin-1 (MER005534), Pepsin-like Napsin A (MER004981), Mername-AA034 Peptidase (MER014038), Pepsin A4 (MER037290), Pepsin A5 (Homo sapiens) (MER037291), hCG1733572 (Homo sapiens) type putative peptidase (MER107386), Pepsin-like aspartic protease B pseudogene (MER004982 ), CYMP g.p. (Homo sapiens) (MER002929), subfamily A1A unspecified peptidase (MER181559), mouse mammary tumor virus retropepsin (retropepsin) (MER048030), rabbit endogenous retrovirus endopeptidase ( MER043650 ), S71-related human endogenous reverse transcriptase pepsin (MER001812), RTVL-H type putative peptidase (MER047117), RTVL-H type putative peptidase (MER047133), RTVL-H type putative peptidase (MER047160), RTVL-H type putative peptidase (MER047206), RTVL-H type putative peptidase (MER047253), RTVL-H type putative peptidase (MER047260), RTVL-H type putative peptidase (MER047291), RTVL-H type putative peptide Enzyme (MER047418), RTVL-H type putative peptidase (MER047440), RTVL-H type putative peptidase (MER047479), RTVL-H type putative peptidase (MER047559), RTVL-H type putative peptidase (MER047583), RTVL - H-Type Putative Peptidase (MER015446), Human Endogenous Retroviral Retroviral Pepsin Homolog 1 (MER015479), Human Endogenous Retroviral Retroviral Pepsin Homolog 2 (MER015481), Endogenous Sexual 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 retroviral retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER030286), Endogenous retroviral retropepsin Pseudogene 3 (Homo sapiens chromosome 17) (MER047144), Endogenous retroviral retropepsin pseudogene 5 (Homo sapiens chromosome 12) (MER029664), Endogenous retroviral retropepsin pseudogene 6 ( Homo sapiens chromosome 7) (MER002094), endogenous retroviral retropepsin pseudogene 7 (Homo sapiens chromosome 6) (MER029776), endogenous retroviral retropepsin pseudogene 8 (Homo sapiens chromosome Y ) (MER030291), endogenous retroviral retropepsin pseudogene 9 (Homo sapiens chromosome 19) (MER029680), endogenous retroviral retropepsin pseudogene 10 (Homo sapiens chromosome 12) (MER002848) , endogenous retroviral retropepsin pseudogene 11 (Homo sapiens chromosome 17) (MER004378), endogenous retroviral retropepsin pseudogene 12 (Homo sapiens chromosome 11) (MER003344), endogenous Retroviral retropepsin pseudogene 13 (Homo sapiens chromosome 2 and similar) (MER029779), endogenous retroviral retropepsin pseudogene 14 (Homo sapiens chromosome 2) (MER029778), endogenous RT Viral retro-pepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047158), endogenous retroviral retro-pepsin pseudo-gene 15 (Homo sapiens chromosome 4) (MER047332), endogenous retroviral retro-pepsin Protease pseudogene 15 (Homo sapiens chromosome 4) (MER003182), Endogenous retroviral retropepsin pseudogene 16 (MER047165), Endogenous retroviral retropepsin pseudogene 16 (MER047178), Endogenous endogenous retroviral retropepsin pseudogene 16 (MER047200), endogenous retroviral retropepsin pseudogene 16 (MER047315), endogenous retroviral retropepsin pseudogene 16 (MER047405), Endogenous retroviral retropepsin pseudogene 16 (MER030292), endogenous retroviral retropepsin pseudogene 17 (Homo sapiens chromosome 8) (MER005305), endogenous retroviral retropepsin pseudogene Gene 18 (Homo sapiens chromosome 4) (MER030288), endogenous retroviral retropepsin pseudogene 19 (Homo sapiens chromosome 16) (MER001740), endogenous retroviral retropepsin pseudogene 21 (Homo sapiens Human) (MER047222), endogenous retroviral pepsin pseudogene 21 (Homo sapiens) (MER047454), endogenous retroviral pepsin pseudogene 21 (Homo sapiens) (MER047477), endogenous Sexual retroviral retropepsin pseudogene 21 (Homo sapiens) (MER004403), endogenous retroviral retroviral pepsin pseudogene 22 (Homo sapiens chromosome X) (MER030287), subfamily A2A non-peptidase homolog Subfamily A2A Nonpeptidase Homolog (MER047046), Subfamily A2A Nonpeptidase Homolog (MER047052), Subfamily A2A Nonpeptidase Homolog (MER047076), Subfamily A2A Nonpeptidase Homolog (MER047080), Subfamily A2A Nonpeptidase Homolog Homolog (MER047088), Subfamily A2A Non-Peptidase Homolog (MER047089), Subfamily A2A Non-Peptidase Homolog (MER047091), Subfamily A2A Non-Peptidase Homolog (MER047092), Subfamily A2A Non-Peptidase Homolog Peptidase Homolog (MER047093), Subfamily A2A Non-Peptidase Homolog (MER047094), Subfamily A2A Non-Peptidase Homolog (MER047097), Subfamily A2A Non-Peptidase Homolog (MER047099), Subfamily A2A non-peptidase homolog MER047101), subfamily A2A non-peptidase homolog (MER047102), subfamily A2A non-peptidase homolog (MER047107), subfamily A2A non-peptidase homolog (MER047108), subfamily Family A2A non-peptidase homolog (MER047109), subfamily A2A non-peptidase homolog (MER047110), subfamily A2A non-peptidase homolog (MER047111), subfamily A2A non-peptidase homolog (MER047114), Subfamily A2A non-peptidase homolog (MER047118), Subfamily A2A non-peptidase homolog (MER047121), Subfamily A2A non-peptidase homolog (MER047122), Subfamily A2A non-peptidase homolog (MER047126 ), subfamily A2A nonpeptidase homolog (MER047129), subfamily A2A nonpeptidase homolog (MER047130), subfamily A2A nonpeptidase homolog (MER047134), subfamily A2A nonpeptidase homolog (MER047135), subfamily A2A nonpeptidase homolog (MER047137), subfamily A2A nonpeptidase homolog (MER047140), subfamily A2A nonpeptidase homolog (MER047141), subfamily A2A nonpeptidase homolog Homolog (MER047142), Subfamily A2A Nonpeptidase Homolog (MER047148), Subfamily A2A Nonpeptidase Homolog (MER047149), Subfamily A2A Nonpeptidase Homolog (MER047151), Subfamily A2A Nonpeptidase Homolog Enzyme Homolog (MER047154), Subfamily A2A Non-Peptidase Homolog (MER047155), Subfamily A2A Non-Peptidase Homolog (MER047156), Subfamily A2A Non-Peptidase Homolog (MER047157), Subfamily A2A Non-peptidase homolog (MER047159), subfamily A2A non-peptidase homolog (MER047161), subfamily A2A non-peptidase homolog (MER047163), subfamily A2A non-peptidase homolog (MER047166), subfamily Family A2A Nonpeptidase Homolog (MER047171), Subfamily A2A Nonpeptidase Homolog (MER047173), Subfamily A2A Nonpeptidase Homolog (MER047174), Subfamily A2A Nonpeptidase Homolog (MER047179) , subfamily A2A nonpeptidase homolog (MER047183), subfamily A2A nonpeptidase homolog (MER047186), subfamily A2A nonpeptidase homolog (MER047190), subfamily A2A nonpeptidase homolog ( MER047191 ), subfamily A2A non-peptidase homolog (MER047196), subfamily A2A non-peptidase homolog (MER047198), subfamily A2A non-peptidase homolog (MER047199), subfamily A2A non-peptidase homolog Subfamily A2A Nonpeptidase Homolog (MER047201), Subfamily A2A Nonpeptidase Homolog (MER047202), Subfamily A2A Nonpeptidase Homolog (MER047203), Subfamily A2A Nonpeptidase Homolog (MER047204), Subfamily A2A Nonpeptidase Homolog Homolog (MER047205), Subfamily A2A Non-Peptidase Homolog (MER047207), Subfamily A2A Non-Peptidase Homolog (MER047208), Subfamily A2A Non-Peptidase Homolog (MER047210), Subfamily A2A Non-Peptidase Homolog Peptidase Homolog (MER047211), Subfamily A2A Non-Peptidase Homolog (MER047212), Subfamily A2A Non-Peptidase Homolog (MER047213), Subfamily A2A Non-Peptidase Homolog (MER047215), Subfamily A2A non-peptidase homolog (MER047216), subfamily A2A non-peptidase homolog (MER047218), subfamily A2A non-peptidase homolog (MER047219), subfamily A2A non-peptidase homolog (MER047221), Subfamily A2A non-peptidase homolog (MER047224), Subfamily A2A non-peptidase homolog (MER047225), Subfamily A2A non-peptidase homolog (MER047226), Subfamily A2A non-peptidase homolog (MER047227 ), subfamily A2A 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 Homolog (MER047240), Subfamily A2A Nonpeptidase Homolog (MER047242), Subfamily A2A Nonpeptidase Homolog (MER047243), Subfamily A2A Nonpeptidase Homolog (MER047249), Subfamily A2A Nonpeptidase Homolog Enzyme Homolog (MER047251), Subfamily A2A Non-Peptidase Homolog (MER047252), Subfamily A2A Non-Peptidase Homolog (MER047254), Subfamily A2A Non-Peptidase Homolog (MER047255), Subfamily A2A Non-peptidase homolog (MER047263), subfamily A2A non-peptidase homolog (MER047265),Subfamily A2A non-peptidase homolog (MER047266), Subfamily A2A non-peptidase homolog (MER047267), Subfamily A2A non-peptidase homolog (MER047268), Subfamily A2A non-peptidase homolog (MER047269 ), subfamily A2A nonpeptidase homolog (MER047272), subfamily A2A nonpeptidase homolog (MER047273), subfamily A2A nonpeptidase homolog (MER047274), subfamily A2A nonpeptidase homolog (MER047275), subfamily A2A nonpeptidase homolog (MER047276), subfamily A2A nonpeptidase homolog (MER047279), subfamily A2A nonpeptidase homolog (MER047280), subfamily A2A nonpeptidase homolog Homolog (MER047281), Subfamily A2A Nonpeptidase Homolog (MER047282), Subfamily A2A Nonpeptidase Homolog (MER047284), Subfamily A2A Nonpeptidase Homolog (MER047285), Subfamily A2A Nonpeptidase Homolog Enzyme Homolog (MER047289), Subfamily A2A Non-Peptidase Homolog (MER047290), Subfamily A2A Non-Peptidase Homolog (MER047294), Subfamily A2A Non-Peptidase Homolog (MER047295), Subfamily A2A Non-peptidase homolog (MER047298), subfamily A2A non-peptidase homolog (MER047300), subfamily A2A non-peptidase homolog (MER047302), subfamily A2A non-peptidase homolog (MER047304), subfamily Family A2A Nonpeptidase Homolog (MER047305), Subfamily A2A Nonpeptidase 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 non-peptidase homolog (MER047321), subfamily A2A non-peptidase homolog (MER047322), subfamily A2A non-peptidase homolog (MER047326), subfamily A2A non-peptidase homolog Subfamily A2A Nonpeptidase Homolog (MER047327), Subfamily A2A Nonpeptidase Homolog (MER047330), Subfamily A2A Nonpeptidase Homolog (MER047333), Subfamily A2A Nonpeptidase Homolog (MER047362), Subfamily A2A Nonpeptidase Homolog Homolog (MER047366), Subfamily A2A Non-Peptidase Homolog (MER047369), Subfamily A2A Non-Peptidase Homolog (MER047370), Subfamily A2A Non-Peptidase Homolog (MER047371), Subfamily A2A Non-Peptidase Homolog Peptidase Homolog (MER047375), Subfamily A2A Non-Peptidase Homolog (MER047376), Subfamily A2A Non-Peptidase Homolog (MER047381), Subfamily A2A Non-Peptidase Homolog (MER047383), Subfamily A2A non-peptidase homolog (MER047384), subfamily A2A non-peptidase homolog (MER047385), subfamily A2A non-peptidase homolog (MER047388), subfamily A2A non-peptidase homolog (MER047389), Subfamily A2A non-peptidase homolog (MER047391), Subfamily A2A non-peptidase homolog (MER047394), Subfamily A2A non-peptidase homolog (MER047396), Subfamily A2A non-peptidase homolog (MER047400 ), subfamily A2A nonpeptidase 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 Homolog (MER047414), Subfamily A2A Nonpeptidase Homolog (MER047416), Subfamily A2A Nonpeptidase Homolog (MER047417), Subfamily A2A Nonpeptidase Homolog (MER047420), Subfamily A2A Nonpeptidase Homolog Enzyme Homolog (MER047423), Subfamily A2A Non-Peptidase Homolog (MER047424), Subfamily A2A Non-Peptidase Homolog (MER047428), Subfamily A2A Non-Peptidase Homolog (MER047429), Subfamily A2A Non-peptidase homolog (MER047431), subfamily A2A non-peptidase homolog (MER047434), subfamily A2A non-peptidase homolog (MER047439), subfamily A2A non-peptidase homolog (MER047442), subfamily Family A2A non-peptidase homolog (MER047445), Subfamily A2A non-peptidase homolog (MER047449), Subfamily A2A non-peptidase homolog (MER047450), Subfamily A2A non-peptidase homolog (MER047452) , subfamily A2A nonpeptidase homolog (MER047455), subfamily A2A nonpeptidase homolog (MER047457), subfamily A2A nonpeptidase homolog (MER047458), subfamily A2A nonpeptidase homolog ( MER047459), subfamily A2A non-peptidase homolog (MER047463), subfamily A2A non-peptidase homolog (MER047468), subfamily A2A non-peptidase homolog (MER047469), subfamily A2A non-peptidase homolog Subfamily A2A Nonpeptidase Homolog (MER047470), Subfamily A2A Nonpeptidase Homolog (MER047476), Subfamily A2A Nonpeptidase Homolog (MER047478), Subfamily A2A Nonpeptidase Homolog (MER047483), Subfamily A2A Nonpeptidase Homolog Homolog (MER047488), Subfamily A2A Non-Peptidase Homolog (MER047489), Subfamily A2A Non-Peptidase Homolog (MER047490), Subfamily A2A Non-Peptidase Homolog (MER047493), Subfamily A2A Non-Peptidase Homolog Peptidase Homolog (MER047494), Subfamily A2A Non-Peptidase Homolog (MER047495), Subfamily A2A Non-Peptidase Homolog (MER047496), Subfamily A2A Non-Peptidase Homolog (MER047497), Subfamily A2A non-peptidase homolog (MER047499), subfamily A2A non-peptidase homolog (MER047502), subfamily A2A non-peptidase homolog (MER047504), subfamily A2A non-peptidase homolog (MER047511), Subfamily A2A non-peptidase homolog (MER047513), Subfamily A2A non-peptidase homolog (MER047514), Subfamily A2A non-peptidase homolog (MER047515), Subfamily A2A non-peptidase homolog (MER047516 ), subfamily A2A 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 Homolog (MER047604), Subfamily A2A Nonpeptidase Homolog (MER047606), Subfamily A2A Nonpeptidase Homolog (MER047609), Subfamily A2A Nonpeptidase Homolog (MER047616), Subfamily A2A Nonpeptidase Homolog Enzyme Homolog (MER047619), Subfamily A2A Non-Peptidase Homolog (MER047648), Subfamily A2A Non-Peptidase Homolog (MER047649), Subfamily A2A Non-Peptidase Homolog (MER047662), Subfamily A2A Non-peptidase homolog (MER048004), subfamily A2A non-peptidase homolog (MER048018), subfamily A2A non-peptidase homolog (MER048019), subfamily A2A non-peptidase homolog (MER048023), subfamily Family A2A non-peptidase homologue (MER048037), subfamily A2A unspecified peptidase (MER047164), subfamily A2A unspecified peptidase (MER047231), subfamily A2A unspecified peptidase (MER047386), skin aspartate Protease (MER057097), Presenilin 1 (MER005221), Presenilin 2 (MER005223), impas 1 peptidase (MER019701), impas 1 peptidase (MER184722), impas 4 peptidase (MER019715), impas 2 peptidase (MER019708) , impas 5 peptidase (MER019712), impas 3 peptidase (MER019711), possible family A22 pseudogene (Homo sapiens chromosome 18) (MER029974), possible family A22 pseudogene (Homo sapiens chromosome 11) (MER023159), cell autolysis Enzyme V (MER004437), Autolysozyme X (MER004508), Autolysozyme F (MER004980), Autolysozyme L (MER000622), Autolysozyme S (MER000633), Autolysozyme O (MER001690) , Autolysozyme K (MER000644), Autolysozyme W (MER003756), Autolysozyme H (MER000629), Autolysozyme B (MER000686), Dipeptidyl-Peptidase I (MER001937), Bray Bleomycin hydrolase (animal) (MER002481), tubulointerstitial nephritis antigen (MER016137), tubulointerstitial nephritis antigen-related protein (MER021799), autolysozyme L-like pseudogene 1 (Homo sapiens ) (MER002789), Autolysozyme B-like pseudogene (chromosome 4, Homo sapiens) (MER029469), Autolysozyme B-like pseudogene (chromosome 1, Homo sapiens) (MER029457), CTSLL2 g.p. (Homo sapiens) ( MER005210), CTSLL3 g.p. (Homo sapiens) (MER005209), Calpain-1 (MER000770), Calpain-2 (MER000964), Calpain-3 (MER001446), Calpain-9 (MER004042), Calpain-8 ( MER021474), Calpain-15 (MER004745), Calpain-5 (MER002939), Calpain-11 (MER005844), Calpain-12 (MER029889), Calpain-10 (MER013510), Calpain-13 (MER020139) , Calpain-14 (MER029744), Mername-AA253 Peptidase (MER005537), Calpamodulin (MER000718), Hypothetical Protein 940251 (MER003201), Ubiquitinyl Hydrolase-L1 (MER000832), Ubiquitinyl Hydrolase -L3 (MER000836), Ubiquitin-based hydrolase-BAP1 (MER003989), Ubiquitin-based hydrolase-UCH37 (MER005539), Ubiquitin-specific peptidase 5 (MER002066), Ubiquitin-specific peptidase 6 (MER000863), Ubiquitin-specific peptidase 4 (MER001795), Ubiquitin-specific peptidase 8 (MER001884), Ubiquitin-specific peptidase 13 (MER002627), Ubiquitin-specific peptidase 2 (MER004834), Ubiquitin-specific peptidase 11 (MER002693), ubiquitin-specific peptidase 14 (MER002667), ubiquitin-specific peptidase 7 (MER002896), ubiquitin-specific peptidase 9X (MER005877), ubiquitin-specific peptidase 10 (MER004439), ubiquitin-specific peptidase Ubiquitin-specific peptidase 1 (MER004978), Ubiquitin-specific peptidase 12 (MER005454), Ubiquitin-specific peptidase 16 (MER005493), Ubiquitin-specific peptidase 15 (MER005427), Ubiquitin-specific peptidase 17 (MER002900),Ubiquitin-specific peptidase 19 (MER005428), Ubiquitin-specific peptidase 20 (MER005494), Ubiquitin-specific peptidase 3 (MER005513), Ubiquitin-specific peptidase 9Y (MER004314), Ubiquitin-specific peptidase 18 (MER005641), ubiquitin-specific peptidase 21 (MER006258), ubiquitin-specific peptidase 22 (MER012130), ubiquitin-specific peptidase 33 (MER014335), ubiquitin-specific peptidase 29 (MER012093), ubiquitin-specific peptidase 29 (MER012093), Ubiquitin-specific peptidase 25 (MER011115), Ubiquitin-specific peptidase 36 (MER014033), Ubiquitin-specific peptidase 32 (MER014290), Ubiquitin-specific peptidase 26 (Homo sapiens) (MER014292), Ubiquitin Specific peptidase 24 (MER005706), Ubiquitin-specific peptidase 42 (MER011852), Ubiquitin-specific peptidase 46 (MER014629), Ubiquitin-specific peptidase 37 (MER014633), Ubiquitin-specific peptidase 28 ( 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), Peptidase 30 (MER014649), Peptidase-AA091 (MER014743), Peptidase 45 (MER030314), Peptidase 51 (MER014769), Ubiquitin-specific peptidase 34 (MER014780), Ubiquitin-specific peptidase 48 (MER064620), Ubiquitin-specific peptidase 40 (MER015483), Ubiquitin-specific peptidase 41 (MER045268), Ubiquitin-specific peptidase 31 (MER015493), Mername-AA129 peptidase (MER016485), Ubiquitin-specific peptidase 49 (MER016486), Mername-AA187 peptidase (MER052579), USP17-like peptidase (MER030192), Ubiquitin-specific peptidase 54 ( [ Misleading] (MER030140), Ubiquitin-specific peptidase 52 [Misleading] (MER030317), NEK2 pseudogene (MER014736), C19 pseudogene (Homo sapiens: chromosome 5) (MER029972), Mername-AA088 peptidase (MER014750), Autophagin-2 (MER013564), Autophagy-1 (MER013561), Autophagy-3 (MER014316), Autophagy-4 (MER064622), Cezanne deubiquitinating peptidase (MER029042), Cezanne-2 peptidase (MER029044), tumor necrosis factor alpha-inducible protein 3 (MER029050), trabid peptidase (MER029052), VCIP135 deubiquitinating peptidase (MER152304), ovarian tumor protein (otubain)-1 (MER029056) , ovarian tumor protein-2 (MER029061), CylD protein (MER030104), UfSP1 peptidase (MER042724), UfSP2 peptidase (MER060306), DUBA deubiquitinase (MER086098), KIAA0459 (Homo sapiens)-like protein (MER122467) , Otud1 protein (MER125457), glycosyltransferase 28-containing domain 1, isoform CRA_c (Homo sapiens)-like (MER123606), hin1L g.p. (Homo sapiens) (MER139816), ataxin-3 (MER099998), ATXN3L putative peptidase (MER115261), Josephin domain-containing 1 (Homo sapiens) (MER125334), Josephin domain-containing 2 (Homo sapiens) (MER124068), YOD1 peptidase (MER116559), pod protein ( Plant α-type) (MER044591), pod protein (MER001800), glycosylphosphatidylinositol:protein transamidase (MER002479), pod protein pseudogene (Homo sapiens) (MER029741), family C13 unspecified peptidase (MER175813 ), Caspase-1 (MER000850), Caspase-3 (MER000853), Caspase-7 (MER002705), Caspase-6 (MER002708), Caspase Enzyme-2 (MER001644), Caspase-4 (MER001938), Caspase-5 (MER002240), Caspase-8 (MER002849), Caspase-9 (MER002707) , Caspase-10 (MER002579), Caspase-14 (MER012083), Para-caspase (MER019325), Mername-AA143 Peptidase (MER021304), Mername-AA186 Peptidase (MER020516) , putative caspase (Homo sapiens) (MER021463), FLIP protein (MER003026), Mername-AA142 protein (MER021316), caspase-12 pseudogene (Homo sapiens) (MER019698), Mername-AA093 half Caspase pseudogene (MER014766), subfamily C14A non-peptidase homolog (MER185329), subfamily C14A non-peptidase homolog (MER179956), separase (Homo sapiens) (MER011775), Protease-like pseudogenes (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), pyroglutaminyl-peptidase I (chordate) (MER011032), Mername-AA073 peptidase (MER029978), Sonic hedgehog ( MER002539), Indian hedgehog protein (MER002538), Desert hedgehog protein (MER012170), dipeptidyl-peptidase III (MER004252), Mername-AA164 protein (MER020410), LOC138971 g.p. (Homo sapiens) (MER020074), Atp23 peptidase (MER060642), prenyl peptidase 1 (MER004246), aminopeptidase N (MER000997), aminopeptidase A (MER001012), leukotriene A4 hydrolase (MER001013), pyro Glutaminyl-peptidase II (MER012221), Cytosolic alanyl aminopeptidase (MER002746), Hemiaminopeptidase (MER002060), Aminopeptidase B (MER001494), Aminopeptidase PILS (MER005331) , arginine aminopeptidase-like 1 (MER012271), leukocyte-derived arginine aminopeptidase (MER002968), aminopeptidase Q (MER052595), aminopeptidase O (MER019730), Tata-binding protein-related factor (MER026493) , ACE peptidase unit 1 (MER004967), ACE peptidase unit 2 (MER001019), ACE-2 (MER011061), Mername-AA153 protein (MER020514), thimet oligopeptidase (MER001737), Neurolysin (MER010991), Mitochondrial Intermediate Peptidase (MER003665), Mername-AA154 Protein (MER021317), Leishmanolysin-2 (MER014492), Leishmania Protease- 3 (MER180031), MMP-1 (MER001063), MMP-8 (MER001084), MMP-2 (MER001080), MMP-9 (MER001085), MMP- 3 (MER001068), MMP-10 (Homo sapiens) (MER001072), MMP-11 (MER001075), MMP-7 (MER001092), MMP-12 (MER001089 ), Matrix metallopeptidase-13 (MER001411), membrane-type matrix metallopeptidase-1 (MER001077), membrane-type matrix metallopeptidase-2 (MER002383), membrane-type matrix metallopeptidase-3 (MER002384), membrane-type matrix metallopeptidase-3 (MER002384), membrane-type matrix metallopeptidase Peptidase-4 (MER002595), MMP-20 (MER003021), MMP-19 (MER002076), MMP-23B (MER004766), MMP-5 (MER005638 ), Membrane-type MMP-6 (MER012071), MMP-21 (MER006101), MMP-22 (MER014098), MMP-26 (MER012072), MMP-28 (MER013587 ), Matrix metallopeptidase-23A (MER037217), macrophage elastase homologue (chromosome 8, Homo sapiens) (MER030035), Mername-AA156 protein (MER021309), matrix metallopeptidase-like 1 (MER045280), sub Family M10A non-peptidase homolog (MER175912), subfamily M10A non-peptidase homolog (MER187997), subfamily M10A non-peptidase homolog (MER187998), subfamily M10A non-peptidase homolog (MER180000) , meprin α subunit (MER001111), meprin β subunit (MER005213), procollagen C-peptidase (MER001113), mammalian tolloid-like 1 protein (MER005124), mammalian tolloid-like 2 protein (MER005866), ADAMTS9 peptide enzyme (MER012092), ADAMTS14 peptidase (MER016700), ADAMTS15 peptidase (MER017029), ADAMTS16 peptidase (MER015689), ADAMTS17 peptidase (MER016302), ADAMTS18 peptidase (MER016090), ADAMTS19 peptidase (MER015663), AD AM8 peptidase (MER003902), ADAM9 peptidase (MER001140), ADAM10 peptidase (MER002382), ADAM12 peptidase (MER005107), ADAM19 peptidase (MER012241), ADAM15 peptidase (MER002386), ADAM17 peptidase (MER003094), ADAM20 peptidase ( MER004725), ADAMDEC1 peptidase (MER000743), ADAMTS3 peptidase (MER005100), ADAMTS4 peptidase (MER005101), ADAMTS1 peptidase (MER005546), ADAM28 peptidase (Homo sapiens) (MER005495), ADAMTS5 peptidase (MER005548 ),ADAMTS8 peptidase (MER005545), ADAMTS6 peptidase (MER005893), ADAMTS7 peptidase (MER005894), ADAM30 peptidase (MER006268), ADAM21 peptidase (Homo sapiens) (MER004726), ADAMTS10 peptidase (MER014331), ADAMTS12 peptidase (MER014337), ADAMTS13 peptidase (MER015450), ADAM33 peptidase (MER015143), ovastacin (MER029996), ADAMTS20 peptidase (Homo sapiens) (MER026906), procollagen I N-peptidase (MER004985), ADAM2 protein (MER003090 ), ADAM6 protein (MER047044), ADAM7 protein (MER005109), ADAM18 protein (MER012230), ADAM32 protein (MER026938), non-peptidase homolog (Homo sapiens chromosome 4) (MER029973), family M12 non-peptidase homolog (Homo sapiens chromosome 16) (MER047654), family M12 non-peptidase homologue (Homo sapiens chromosome 15) (MER047250), ADAM3B protein (Homo sapiens) (MER005199), ADAM11 protein (MER001146), ADAM22 protein (MER005102) , ADAM23 protein (MER005103), ADAM29 protein (MER006267), protein similar to ADAM21 peptidase preproprotein (Homo sapiens) (MER026944), Mername-AA225 peptidase homologue (Homo sapiens) (MER047474), putative ADAM pseudo Genes (chromosome 4, Homo sapiens) (MER029975), ADAM3A g.p. (Homo sapiens) (MER005200), ADAM1 g.p. (Homo sapiens) (MER003912), subfamily M12B non-peptidase homologue (MER188210), subfamily M12B non-peptidase Enzyme homolog (MER188211), subfamily M12B non-peptidase homolog (MER188212), subfamily M12B non-peptidase homolog (MER188220), neprilysin (MER001050), endothelin converting enzyme 1 (MER001057), endothelin converting enzyme 2 (MER004776), DINE peptidase (MER005197), enkephalinase-2 (MER013406), Kell blood group protein (MER001054), PHEX peptidase (MER002062), i-AAA peptidase ( MER001246), i-AAA peptidase (MER005755), paraplegin (MER004454), Afg3-like protein 2 (MER005496), Afg3-like protein 1A (MER014306), pregnancy-associated plasma protein (pappalysin)-1 (MER002217), Pregnancy-associated plasma protein-2 (MER014521), farnesylated protein convertase 1 (MER002646), metalloproteinase-associated protein-1 (MER030873), aminopeptidase AMZ2 (MER011907), aminopeptidase AMZ1 (MER058242) , carboxypeptidase A1 (MER001190), carboxypeptidase A2 (MER001608), carboxypeptidase B (MER001194), carboxypeptidase N (MER001198), carboxypeptidase E (MER001199), carboxypeptidase M (MER001205), carboxypeptidase Peptidase U (MER001193), Carboxypeptidase A3 (MER001187), Metallocarboxypeptidase D Peptidase Unit 1 (MER003781), Metallocarboxypeptidase Z (MER003428), Metallocarboxypeptidase D Peptidase Unit 2 (MER004963), Carboxypeptidase A4 (MER013421), Carboxypeptidase A6 (MER013456), Carboxypeptidase A5 (MER017121), Metallocarboxypeptidase O (MER016044), Cytosolic Carboxypeptidase-like Protein 5 (MER033174), Cytosolic Carboxypeptidase 3 (MER033176), cytosolic carboxypeptidase 6 (MER033178), cytosolic carboxypeptidase 1 (MER033179), cytosolic carboxypeptidase 2 (MER037713), metallocarboxypeptidase D non-peptidase unit (MER004964), adipocyte Enhancer-binding protein 1 (MER003889), carboxypeptidase-like protein X1 (MER013404), carboxypeptidase-like protein X2 (MER078764), cytosolic carboxypeptidase (MER026952), family M14 non-peptidase homolog (MER199530), Insulin lysin (MER001214), mitochondrial processing peptidase β-subunit (MER004497), phenelzine lyase (nardilysin) (MER003883), eupitrilysin (MER004877), mitochondrial processing peptidase non-peptidase α subunit (MER001413), Ubiquinol-cytochrome c reductase core protein I (MER003543), Ubiquinol-cytochrome c reductase core protein II (MER003544), Ubiquinol-cytochrome c reductase core protein domain 2 (MER043998) , insulin lysin unit 2 (MER046821), nadilysin (nardilysin) unit 2 (MER046874), insulin lysin unit 3 (MER078753), mitochondrial processing peptidase subunit α unit 2 (MER124489), nadilysin (MER142856), LOC133083 g.p. (Homo sapiens) (MER021876), subfamily M16B non-peptidase homologue (MER188757), leucyl aminopeptidase (animal) (MER003100), Mername-AA040 peptidase ( MER003919), leucyl aminopeptidase-1 (neobaculotype) (MER013416), methionyl aminopeptidase 1 (MER001342), methionyl aminopeptidase 2 (MER001728), amine Peptidase P2 (MER004498), Xaa-Pro Dipeptidase (Eukaryotes) (MER001248), Aminopeptidase P1 (MER004321), Mitochondrial Intermediate Cleavage Peptidase 55 kDa (MER013463), Mitochondrial Methionine Acyl aminopeptidase (MER014055), Mername-AA020 peptidase homologue (MER010972), proliferation-associated protein 1 (MER005497), chromatin-specific transcription elongation factor 140 kDa subunit (MER026495), proliferation-associated protein 1-like ( Homo sapiens chromosome X) (MER029983), Mername-AA226 peptidase homolog (Homo sapiens) (MER056262), Mername-AA227 peptidase homolog (Homo sapiens) (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 ), aminoylase (MER001271), glutamine carboxypeptidase II (MER002104), NAALADASE L peptidase (MER005239), glutamic carboxypeptidase III (MER005238), plasma glutamine carboxypeptidase (MER005244 ), Mername-AA103 peptidase (MER015091), Fxna peptidase (MER029965), transferrin receptor protein (MER002105), transferrin receptor 2 protein (MER005152), glutaminyl cyclase (glutaminyl cyclase ) (MER015095), glutamic carboxypeptidase II (Homo sapiens) type non-peptidase homologue (MER026971), nicalin (MER044627), membrane dipeptidase (MER001260), membrane-bound dipeptidase-2 (MER013499) , Membrane-bound dipeptidase-3 (MER013496), dihydroorotase (MER005767), dihydropyrimidinase (MER033266), dihydropyrimidinase-related protein-1 (MER030143), dihydropyrimidinase-related protein-2 (MER030155), dihydropyrimidinase-related protein-3 (MER030151), dihydropyrimidinase-related protein-4 (MER030149), dihydropyrimidinase-related protein-5 (MER030136), putative protein-like 5730457F11RIK (MER033184), 1300019j08rik protein (MER033186)), Guanine aminohydrolase (MER037714), Kae1 putative peptidase (MER001577), OSGEPL1-like protein (MER013498), S2P peptidase (MER004458), subfamily M23B non-peptidase homologue (MER199845), Subfamily M23B non-peptidase homolog (MER199846), subfamily M23B non-peptidase homolog (MER199847), subfamily M23B non-peptidase homolog (MER137320), subfamily M23B non-peptidase homolog (MER201557 ), subfamily M23B non-peptidase homolog (MER199417), subfamily M23B non-peptidase homolog (MER199418), subfamily M23B non-peptidase homolog (MER199419), subfamily M23B non-peptidase homolog (MER199420), subfamily M23B non-peptidase homolog (MER175932), subfamily M23B non-peptidase homolog (MER199665), Poh1 peptidase (MER020382), Jab1 / MPN domain metalloenzyme (MER022057), Mername- AA165 peptidase (MER021865), Brcc36 isopeptidase (MER021890), Histone H2A deubiquitinase 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 homologue (MER191181), subfamily M67A unspecified peptidase (MER191144), granzyme B (Homo sapiens) (MER000168), testisin (MER005212 ), Neutralase β (MER000136), Kallikrein-related Peptidase 5 (MER005544), corin (MER005881), Kallikrein-related Peptidase 12 (MER006038), DESC1 Peptidase (MER006298), Neutralase γ1 (MER011036), kallikrein-related peptidase 14 (MER011038), hyaluronic acid-binding peptidase (MER003612), transmembrane peptidase serine 4 (MER011104), intestinal serine peptidase (rodent) (MER016130), Adrenal secreted serine peptidase (MER003734), tryptase δ1 (Homo sapiens) (MER005948), protein lyase-3 (MER029902), marapsin (MER006119), neutral proteinase-6 (MER006118), Ovochymase-1 domain 1 (MER099182), transmembrane peptidase serine 3 (MER005926), kallikrein-related peptidase 15 (MER000064), Mername-AA031 peptidase (MER014054), TMPRSS13 peptide Enzyme (MER014226), Mername-AA038 Peptidase (MER062848),Mername-AA204 peptidase (MER029980), cationic trypsin (Homo sapiens) (MER000020), elastase-2 (MER000118), mannan-binding lectin-related serine peptidase-3 (MER031968), cell autologous Lysozyme G (MER000082), Myeloblastic Protease (MER000170), Granzyme A (MER001379), Granzyme M (MER001541), Chymosin (Homo sapiens) (MER000123), Dispase Alpha (MER000135), Granzyme Enzyme K (MER001936), Granzyme H (MER000166), Chymotrypsin B (MER000001), Elastase-1 (MER003733), Pancreatic Endopeptidase E (MER000149), Pancreatic Elastase II (MER000146), Intestinal Peptidase (MER002068), Chymotrypsin C (MER000761), Prostate Protease (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 activating C1r (MER000238), complement component activating 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 (activated) (MER000222), acrosin (MER000078), serine hepsin (MER000156), hepatocyte growth factor activator (MER000186), mannan Carbohydrate-binding lectin-related serine peptidase 2 (MER002758), u-plasminogen activator (MER000195), t-plasminogen activator (MER000192), plasmin (MER000175), kinase Peptidrein-related peptidase 6 (MER002580), neurotrypsin (MER004171), kallikrein-related peptidase 8 (MER005400), kallikrein-related peptidase 10 (MER003645), epitheliasin (MER003736), kallikrein Enzyme-related peptidase 4 (MER005266), prosemin (MER004214), chymopasin (MER001503), kallikrein-related peptidase 11 (MER004861), kallikrein-related peptidase 11 (MER216142), trypsin-2 type A ( MER000021), HtrA1 peptidase (Homo sapiens) (MER002577), HtrA2 peptidase (MER208413), HtrA2 peptidase (MER004093), HtrA3 peptidase (MER014795), HtrA4 peptidase (MER016351), Tysnd1 peptidase (MER050461), TMPRSS12 peptidase (MER017085), HAT-like putative peptidase 2 (MER021884), trypsin C (MER021898), kallikrein-related peptidase 7 (MER002001), proteolytic enzyme (MER003735), kallikrein-related peptidase 13 (MER005269), kallikrein-related peptidase 9 (MER005270), protease-2 (MER005278), umbilical vein peptidase (MER005421), LCLP peptidase (MER001900), spinesin (MER014385) , marapsin-2 (MER021929), complement factor D-like putative peptidase (MER056164), oozyme-2 (MER022410), HAT-like 4-peptidase (MER044589), oozyme 1 domain 1 (MER022412), epidermis-specific Sexual SP-like putative peptidase (MER029900), testicular serine peptidase 5 (MER029901), Mername-AA258 peptidase (MER000285), polyserine enzyme (polyserine enzyme)-IA unit 1 (MER030879), Polyserine-IA unit 2 (MER030880), testicular serine peptidase 2 (human type) (MER033187), putative acrosin-like peptidase (Homo sapiens) (MER033253), HAT-like pentapeptidase (MER028215 ), polyserinase-3 unit 1 (MER061763), polyserinase-3 unit 2 (MER061748), peptidase-like tryptophan / serine protease (MER056263), polyserinase- 2 unit 1 (MER061777), Mername-AA123 peptidase (MER021930), HAT-like 2-peptidase (MER099184), hCG2041452-like protein (MER099172), hCG22067 (Homo sapiens) (MER099169), brain rescue factor 1 (Homo sapiens) ( MER098873), hCG2041108 (Homo sapiens) (MER099173), polyserinase-2 unit 2 (MER061760), polyserinase-2 unit 3 (MER065694), Mername-AA201 (peptidase homolog) MER099175, Secreted trypsin-like serine peptidase homolog (MER030000), polyserinease-1A unit 3 (MER029880), azucidin (MER000119), haptoglobin-1 (MER000233), Binding globulin-associated 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), lipoprotein element (MER000183), ψ-KLK1 pseudogene (Homo sapiens) (MER033287), dispase pseudogene I (MER015077), Protease pseudogene II (MER015078), neutral protease pseudogene III (MER015079), subfamily S1A unassigned peptidase (MER216982), subfamily S1A unassigned peptidase (MER216148), amide phosphoribosyltransferase precursor (MER003314), glutamine-fructose-6-phosphate aminotransferase 1 (MER003322), glutamine:fructose-6-phosphate aminotransferase (MER012158), Mername-AA144 protein (MER021319), day Paragine Synthase (MER033254), Family C44 Non-Peptidase Homologue (MER159286), Family C44 Unspecified Peptidase (MER185625), Family C44 Unspecified Peptidase (MER185626), Secernin 1 (MER045376) , Isolate 2 (MER064573), Isolate 3 (MER064582), Acid Ceramidase Precursor (MER100794), N-Acyl Ethanolamine Enzyme Precursor (MER141667), Proteasome Catalytic Subunit 1 (MER000556) , Proteasome Catalytic Subunit 2 (MER002625), Proteasome Catalytic Subunit 3 (MER002149), Proteasome Catalytic Subunit 1i (MER000552), Proteasome Catalytic Subunit 2i (MER001515), Proteasome Catalytic Subunit 3i (MER000555) , proteasome catalytic subunit 5t (MER026203), protein serine kinase c17 (MER026497), proteasome subunit α6 (MER000557), proteasome subunit α2 (MER000550), proteasome subunit α4 (MER000554), proteasome Subunit α7 (MER033250), Proteasome Subunit α5 (MER000558), Proteasome Subunit α1 (MER000549), Proteasome Subunit α3 (MER000553), Proteasome Subunit XAPC7 (MER004372), Proteasome Subunit β3 (MER001710 ), Proteasome Subunit β2 (MER002676), Proteasome Subunit β1 (MER000551), Proteasome Subunit β4 (MER001711), Mername-AA230 Peptidase Homolog (Homo sapiens) (MER047329), Mername-AA231 Pseudogene (Homo sapiens) (MER047172), Mername-AA232 pseudogene (Homo sapiens) (MER047316), glycosylasparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622), threonine aspartase (taspase)-1 (MER016969), γ-glutamyltransferase 5 (mammalian) (MER001977), γ-glutamyltransferase 1 (mammal Animal type) (MER001629), γ-glutamyltransferase 2 (Homo sapiens) (MER001976), γ-glutamyltransferase-like protein 4 (MER002721), γ-glutamyltransferase-like protein 3 (MER016970), similar to γ-glutamyltransferase 1 precursor (Homo sapiens) (MER026204), similar to γ-glutyltransferase 1 precursor (Homo sapiens) (MER026205), Mername-AA211 putative Peptidase (MER026207), γ-glutamyltransferase 6 (MER159283), γ-glutamyltransferase homologue (chromosome 2, Homo sapiens) (MER037241), polycystin-1 (MER126824), KIAA1879 protein (MER159329), polycystic kidney disease 1-like 3 (MER172554), γ-glutaminyl hydrolase (MER002963), guanine 5″-monophosphate synthase (MER043387), carbamoyl Phosphate synthase (Homo sapiens) (MER078640), Dihydroorotase (N-terminal unit) (Homo sapiens) (MER060647), DJ-1 putative peptidase (MER003390), Mername-AA100 putative peptidase (MER014802 ), Mername-AA101 non-peptidase homologue (MER014803), KIAA0361 protein (Homo sapiens) (MER042827), F1134283 protein (Homo sapiens) (MER044553), non-peptidase homolog chromosome 21 open reading frame 33 (Homo sapiens Human) (MER160094), family C56 non-peptidase homolog (MER177016), family C56 non-peptidase homolog (MER176613), family C56 non-peptidase homolog (MER176918), mucin containing EGF-like modules Hormone receptor-like 2 (MER037230), CD97 antigen (human type) (MER037286), mucin-like hormone receptor-like 3 with EGF-like module (MER037288), mucin-like hormone receptor with EGF-like module MUC-like 1 (MER037278), mucin-like hormone receptor-like 4 with EGF-like module (MER037294),Cadherin EGF LAG seven channel G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (Mus musculus) type protein (MER123205), GPR56 (Homo sapiens) type protein (MER122057), spider Toxin affinity protein (latrophin) 2 (MER122199), spider toxin affinity protein-1 (MER126380), spider toxin affinity protein 3 (MER124612), protocadherin flamingo (Flamingo) 2 (MER124239), ETL protein ( MER126267), G protein-coupled receptor 112 (MER126114), seven-transmembrane helix receptor (MER125448), Gpr114 protein (MER159320), GPR126 blood vessel-inducible G protein-coupled receptor (MER140015), GPR125 (Homo sapiens) type protein (MER159279), GPR116 (Homo sapiens) type G protein-coupled receptor (MER159280), GPR128 (Homo sapiens) type G protein-coupled receptor (MER162015), GPR133 (Homo sapiens) type protein (MER159334), GPR110 G protein-coupled receptor (MER159277), GPR97 protein (MER159322), KPG_006 protein (MER161773), KPG_008 protein (MER161835), KPG_009 protein (MER159335), unspecified homologue (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), dystrophin (MER054741), protein Proconvertase 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 homologue ( MER201339), subfamily S8A non-peptidase homologue (MER191613), subfamily S8A unassigned peptidase (MER191611), subfamily S8A unassigned peptidase (MER191612), subfamily S8A unassigned peptidase (MER191614), three Peptidyl-peptidase I (MER003575), prolyl oligopeptidase (MER000393), dipeptidyl-peptidase IV (eukaryotes) (MER000401), acylaminoacyl-peptidase (MER000408), Fibroblast activation protein alpha subunit (MER000399), PREPL A protein (MER004227), dipeptidyl-peptidase 8 (MER013484), dipeptidyl-peptidase 9 (MER004923), FLJ1 putative peptidase (MER017240), Mername - AA194 putative peptidase (MER017353), Mername-AA195 putative peptidase (MER017367), Mername-AA196 putative peptidase (MER017368), Mername-AA197 putative peptidase (MER017371), C14orf29 protein (MER033244), hypothetical protein (MER033245) , putative esterase / lipase / thioesterase (MER047309), protein bat5 (MER037840), putative flj40219 (MER033212), putative flj37464 (MER033240), putative flj33678 (MER033241), dipeptidyl peptidase homologue DPP6 (MER000403 ), dipeptidyl peptidase homologue DPP10 (MER005988), protein similar to open reading frame 135 of mouse chromosome 20 (MER037845), kynurenine formylase (MER046020), thyroglobulin Precursor (MER011604), Acetylcholine Ester (MER033188), Cholinesterase (MER033198), Carboxylesterase D1 (MER033213), Liver Carboxylesterase (MER033220), Carboxylesterase 3 (MER033224), Carboxylesterase 2 (MER033226 ), bile salt-dependent lipase (MER033227), carboxyl esterase-related protein (MER033231), neuroligin 3 (MER033232), neuroligin 4, X-linked (MER033235), neuroligin 4 , Y-linked (MER033236), esterase D (MER043126), aryl acetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone-sensitive lipase (MER033274), neuronexin 1 (MER033280 ), Neuronexin 2 (MER033283), Family S9 Non-Peptidase Homolog (MER212939), Family S9 Non-Peptidase Homolog (MER211490), Subfamily S9C Unassigned Peptidase (MER192341), Family S9 Unassigned Peptide Enzyme (MER209181), Family S9 Unassigned Peptidase (MER200434), Family S9 Unassigned Peptidase (MER209507), Family S9 Unassigned Peptidase (MER209142), Serine Carboxypeptidase A (MER000430), Egg Yolk Carboxypeptidase Like protein (MER005492), RISC peptidase (MER010960), family S15 unspecified peptidase (MER199442), family S15 unspecified peptidase (MER200437), family S15 unspecified peptidase (MER212825), lysosomal Pro-Xaa carboxyl Peptidase (MER000446), dipeptidyl-peptidase II (MER004952), thymus-specific serine peptidase (MER005538), epoxide hydrolase-like putative peptidase (MER031614), Loc328574-like protein (MER033246), containing α / β-abhydrolase domain protein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm-specific transcript protein (MER199890), mesoderm-specific transcript protein (MER017123), cell Solute epoxide hydrolase (MER029997), cytosolic epoxide hydrolase (MER213866), hypothetical protein FLJ22408 (MER031608), CGI-58 putative peptidase (MER030163), Williams-Beuren syndrome critical region protein 21 epoxy Compound hydrolase (MER031610), epoxide hydrolase (MER031612), hypothetical protein 922408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), hypothetical protein (MER033249), valacyclovir ( valacyclovir) hydrolase (MER033259), Ccg1 interactor b (MER210738), glycosylasparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622). Threonine aspartase-1 (MER016969), γ-glutamyltransferase 5 (mammalian) (MER001977), γ-glutamyltransferase 1 (mammalian) (MER001629), Gamma-glutamyltransferase 2 (Homo sapiens) (MER001976), Gamma-glutamyltransferase-like protein 4 (MER002721). Gamma-glutamyltransferase-like protein 3 (MER016970). Similar to γ -Glutaminyltransferase 1 precursor (Homo sapiens) (MER026204). Similar to γ-glutaminyltransferase 1 precursor (Homo sapiens) (MER026205). Mername-AA211 putative peptidase (MER026207).γ -Glutaminyltransferase 6 (MER159283). Gamma-glutaminyltransferase homologue (chromosome 2, Homo sapiens) (MER037241). Polycystin-1 (MER126824), KIAA1879 protein (MER159329). Polycystic kidney disease 1-like 3 (MER172554). γ-Glutaminyl hydrolase (MER002963). Guanine 5″-monophosphate synthase (MER043387). Carbamoyl-phosphate synthase (Homo sapiens) ( MER078640). Dihydroorotase (N-terminal unit) (Homo sapiens) (MER060647). DJ-1 putative peptidase (MER003390). Mername-AA100 putative peptidase (MER014802). Mername-AA101 non-peptidase same KIAA0361 protein (Homo sapiens) (MER042827). F1134283 protein (Homo sapiens) (MER044553). Non-peptidase homologue chromosome 21 open reading frame 33 (Homo sapiens) (MER160094). Family C56 non Peptidase homolog (MER177016), family C56 non-peptidase homolog (MER176613). Family C56 non-peptidase homolog (MER176918). Mucin-like hormone receptor-like 2 with EGF-like module (MER037230) . CD97 antigen (human type) (MER037286). Mucin-like hormone receptor-like 3 with EGF-like module (MER037288). Mucin-like hormone receptor-like 1 with EGF-like module (MER037278). EGF-like Module mucin-like hormone receptor-like 4 (MER037294). Cadherin EGF LAG seven channel G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (house mouse) type protein (MER123205). GPR56 (Homo sapiens) type protein (MER122057). Spider toxin affinity protein 2 (MER122199). Spider toxin affinity protein-1 (MER126380). Spider toxin affinity protein 3 (MER124612). Protocadherin flamingo 2 (MER124239 ). ETL protein (MER126267 ). G protein-coupled receptor 112 (MER126114). Seven transmembrane helix receptor (MER125448). Gpr114 protein (MER159320). GPR125 (Homo sapiens) type protein (MER159279). GPR116 (Homo sapiens) type G protein-coupled receptor (MER159280). GPR128 (Homo sapiens) type G protein-coupled receptor (MER162015). GPR133 (Homo sapiens) type protein (MER159334) GPR110 G protein-coupled receptor (MER159277), GPR97 protein (MER159322), KPG_006 protein (MER161773) KPG_008 protein (MER161835), KPG_009 protein (MER159335), unspecified homologue (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), dystrophin (MER054741 ), Proprotein Convertase 9 (MER022416), Site-1 Peptidase (MER001948), Furin (MER000375), Proprotein Convertase 1 (MER000376), Proprotein Convertase 2 (MER000377), Proprotein Convertase 4 (MER028255),PACE4 Proprotein Convertase (MER000383), Proprotein Convertase 5 (MER002578), Proprotein Convertase 7 (MER002984), Tripeptidyl-Peptidase II (MER000355), Subfamily S8A Non-Peptidase Homolog (MER201339) , subfamily S8A non-peptidase homologue (MER191613), subfamily S8A unspecified peptidase (MER191611), subfamily S8A unspecified peptidase (MER191612), subfamily S8A unspecified peptidase (MER191614), tripeptidyl -Peptidase I (MER003575), Prolyl Oligopeptidase (MER000393), Dipeptidyl-Peptidase IV (Eukaryotes) (MER000401), Acylaminoacyl-Peptidase (MER000408), Cellulose Cell activation protein alpha subunit (MER000399), PREPL A protein (MER004227), dipeptidyl-peptidase 8 (MER013484), dipeptidyl-peptidase 9 (MER004923), FLJ1 putative peptidase (MER017240), Mername-AA194 Putative peptidase (MER017353), Mername-AA195 putative peptidase (MER017367), Mername-AA196 putative peptidase (MER017368), Mername-AA197 putative peptidase (MER017371), C14orf29 protein (MER033244), hypothetical protein (MER033245), putative Esterase / lipase / thioesterase (MER047309), protein bat5 (MER037840), hypothetical flj40219 (MER033212), hypothetical flj37464 (MER033240), hypothetical flj33678 (MER033241), dipeptidyl peptidase homologue DPP6 ( MER000403), dipeptidyl peptidase homologue DPP10 (MER005988), house mouse chromosome 20 open reading frame 135-like protein (MER037845), kynurenine carbamidase (MER046020), thyroglobulin precursor (MER011604), acetylcholine ester (MER033188), cholinesterase (MER033198), carboxylesterase D1 (MER033213), liver carboxylesterase (MER033220), carboxylesterase 3 (MER033224), carboxylesterase 2 ( MER033226), bile salt-dependent lipase (MER033227), carboxyl esterase-related protein (MER033231), neuronexin 3 (MER033232), neuronexin 4, X-linked (MER033235), neuronexin 4, Y-linked (MER033236 ), esterase D (MER043126), aryl acetylamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone-sensitive lipase (MER033274), neuronexin 1 (MER033280), neuronexin 2 (MER033283), family S9 non-peptidase homolog (MER212939), family S9 non-peptidase homolog (MER211490), subfamily S9C unspecified peptidase (MER192341), family S9 unspecified peptidase (MER209181), Family S9 unassigned peptidase (MER200434), family S9 unassigned peptidase (MER209507), family S9 unassigned peptidase (MER209142), serine carboxypeptidase A (MER000430), egg yolk carboxypeptidase-like protein (MER005492) , RISC peptidase (MER010960), family S15 unspecified peptidase (MER199442), family S15 unspecified peptidase (MER200437), family S15 unspecified peptidase (MER212825), lysosomal Pro-Xaa carboxypeptidase (MER000446) , dipeptidyl-peptidase II (MER004952), thymus heteroserine peptidase (MER005538), epoxide hydrolase-like putative peptidase (MER031614), Loc328574-like protein (MER033246), containing α / β-hydrolyzed Enzyme domain protein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm-specific transcript protein (MER199890), mesoderm-specific transcript protein (MER017123), cytosolic epoxide hydrolase (MER029997 ), cytosolic epoxide hydrolase (MER213866), similar hypothetical protein FLJ22408 (MER031608), CGI-58 putative peptidase (MER030163), Williams-Beuren syndrome critical region protein 21 epoxide hydrolase (MER031610), cyclic Oxide hydrolase (MER031612), hypothetical flj22408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), hypothetical protein (MER033249), valacyclovir (valacyclovir) hydrolase (MER033259), Ccg1 interactor b (MER210738). ,

[0087] The enzymatic activity of a protease can be modulated. For example, certain proteases can be inactivated by the presence or absence of specific agents (eg, agents that bind to the protease, such as specific small molecule inhibitors). Such proteases may be referred to as "repressor proteases". Exemplary inhibitors for certain proteases are listed in Table 4B. For example, NS3 protease can be blocked by protease inhibitors including but not limited to: simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, Riprevir, telaprevir, glaprevir, gecaprevir, and vociprevir. In another example, protease activity can be regulated by modulating the expression of the protease itself, such as by engineering cells to use an inducible promoter system (e.g., a Tet On / Off system) or a cell-specific promoter (which can be used to express Promoters for heterologous proteases are described in more detail herein in the section entitled "Promoters") expressing proteases. Proteases may also contain degrons, such as any of the degrons described herein, and may be regulated using any of the degron systems described herein.

[0088] The enzymatic activity of a protease can also be regulated by selecting a specific protease cleavage site. For example, a protease cleavage site can be selected and / or engineered such that the sequence exhibits a desired rate of cleavage by the desired protease, such as a reduced cleavage kinetics relative to the endogenous sequence of the substrate naturally cleaved by the desired protease study. As another example, a protease cleavage site can be selected and / or engineered such that the sequence exhibits a desired rate of cleavage in a cell state-specific manner. For example, various cellular states (eg, following cell signaling, such as immune cell activation) can affect the expression and / or localization of certain proteases. As an illustrative example, ADAM17 protein levels and localization are known to be affected by signaling, such as via the protein kinase C (PKC) signaling pathway (e.g., by the PKC activator (phorbol-12-myristate-13-acetate [Phorbol-12-myristat-13-acetat, PMA]) activates) signaling. Thus, protease cleavage sites can be selected and / or engineered such that cleavage of the protease cleavage site and subsequent release of effector molecules is increased or decreased as desired, depending on the properties of the protease (e.g., expression and / or or positioning). As another example, protease cleavage sites, especially in combination with specific membrane tether domains, can be selected and / or engineered for optimal protein expression of the chimeric protein. membrane tether domain

[0089] The membrane cleavable chimeric proteins provided herein contain a cell membrane tether domain (referred to as "MT" in the formula S-C-MT or MT-C-S). In general, a cell membrane tethering domain can be any amino acid sequence motif capable of directing localization (eg, insertion) of a chimeric protein into or otherwise associated with the cell membrane of a cell expressing the chimeric protein. The cell membrane tether domain may be a transmembrane intracellular domain. A cell membrane tether domain can be a transmembrane domain. A cell membrane tether domain can be an integral membrane protein domain (eg, a transmembrane domain). Cell membrane tether domains can be derived from type I, type II or type III transmembrane proteins. The cell membrane tether domain can include a post-translational modification tag, or a motif capable of post-translational modification to modify the chimeric protein to include a post-translational modification tag that allows association with the cell membrane. Examples of post-translational modification tags include, but are not limited to, lipid anchor domains (eg, GPI lipid anchors, myristoylation tags, or palmitoylation tags). Examples of cell membrane tether domains include, but are not limited to, those derived from PDGFR-β, CD8, CD28, CD3ζ chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, Transmembrane-intracellular domain and / or transmembrane domain of NKG2D, EpoR, TNFR2, B7-1 or BTLA. A cell membrane tethering domain can be a cell surface receptor or a cell membrane-associated portion thereof.

[0090] In some embodiments, the cell membrane tether domain comprises a transmembrane domain derived from a B-71 polypeptide. In some embodiments, the transmembrane domain comprises the sequence LLPSWAITLISVNGIFVICCLTYCFAPRCRERRNERLRRESVRPV (SEQ ID NO: 204).

[0091] In some embodiments, the cell membrane tether domain comprises a transmembrane domain derived from a CD8 polypeptide. Any suitable CD8 polypeptide may be used. Exemplary CD8 polypeptides include, but are not limited to, NCBI reference numbers NP_001139345 and AAA92533.1. Examples of CD8 transmembrane domains include IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO:205), IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO:206) and IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO:207). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 205). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 206). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 207). In some embodiments, the cell membrane tether domain comprises a hinge and transmembrane domain derived from CD8. In some embodiments, the CD8 hinge comprises the sequence TTTPARPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 208). In some embodiments, the CD8 hinge comprises the sequence AAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 209).

[0092] In general, for all membrane cleavable chimeric proteins described herein, the cell membrane tether domain is: (1) C-terminal to the protease cleavage site and N-terminal to any intracellular domain (if present) (that is, the cell membrane tether domain is between the protease cleavage site and the intracellular domain (if present); or (2) N-terminal to the protease cleavage site and C-terminal to any intracellular domain (if present) present) (also between the protease cleavage site and the inverted domain-directed intracellular domain, if present). In embodiments featuring a degron associated with a chimeric protein, the degron domain is a terminal cytoplasmic targeting domain, specifically with respect to a cell membrane tether (in other words, a cell membrane tether domain at the protease cleavage site point and the degron). The cell membrane tether domain can be linked to the protease cleavage site by a polypeptide linker (ie, a polypeptide sequence not generally considered part of the cell membrane tether domain or the protease cleavage site). The cell membrane tethering domain can be linked to the intracellular domain (if present) by a polypeptide linker (ie, a polypeptide sequence not generally considered part of the cell membrane tethering domain or the intracellular domain). A cell membrane tether domain can be linked to a degron, if present, by a polypeptide linker (ie, a polypeptide sequence not normally considered part of the cell membrane tether domain or degron). The polypeptide linker can be any amino acid sequence linking the first polypeptide sequence and the second polypeptide sequence. A polypeptide linker can be a flexible linker (eg, a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS]4GG [SEQ ID NO: 182]), A(EAAAK)3A (SEQ ID NO: 183), and Whitlow linkers (e.g., "KEGS" Linkers, such as the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), eGK linkers, such as the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 185), and are described in more detail in issued U.S. Patent No. 5,990,275 linker, the U.S. patent is incorporated herein by reference). Additional polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, and SEQ ID NO: 197. Other polypeptide linkers can be selected based on desired properties (eg, length, flexibility, amino acid composition, etc.) and are known to those skilled in the art.

[0093] In general, the cell membrane tethering domain is oriented such that the secreted effector molecule and the protease cleavage site are exposed extracellularly after insertion into or association with the cell membrane such that the protease cleavage site can be detected by its respective protease. Cleaves and releases ("secretes") the effector molecule into the extracellular space. Degron systems and domains

[0094] In some embodiments, any of the proteins described herein may include a degron domain, including but not limited to, a protease, a transcription factor, a promoter or part of a promoter system (e.g., ACP), and / or Any of the membrane cleavable chimeric proteins described herein. In general, a degron domain can be any amino acid sequence motif capable of directing regulated degradation, such as via an ubiquitin-mediated pathway. The degron domain directs ubiquitin-mediated degradation of the degron fusion protein in the presence of an immunomodulatory drug (IMiD).

[0095] The degron domain can be a substrate domain of a hydroxycerebroside (CRBN) polypeptide capable of binding CRBN in response to an immunomodulatory drug (IMiD), including but not limited to IKZF1, IKZF3, CK1a, ZFP91, GSPT1, MEIS2 , GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787 and ZN827 or fragments thereof capable of drug-inducible binding to CRBN. The CRBN polypeptide substrate domain can be a chimeric fusion product of a native CRBN polypeptide sequence, such as an IKZF3 / ZFP91 / IKZF3 chimeric fusion product having the amino acid sequence of FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 175). Degron domains, and in particular the CRBN degron system, are described in more detail in International Application Publication No. WO2019 / 089592A1, which is hereby incorporated by reference for all purposes. Other examples of degron domains include, but are not limited to, HCV NS4 degron, PEST (two copies of residues 277-307 of human 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 (SP2 and tandem repeat of 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 (split repeats of SP1 and SP2 (SP2-SP1- SP2-SP1-SP2; SEQ ID NO: 166), NS2 (three copies of residues 79-93 of the NS protein of influenza A virus; SEQ ID NO: 167), ODC (residues of ornithine decarboxylase 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, COP1 E3 ligase binding degron motif, CRL4-Cdt2 binding PIP degron, actinfilin binding degron, KEAP1 binding degron, KLHL2 and KLHL3 binding degron, MDM2 binding pattern N-degron, hydroxyproline modification in hypoxic signaling, phytohormone-dependent SCF-LRR binding degron, SCF ubiquitin ligase-binding phosphorylation degron, phytohormone-dependent SCF-LRR - Binding degron, DSGxxS phosphate-dependent degron, Siah binding motif, SPOP SBC docking motif or PCNA binding PIP box.

[0096] The regulated degradation can be drug-inducible. Drugs capable of mediating / regulating degradation can be small molecule compounds. Drugs capable of mediating / modulating degradation may include "immunomodulatory drugs" (IMiDs). In general, as used herein, IMiDs refer to a class of small molecule immunomodulatory drugs containing imide groups. Hydroxycerebroside (CRBN) is a known target of IMiD, and binding of IMiD to CRBN or the CRBN polypeptide substrate domain alters the substrate specificity of the CRBN E3 ubiquitin ligase complex, resulting in a protein with a CRBN polypeptide substrate domain. Degradation of proteins (eg, any of the secretable effector molecules described herein or other proteins of interest). Examples of imide-containing IMiDs for a degron domain with a CRBN polypeptide receptor domain include, but are not limited to, thalidomide, ralidome, or pomatomaide. An IMiD may be an FDA-approved drug.

[0097] The chimeric proteins described herein may contain a degron domain (e.g., in the formula S-C-MT-D or D-MT-C-S for the membrane cleavable chimeric proteins described herein , referred to as "D"). In the absence of IMiDs, degron / ubiquitin-mediated degradation of the chimeric protein does not occur. Following expression and localization of the chimeric protein into the cell membrane, the protease cleavage site directs cleavage of the chimeric protein, allowing release ("secretion") of the effector molecule into the extracellular space. In the presence of an immunomodulatory drug (IMiD), the degron domain directs ubiquitin-mediated degradation of the chimeric protein, resulting in reduced or eliminated secretion of effector molecules. In general, for a membrane-cleavable chimeric protein fused to a degron domain, the degron domain is the terminal cytoplasmic targeting domain, specifically the cell membrane tether domain, e.g., Formulas S-C - the most C-terminal domain in MT - D or the most N-terminal domain in the formula D - MT - C - S. A degron domain can be linked to a cell membrane tether domain by a polypeptide linker (ie, a polypeptide sequence not generally considered a cell membrane tether domain or part of a degron domain). The polypeptide linker can be any amino acid sequence linking the first polypeptide sequence and the second polypeptide sequence. A polypeptide linker can be a flexible linker (eg, a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS]4GG [SEQ ID NO: 182]), A(EAAAK)3A (SEQ ID NO: 183), and Whitlow linkers (e.g., "KEGS" Linkers, such as the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), eGK linkers, such as the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 185), and are described in more detail in issued U.S. Patent No. 5,990,275 linker, the U.S. patent is incorporated herein by reference). Additional polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, and SEQ ID NO: 197. Other polypeptide linkers can be selected based on desired properties (eg, length, flexibility, amino acid composition, etc.) and are known to those skilled in the art. In general, degrons are oriented relative to the cell membrane tether domain such that after localization to the cell membrane the degron is exposed to the cytosol such that the degron domain is capable of mediating degradation (e.g., exposure to the cytosol and cytosolic ) and can mediate ubiquitin-mediated degradation.

[0098] For a degron fusion protein, the degron domain can be N-terminal or C-terminal to the protein of interest (eg, effector molecule). A degron domain can be linked to a protein of interest by a polypeptide linker (ie, a polypeptide sequence not normally considered part of the protein of interest or the degron domain). The polypeptide linker can be any amino acid sequence linking the first polypeptide sequence and the second polypeptide sequence. A polypeptide linker can be a flexible linker (eg, a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS]4GG [SEQ ID NO: 182]), A(EAAAK)3A (SEQ ID NO: 183), and Whitlow linkers (e.g., "KEGS" Linkers, such as the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), eGK linkers, such as the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 185), and are described in more detail in issued U.S. Patent No. 5,990,275 linker, the U.S. patent is incorporated herein by reference). Additional polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, and SEQ ID NO: 197. Other polypeptide linkers can be selected based on desired properties (eg, length, flexibility, amino acid composition, etc.) and are known to those skilled in the art. A polypeptide linker can be cleavable, eg, by any of the protease cleavage sites described herein. Homing Molecule

[0099] "Tumor microenvironment" is the cellular environment in which a tumor exists, including peripheral blood vessels, immune cells, fibroblasts, inflammatory cells of bone marrow origin, lymphocytes, signaling molecules, and the extracellular matrix (ECM) (see, e.g., Pattabiraman, D.R. and Weinberg, R.A. Nature Reviews Drug Discovery13, 497-512 (2014); Balkwill, F.R. et al. J Cell Sci125, 5591-5596, 2012; and Li, H. et al. J Cell Biochem101(4), 805-15, 2007 ).

[0100] In some embodiments, the engineered nucleic acid is configured to produce at least one homing molecule. For example, in the membrane cleavable chimeric proteins described herein that contain a secreted effector molecule, the secreted effector molecule can be a homing molecule. "Homing" refers to the active navigation (migration) of cells to a target site (eg, cell, tissue (eg, tumor) or organ). "Homing molecule" refers to a molecule that directs a cell to a target site. In some embodiments, the function of the homing molecule is to recognize and / or initiate the interaction of the engineered cell with the target site. Non-limiting examples of homing molecules include CXCR1, CCR9, CXCR2, CXCR3, CXCR4, CCR2, CCR4, FPR2, VEGFR, IL6R, CXCR1, CSCR7, and PDGFR.

[0101] 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 induce directional chemotaxis in cells. Chemokines can be divided into four major subfamilies: CXC, CC, CX3C, and XC, all of which exert biological effects by selectively binding to chemokines receptors located on the surface of target cells. In some embodiments, the engineered nucleic acid is configured to produce CXCR4, a chemokine receptor that allows engineered cells to express stromal cell-derived factor 1 along a chemokine gradient (also known as SDF1, C-X-C motif chemokine 12 and CXCL12) for cell, tissue or tumor homing. Non-limiting examples of chemokine receptors that can be encoded by engineered nucleic acids of the disclosure include: CXC chemokine receptors (e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7), CC chemokine receptors (CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, and CCR11), CX3C chemokine receptors (e.g., CX3CR1, which binds to CX3CL1), and XC Chemokine receptors (eg, XCR1). In some embodiments, the chemokine receptor is a G protein-linked transmembrane receptor, or a member of the tumor necrosis factor (TNF) receptor superfamily (including but not limited to TNFRSF1A, TNFRSF1B). In some embodiments, the engineered nucleic acid is configured to produce CXCL8, CXCL9 and / or CXCL10 (promote T cell recruitment), CCL3 and / or CXCL5, CCL21 (Th1 recruitment and polarization).

[0102] In some embodiments, engineered nucleic acids are configured to generate G protein-coupled receptors (GPCRs) that detect N-formylated oligopeptides (including but not limited to FPR2 and FPRL1) .

[0103] In some embodiments, the engineered nucleic acid is configured to generate a receptor (including but not limited to IL6R) that detects interleukin.

[0104] In some embodiments, the engineered nucleic acid is configured to produce receptors that detect growth factors secreted from other cells, tissues, or tumors (including, but not limited to, the FGFR, PDGFR, EGFR, and VEGF families (including But not limited to VEGF-C and VEGF-D) receptors).

[0105] In some embodiments, the homing molecule is an integrin. Integrins are transmembrane receptors that promote cell-extracellular matrix (ECM) adhesion. Integrins are obligate heterodimers with two subunits, alpha (alpha) and beta (beta). The alpha subunit of an integrin can be, but is not limited to: ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, IGTA7, ITGA8, ITGA9, IGTA10, IGTA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGA2B, ITGAX. The β subunit of integrin can be, but not limited to: ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7 and ITGB8. Engineered nucleic acids can be configured to produce any combination of integrin alpha and beta subunits.

[0106] 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 MMPs. In some embodiments, engineered nucleic acids are configured to produce inhibitors of molecules (eg, proteins) that inhibit MMPs. For example, an engineered nucleic acid can be configured to express an inhibitor of membrane type 1 MMP (MT1-MMP) (eg, an RNAi molecule) or TIMP metallopeptidase inhibitor 1 (TIMP-1).

[0107] In some embodiments, the homing molecule is, for example, a ligand that binds to selectin on the endothelium of the target tissue (e.g., hematopoietic E- / L-selectin ligand (HCELL), Dykstran et al. , Stem Cells. 2016 Oct;34(10):2501-2511).

[0108] The term "homing molecule" also encompasses transcription factors that regulate the production of molecules that improve / enhance cell homing.

[0109] In some embodiments, the homing molecule comprises an antibody, such as anti-integrin α4, β7 or anti-MAdCAM. engineered nucleic acid

[0110] Provided herein are engineered nucleic acids encoding at least one chimeric protein of the disclosure, such as the membrane cleavable chimeric proteins described herein having the formula S-C-MT or MT-C-S. Provided herein are engineered nucleic acids encoding two or more chimeric proteins.

[0111] 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, the membrane-cleavable Chimeric proteins are oriented from N-terminus to C-terminus and have the following formula: S - C - MT or MT - C - S. S refers to secretable effector molecule. C refers to the protease cleavage site. MT refers to the cell membrane tether domain. The promoter is operably linked to the exogenous polynucleotide sequence, and the S-C-MT or MT-C-S is configured to represent a single polypeptide.

[0112] 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, the membrane-cleavable A chimeric protein has a protein of interest (eg, any effector molecule described herein). A promoter is operably linked to an exogenous polynucleotide sequence, and the membrane-cleavable chimeric protein is configured to represent a single polypeptide.

[0113] "Engineered nucleic acid" refers to a nucleic acid that does not occur in nature. It should be understood, however, that although the engineered nucleic acid as a whole is not naturally occurring, it may include naturally occurring nucleotide sequences. In some embodiments, the engineered nucleic acid comprises nucleotide sequences from different organisms (eg, from different species). For example, in some embodiments, engineered nucleic acids include murine nucleotide sequences, bacterial nucleotide sequences, human nucleotide sequences, and / or viral nucleotide sequences. The term "engineered nucleic acid" includes recombinant nucleic acids as well as synthetic nucleic acids. "Recombinant nucleic acid" refers to a molecule constructed by conjugating nucleic acid molecules and, in some embodiments, replicable in living cells. "Synthetic nucleic acid" refers to a molecule that is amplified or synthesized chemically or by other means. Synthetic nucleic acids include those that have been chemically or otherwise modified, but are capable of base pairing with naturally occurring nucleic acid molecules. Modifications include, but are not limited to, one or more modified internucleotide linkages and non-natural nucleic acids. Modifications are described in further detail in US Patent No. 6,673,611 and US Application Publication 2004 / 0019001, each of which is incorporated by reference in its entirety. Modified internucleotide linkages may be phosphorodithioate or phosphorothioate linkages. Unnatural nucleic acids can be locked nucleic acid (LNA), peptide nucleic acid (PNA), diol nucleic acid (GNA), phosphorodiamidic morpholino oligomer (PMO or "morpholino"), and threose nucleic acid (TNA) . Non-natural nucleic acids are described in further detail in International Application WO 1998 / 039352, U.S. Application Publication No. 2013 / 0156849, and U.S. Patent Nos. 6,670,461, 5,539,082, 5,185,444, each of which is incorporated herein by reference in its entirety . Recombinant nucleic acids and synthetic nucleic acids also include those molecules resulting from the replication of any of the foregoing. The engineered nucleic acids of the disclosure can be encoded by a single molecule (eg, included in the same plastid or another vector) or by multiple different molecules (eg, multiple different molecules that replicate independently). An engineered nucleic acid can be an isolated nucleic acid. Isolated nucleic acids include, but are not limited to, cDNA polynucleotides, RNA polynucleotides, RNAi oligonucleotides (e.g., siRNA, miRNA, antisense oligonucleotides, shRNA, etc.), mRNA polynucleotides, circular plastids, Linear DNA fragments, vectors, minicircles, ssDNA, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC), and oligonucleotides.

[0114] The engineered nucleic acids of the disclosure can be produced using standard molecular biology methods (see, eg, Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, the engineered nucleic acid constructs use GIBSON ASSEMBLY® Cloning (see, e.g., Gibson, D.G. et al. Nature Methods, 343-345, 2009; and Gibson, D.G. et al. Nature Methods, 901-903, 2010, each incorporated herein by reference). GIBSON ASSEMBLY® usually uses three enzyme activities in a single-tube reaction: 5' exonuclease, DNA polymerase Ύ extension activity and DNA ligase activity. The 5' exonuclease activity chews back the 5' end sequence and exposes the complementary sequence for annealing. Polymerase activity then fills the gap over the annealed region. DNA ligase then seals the nick and covalently joins the DNA fragments together. The overlapping sequences of contiguous fragments are much longer than the sequences used in Golden Gate Assembly and thus yield a higher percentage of correct assemblies. In some embodiments, engineered nucleic acid constructs are generated using IN-FUSION® cloning (Clontech). Promoter

[0115] In general, in all of the embodiments described herein, the engineered nucleic acid encoding one or more membrane-cleavable chimeric proteins encodes an expression cassette comprising a promoter. In some embodiments, engineered nucleic acids (e.g., engineered nucleic acids comprising expression cassettes) comprise nucleotide sequences (e.g., exogenous polynucleotides) operably linked to encoding at least two different proteins sequence) promoter. For example, an engineered nucleic acid can comprise a DNA sequence operably linked to a gene encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least Promoters of nucleotide sequences of 10 different proteins. In some embodiments, the engineered nucleic acid comprises operably linked to a gene encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Promoters of nucleotide sequences of different proteins. In some embodiments, the engineered nucleic acid (e.g., an engineered nucleic acid comprising an expression cassette) comprises a nucleotide sequence (e.g., exogenous Sexual polynucleotide sequence) promoter. For example, an engineered nucleic acid can comprise a DNA sequence operably linked to a gene encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least Promoters of nucleotide sequences of 10 membrane-cleavable chimeric proteins. In some embodiments, the engineered nucleic acid comprises operably linked to a gene encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more The seed membrane can cleave the promoter of the nucleotide sequence of the chimeric protein.

[0116] "Promoter" refers to the control region of a nucleic acid sequence that controls the initiation and rate of transcription of the remainder of the nucleic acid sequence. A promoter may also contain subregions to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. A promoter can be constitutive, inducible, repressible, tissue specific, or any combination thereof. A promoter drives the expression or transcription of the nucleic acid sequence it regulates. Herein, a promoter is said to be "operably linked" when it is in the correct functional position and orientation relative to the nucleic acid sequence it regulates to control ("drive") the transcriptional initiation and / or expression of that sequence.

[0117] A promoter may be one that is naturally associated with a gene or sequence, such as may be obtained by isolating the 5' non-coding sequence upstream of the coding segment of a given gene or sequence. The promoter can be referred to as "endogenous". In some embodiments, a coding nucleic acid sequence may be under the control of a recombinant or heterologous promoter, which refers to a promoter not normally associated with a coding sequence in its natural environment. Such promoters may include those of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that do not "occur in nature", such as, for example, different elements containing different transcriptional regulatory regions and / or by means of which Genetic engineering methods known in the art alter the expression of the mutants. In addition to synthetically generating nucleic acid sequences for promoters and enhancers, sequences can also be generated using recombinant cloning and / or nucleic acid amplification techniques, including polymerase chain reaction (PCR) (see, e.g., U.S. Patent No. 4,683,202 and U.S. Patent No. 5,928,906).

[0118] The promoter of the engineered nucleic acid may be an "inducible promoter", which refers to an initiation characterized by regulation (e.g., initiation or activation) of transcriptional activity when a signal is present, influenced by, or contacted by a signal son. Signals can be endogenous or often exogenous conditions (e.g., light), compounds (e.g., chemical or non-chemical compounds), or proteins (e.g., cytokines) that act to regulate transcriptional activity in an autoinducible promoter The active form contacts the inducible promoter. Activation of transcription may involve acting directly on the promoter to drive transcription, or indirectly by inactivating a repressor that prevents the promoter from driving transcription. Conversely, inactivation of transcription may involve acting directly on the promoter to prevent transcription, or indirectly by activating a repressor that subsequently acts on the promoter.

[0119] A promoter is "responsive" to a local tumor state (eg, inflammation or hypoxia) or signal if transcription from the promoter is activated, deactivated, increased or decreased in the presence of that state or signal. or "regulated by it". In some embodiments, the promoter comprises a response element. A "response element" is a short DNA sequence within a promoter region that binds specific molecules (eg, transcription factors) that regulate (regulate) gene expression from the promoter. Response elements that can be used in accordance with the present disclosure include, but are not limited to, the phloretin regulatable control element (PEACE), the zinc finger DNA binding domain (DBD), the interferon-γ-activating sequence (GAS) (Decker, T. et al. 1997 Mar; 17(3):121-34, which is incorporated herein by reference), Interferon Stimulated Response Element (ISRE) (Han, K. J. et al., J Biol Chem. 2004 Apr 9; 279(15):15652-61, which is incorporated herein by reference), NF-κB response element (Wang, V. et al., Cell Reports. 2012; 2(4): 824 -839, which is incorporated herein by reference) and the STAT3 response element (Zhang, D. et al., J of Biol Chem. 1996; 271: 9503-9509, which is incorporated herein by reference). Other reactive elements are also contemplated herein. Response elements may also contain tandem repeats (eg, consecutive repeats of the same nucleotide sequence encoding the response element) to substantially increase the sensitivity of the response element to its cognate binding molecule. Tandem repeats can be labeled 2X, 3X, 4X, 5X, etc. to indicate the number of repeats present.

[0120] Non-limiting examples of responsive promoters (also referred to as "inducible promoters") (e.g., TGF-β responsive promoters) are listed in Table 5A, which shows the design of promoters and transcription factors , and showing the effect of inducible molecules on transcription factor (TF) and transgene transcription (T) (B, binding; D, dissociation; n.d., not determined) (A, activation; DA, inactivation; DR, derepression) ( See Horner, M. and Weber, W. FEBS Letters 586 (2012) 20784-2096m, and references cited therein). Non-limiting examples of components of an inducible promoter include those presented in Table 5B. Table 5A. Examples of responsive promoters system promoters and operators transcription factor (TF) inducer molecule response to inducer TF T transcriptional activator responsive promoter AIR PAIR (OalcA-PhCMVmin) AlcR Acetaldehyde n.d. A ART PART (OARG-PhCMVmin) ArgR-VP16 l-arginine B A BIT PBIT3 (OBirA3-PhCMVmin) BIT (BirA-VP16) Biotin B A Cumate - Activator PCR5 (OCuO6-PhCMVmin) cTA (CymR-VP16) Cumate D. DA Cumate - reverse activator PCR5 (OCuO6-PhCMVmin) rcTA (rCymR-VP16) Cumate B A E-OFF PETR (OETR-PhCMVmin) ET (E-VP16) Erythromycin D. DA NICE-OFF PNIC (ONIC-PhCMVmin) NT (HdnoR-VP16) 6-Hydroxy-nicotine D. DA PEACE PTtgR1 (OTtgR-PhCMVmin) TtgA1 (TtgR-VP16) Phloretin D. DA PIP-OFF PPIR (OPIR-Phsp70min) PIT (PIP-VP16) Pristinamycin I D. DA QuoRex PSCA (OscbR-PhCMVmin)PSPA (OpapRI-PhCMVmin) SCA (ScbR-VP16) SCB1 D. DA redox PROP (OROP-PhCMVmin) REDOX (REX-VP16) NADH D. DA TET-OFF PhCMV*-1 (OtetO7-PhCMVmin) tTA (TetR-VP16) tetracycline D. DA TET-ON PhCMV*-1 (OtetO7-PhCMVmin) rtTA (rTetR-VP16) Deoxycycline B A TIGR PCTA (OrheO-PhCMVmin) CTA (RheA-VP16) hot D. DA TrR O7x(tra box)-PhCMVmin p65-TraR 3-oxo-C8-HSL B A VAC-OFF P1VanO2 (OVanO2-PhCMVmin) VanA1 (VanR-VP16) vanillic acid D. DA transcription repressor responsive promoter Cumate-repressor PCuO (PCMV5-OCuO) CymR Cumate D. DR E-ON PETRON8 (PSV40-OETR8) E-KRAB Erythromycin D. DR NICE-ON PNIC (PSV40-ONIC8) NS (HdnoR-KRAB) 6-Hydroxy-nicotine D. DR PIP-ON PPIRON (PSV40-OPIR3) PIT3 (PIP-KRAB) Protomycin I D. DR Q-ON PSCAON8 (PSV40-OscbR8) SCS (ScbR-KRAB) SCB1 D. DR Based on TET-ON repressor OtetO-PHPRT tTS-H4 (TetR-HDAC4) Deoxycycline D. DR T-REX PTetO (PhCMV-OtetO2) TetR tetracycline D. DR UREX PUREX8 (PSV40-OhucO8) mUTS (KRAB-HucR) uric acid D. DR VAC-ON PVanON8 (PhCMV-OVanO8) VanA4 (VanR-KRAB) vanillic acid D. DR hybrid promoter QuoRexPIP-ON (NOT IF gate) OscbR8-OPIR3-PhCMVmin SCAPIT3 SCB1 protomycin I DD DADR QuoRexE-ON (reverse gate) OscbR-OETR8-PhCMVmin SCAE-KRAB SCB1 erythromycin DD DADR TET-OFFE-ON (reverse if the gate) OtetO7-OETR8-PhCMVmin tTAE-KRAB tetracycline erythromycin DD DADR TET-OFF PIP-ONE-ON OtetO7-OPIR3-OETR8-PhCMVmin tTAPIT3E-KRAB tetracycline primitive mycin I erythromycin DDD DADRDR Table 5B. Exemplary components of inducible promoters name dna sequence Minimal promoter; minP AGAGGGTATATAATGGAAGCTCGACTTCCAG (SEQ ID NO: 1) NFkB response element protein promoter; 5× NFkB-RE GGGAATTTCCGGGGACTTTCCGGGAATTTCCGGGGACTTTCCGGGAATTTCC (SEQ ID NO: 2) CREB response element protein promoter; 4×CRE CACCAGACAGTGACGTCAGCTGCCAGATCCCATGGCCGTCATACTGTGACGTCTTTTCAGACACCCCATTGACGTCAATGGGAGAA (SEQ ID NO: 3) NFAT response element protein promoter; 3× NFAT binding sites GGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGT (SEQ ID NO: 4) SRF response element protein promoter; 5× SRE AGGATGTCCATATTAGGACATCTAGGATGTCCATATTAGGACATCTAGGATGTCCATATTAGGACATCTAGGATGTCCATATTAGGACATCTAGGATGTCCATATTAGGACATCT (SEQ ID NO: 5) SRF response element protein promoter 2; 5 × SRF-RE AGTATGTCCATATTAGGACATCTACCATGTCCATATTAGGACATCTACTATGTCCATATTAGGACATCTTGTATGTCCATATTAGGACATCTAAAATGTCCATATTAGGACATCT (SEQ ID NO: 6) AP1 response element protein promoter; 6× AP1-RE TGAGTCAGTGACTCAGTGAGTCAGTGACTCAGTGAGTCAGTGACTCAG (SEQ ID NO: 7) TCF-LEF response element promoter; 8× TCF-LEF-RE AGATCAAAGGGTTTAAGATCAAAGGGCTTAAGATCAAAGGGTATAAGATCAAAGGGCCTAAGATCAAAGGGACTAAGATCAAAGGGTTTAAGATCAAAGGGCTTAAGATCAAAGGGCCTA (SEQ ID NO: 8) SBEx4 GTCTAGACGTCTAGACGTCTAGACGTCTAGAC (SEQ ID NO: 9) SMAD2 / 3 - CAGACA × 4 CAGACACAGACACAGACACAGACA (SEQ ID NO: 10) STAT3 binding site Ggatccggtactcgagatctgcgatctaagtaagcttggcattccggtactgttggtaaagccac (SEQ ID NO: 11) minCMV taggcgtgtacggtgggaggcctatataagcagagctcgtttagtgaaccgtcagatcgcctgga (SEQ ID NO: 170) YB_TATA TCTAGAGGGTATATAATGGGGGCCA (SEQ ID NO: 171) wxya Ttcgcatattaaggtgacgcgtgtggcctcgaacaccgagcgaccctgcagcgacccgcttaa (SEQ ID NO: 172)

[0121] Non-limiting examples of promoters include cytomegalovirus (CMV) promoter, elongation factor 1-alpha (EF1a) promoter, elongation factor (EFS) promoter, MND promoter (containing myeloproliferative sarcoma virus A synthetic promoter of the U3 region of the modified MoMuLV LTR of the enhancer), the phosphoglycerate kinase (PGK) promoter, the spleen focus forming virus (SFFV) promoter, the simian virus 40 (SV40) promoter, and the ubiquitin C ( UbC) promoter (see Table 5C). Table 5C. Exemplary constitutive promoters name dna sequence CMV GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGG TAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTAGTCATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACG GGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTC (SEQ ID NO: 12) EF1a GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCGAGAAGTTGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGCCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTT TTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGA GGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGT GCGTCCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGACCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGTCCTCGCGCCGCCGTGTATCGCCCCGCCCCGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGTCCTGCTGCAGG GAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACA CTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA (SEQ ID NO: 13) EFS GGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCCACATCGCCCACAGTCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCC GTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGC TCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAAGATCCAAGCTGTGACCGGCGCCTAC (SEQ ID NO: 14) MND TTTATTTTAGTCTCCAGAAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTC AGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCA (SEQ ID NO: 15) PGK GGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGG GAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCGGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGC GGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAG (SEQ ID NO: 16) SFFV GTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGGCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGTCCCCAAGATATGGCCCAACCC TCAGCATTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCTCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGCCCGGG (SEQ ID NO: 17) SV40 CTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCC ATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCT (SEQ ID NO: 18) UBC GCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCGCTGCCACGTCAGACGAAGGGCGCAGGAGCGTTCCTGATCCTTCCGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTC CAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGGTGAGTTGCGGGCTGCTGGGCTGGCC GGGGCTTTCGTGGCCGCCGGGCCGCTCGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAGGGCTGTAGTCTGGGTCCGCGAGCAAGGTTGCCCTGAACTGGGGGTTGGGGGGAGCGCACAAAATGGCGGCTGTTCCCGAGTCTTGAATGGAAGACGCTTGTAAGGCGGGCTGTGAGGTCGTTGAAACAAGGTGGG GGGCATGGTGGGCGGCAAGAACCCCAAGGTCTTGAGGCCTTCGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGGCTGGGGCACCATCTGGGGACCCTGACGTGAAGTTTGTCACTGACTGGAGAACTCGGGTTTGTCGTCTGGTTGCGGGGGCGGCAGTTATGCGGTGCCGTTGGGCAGTGCACCCGTACCTTTGGGA GCGCGCGCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAATGCAGGGTGGGGCCACCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGACGCAGGGTTCGGGCCTAGGGTAGGCTCTCCTGAATCGACAGGCGCCGGACCTCTGGTGAGGGGAGGGATAAGTGAGGCGTCAGTTTCTTTGGTCGGTTTTAT GTACCTATCTTTCTTTAAGTAGCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTGTTTTGTGAAGTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATATGTAATTTTCAGTGTTAGACTAGTAAAGCTTCTGCAGGTCGACTCTAGAAAATTGTCCGCTAAATTCTGGCCGTTTTTGGCTTTTTTGTTAGAC (SEQ ID NO: 1 9) hEF1aV1 GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTT TTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGA GGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGT GCGTCCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGTCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAG GGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCA CACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA (SEQ ID NO: 20) hCAGG ACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGC AGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGTTTCACTCTCCCCATCTCCCCCCCTCCCCCCCCAATTTTGTATTATTTAT TTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGC GCGCGGCGGGCGGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCCAGGTGAGCGGGCGGGACGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGG GGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGCGGTGCCCCGCGGTGCGGGG GGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGTG GCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGG GAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGT GTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTC (SEQ ID NO: 21) hEF1aV2 Gggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcg caacgggtttgccgccagaacacag (SEQ ID NO: 22) wxya CCACTAGTTCCATGTCCTTATATGGACTCATCTTTGCCTATTGCGACACCTACTCAATGAACACCTACTACGCGCTGCAAAGAGCCCCGCAGGCCTGAGGTGCCCCCACTCACCACTCTTCCTATTTTTGTGTAAAAAATCCAGCTTCTTGTCACCACCTCCAAGGAGGGGGAGGAGGAGGAAGGCAGGTTCCTCTAGGCTGAGCCGAATGCCCCTCTGTGGTC CCACGCCACTGATCGCTGCATGCCCCACCACCTGGGGTACACACAGTCTGTGATTCCCGGAGCAGAACGGACCCTGCCCACCCGGTCTTGTGTGCTACTCAGTGGACAGACCCAAGGCAAGAAAGGGTGACAAGGACAGGGTCTTCCCAGGCTGGCTTTGAGTTCCTAGCACCGCCCCGCCCCCAATCCTCTGTGGCACATGGAGTCTTGGTCCCCAGAGTCC CCCAGCGGCCTCCAGATGGTCTGGGAGGGCAGTTCAGCTGTGGCTGCGCATAGCAGCAGACATACAACGGACGGTGGGCCCAGACCCAGGCTGTGTAGACCCAGCCCCCCCGCCCCGCAGTGCCTAGGTCACCCACTAACGCCCCAGGCCTGGTCTTGGCTGGGCGTGACTGTTACCCTCAAAAAGCAGGCAGCTCCAGGGTAAAAGGTGCCCTGCCCT GTAGAGCCCACCTTCCTTCCCAGGGCTGCGGCTGGGTAGGTTTGTAGCTTCATCACGGGCCACCTCCAGCCACTGGACCGCTGGCCCCTGCCCTGTCCTGGGGAGTGTGGTCCTGCGACTTCTAAGTGGCCGCAAGCCACCTGACTCCCCCAACACCACACTCTACCTTCAAAGCCCAGGTCTCTCCCTAGTGACCCACCCAGCACATTTAGCTAGCTGA GCCCCACAGCCAGAGGTCCTCAGGCCCTGCTTTCAGGGCAGTTGCTCTGAAGTCGGCAAGGGGGAGTGACTGCCTGGCCACTCCATGCCCTCCAAGAGCTCCTTCTGCAGGAGCGTACAGAACCCAGGGCCCTGGCACCCGTGCAGACCCTGGCCCACCCCACCTGGGCGCTCAGTGCCCAAGAGATGTCCAACACCTAGGATGTCCCGCGGTGGGTGG GGGGCCCGAGAGACGGGCAGGCCGGGGGCAGGCCTGGCCATGCGGGGCCGAACCGGGCACTGCCCAGCGTGGGGCGCGGGGGCCACGGCGCGCGCCCCCAGCCCCCGGGCCCAGCACCCCAAGGCGGCCAACGCCAAAACTTCCCTCCTCCTCTTCCTCAATCTCGCTCTTTTTTTTTTTCGCAAAAGGAGGGGAGAG GGGGTAAAAAAAATGCTGCACTGTGCGGCGAAGCCGGTGAGTGAGCGGCGCGGGGCCAATCAGCGTGCGCCGTTCCGAAAGTTGCCTTTTATGGCTCGAGCGGCCGCGGCGGCGCCCTATAAAACCCAGCGGCGCGACGCGCCACCACCGCCGAGACCGCGTCCGCCCCGCGAGCACAGAGCCTCGCCTTTGCCGATCCGCCGCCCGTCCACACCCG CCGCCAGgtaagcccggccagccgaccggggcaggcggctcacggcccggccgcaggcggccgcggccccttcgcccgtgcagagccgccgtctgggccgcagcggggggcgcatggggggggaaccggaccgccgtggggggcgcgggagaagcccctgggcctccggagatgggggacaccccacgccagtcggaggc gcgaggccgcgctcgggaggcgcgctccgggggtgccgctctcggggcgggggcaaccggcggggtctttgtctgagccggggctcttgccaatggggatcgcagggtgggcgcggcggagcccccgccaggcccggtgggggctggggcgccattgcgcgtgcgcggtggtcctttgggcgc taactgcgtgcgcgctgggaattggcgctaattgcgcgtgcgcgctgggactcaaggcgctaactgcgcgtgcgttctggggcccggggtgccgcggcctgggctggggcgaaggcgggctcggccggaaggggtggggtcgccgcggctcccgggcgcttgcgcgcacttcctgccc gagccgctggccgcccgagggtgtggccgctgcgtgcgcgcgcgccgacccggcgctgtttgaaccgggcggaggcggggctggcgcccggttgggaggggggttggggcctggcttcctgccgcgcgccgcggggacgcctccgaccagtgtttgccttttatggtaataacgcggccgg cccggcttcctttgtccccaatctgggcgcgcgccggcgccccctggcggcctaaggactcggcgcgccggaagtggccagggcgggggcgacctcggctcacagcgcgcccggctat (SEQ ID NO: 23) heIF4A1 GTTGATTTCCTTCATCCCTGGCACACGTCCAGGCAGTGTCGAATCCATCTCTGCTACAGGGGAAAACAAATAACATTTGAGTCCAGTGGAGACCGGGAGCAGAAGTAAAGGGAAGTGATAACCCCCAGAGCCCGGAAGCCCTTGGAGGCTGAGACCTCGCCCCCCTTGCGTGATAGGGCCTACGGAGCCACATGACCAAGGCACTGTCGCCTCCGC ACGTGTGAGAGTGCAGGGCCCCAAGATGGCTGCCAGGCCTCGAGGCCTGACTCTTCTATGTCACTTCCGTACCGGCGAGAAAGGCGGGCCCTCCAGCCAATGAGGCTGCGGGGCGGGCCTTCACCTTGATAGGCACTCGAGTTATTCCAATGGTGCCTGCGGGCCGGAGCGACTAGGAACTAACGTCATGCCGAGTTGCTGAGCGCCGGCAGGC GGGGCCGGGGCGGCCAAACCAATGCGATGGCCGGGGCGGAGTCGGGCGTCCTATAAGTTGTCGATAGGCGGGCACTCCGCCCTAGTTTCTAAGGACCATG (SEQ ID NO: 24) hGAPDH AGTTCCCCCAACTTTCCCGCCTCTCCAGCCTTTGAAAGAAAGAAAGGGGAGGGGGCAGGCCGCGTGCAGTCGCGAGCGGTGCTGGGCTCCGGCTCCAATTCCCCATCTCAGTCGCTCCCAAAGTCCCTTCTGTTTCATCCAAGCGTGTAAGGGTCCCTTGACTCCCTAGTGTCCTGCTGCCCACAGTCCAGTCCTGGGAACCAGCACCGATCACCT CCCATCGGGCCAATCTCAGTCCCTCCCCCCTACGTCGGGGCCCACACGCTCGGTGCGTGCCCAGTTGAACCAGGCGGCTGCGGAAAAAAAAAGCGGGGAGAAAGTAGGGCCCGGCTACTACGCGGTTTTACGGGCGCACGTAGCTCAGGCCTCAAGACCTGGGCTGGGACTGGCTGAGCCTGGCGGGAGGCGGGGTCCGAGTCACC GCCTGCCGCCGCGCCCCCGGTTTCTATAAATTGAGCCCGCAGCCTCCCGCTTCGCTCTCTGCTCCTCCTGTTCGACAGTCAGCCGCATCTTCTTTTGCGTCGCCAGgtgaagacgggcggagagaaacccgggaggctagggacggcctgaaggcggcaggggcgggcgcaggccggatgtgttcgcgccgctgcggggtggccccg ggcggcctccgcattgcaggggcgggcggaggacgtgatgcggcgcgggctgggcatggaggcctggtgggggaggggaggggaggcgtgggtgtcggccggggccactaggcgctcactgttctctccctccgcgcagCCGAGCCACATCGCTGAGACAC (SEQ ID NO: 25) hGRP78 AGTGCGGTTACCAGCGGAAATGCCTCGGGGTCAGAAGTCGCAGGAGAGATAGACAGCTGCTGAACCAATGGGACCAGCGGATGGGGCGGATGTTATTCTACCATTGGTGAACGTTAGAAACGAATAGCAGCCAATGAATCAGCTGGGGGGGCGGAGCAGTGACGTTTATTGCGGAGGGGGCCGCTTCGAATCGGCGGCG GCCAGCTTGGTGGCCTGGGCCAATGAACGGCCTCCAACGAGCAGGGCCTTCACCAATCGGCGGCCTCCACGACGGGGCTGGGGGAGGGTATATAAGCCGAGTAGGCGACGGTGAGGTCGACGCCGGCCAAGACAGCACAGAGATTGACCTATTGGGGTGTTTCGCGAGTGTGAGAGGGAAGCGCCGCGGCCTGTATTTCTAGACCT GCCCTTCGCCTGGTTCGTGGCGCCTTGTGACCCCGGGCCCCTGCCGCCTGCAAGTCGGAAATTGCGCTGTGCTCCTGTGCTACGGCCTGTGGCTGGACTGCCTGCTGCTGCCCAACTGGCTGGCAC (SEQ ID NO: 26) hGRP94 TAGTTTCATCACCACCCGCCACCCCCCCGCCCCCCCGCCATCTGAAAGGGTTCTAGGGGATTTGCAACCTCTCTCGTGTGTTTCTTCTTTCCGAGAAGCGCCGCCACACGAGAAAGCTGGCCGCGAAAGTCGTGCTGGAATCACTTCCAACGAAACCCCAGGCATAGATGGGAAAGGGTGAAGAACACGTTGCCATGGCTACCGTTTCCCCGGTC ACGGAATAAACGCTCTCTAGGATCCGGAAGTAGTTCCGCCGCGACCTCTCTAAAAGGATGGATGTGTTCTCTGCTTACATTCATTGGACGTTTCCCTTAGAGGCCAAGGCCGCCCAGGCAAAGGGGCGGTCCCACGCGTGAGGGGCCCGCGGAGCCATTTGATTGGAGAAAAGCTGCAAACCCTGACCAATCGGAAGGAGCCACGCTTCGGGCA TCGGTCACCGCACCTGGACAGCTCCGATTGGTGGACTTCCGCCCCCCCTCACGAATCCTCATTGGGTGCCGTGGGTGCGTGGTGCGGCGCGATTGGTGGGTTCATGTTTCCCGTCCCCCGCCCGAGAAGTGGGGGTGAAAAGCGGCCCGACCTGCTTGGGGTGTAGTGGGCGGACCGCGCGGCTGGAGGTGTGAGGATCCGAAC CCAGGGGTGGGGGGTGGAGGCGGCTCCTGCGATCGAAGGGGACTTGAGACTCACCGGCCGCACGTC (SEQ ID NO: 27) hHSP70 GGGCCGCCCACTCCCCTTCCTCTCTCAGGGTCCCCTGTCCCTCCAGTGAATCCCAGAAGACTCTGGAGAGTTCTGAGCAGGGGGCGGCACTCTGGCCTCTGATTGGTCCAAGGAAGGCTGGGGGGCAGGACGGGAGGCGAAAACCCTGGAATATTCCCGACCTGGCAGCCTCATCGAGCTCGGTGATTGGCTCAGAAGGGAAAAGGCGG GTCTCCGTGACGACTTATAAAAGCCCAGGGGCAAGCGGTCCGGATAACGGCTAGCCTGAGGAGCTGCTGCGACAGTCCACTACCTTTTTCGAGAGTGACTCCCGTTGTCCCAAGGCTTCCCAGAGCGAACCTGTGCGGCTGCAGGCACCGGCGCGTCGAGTTTCCGGCGTCCGGAAGGACCGAGCTCTTCTCGCGGATCCAGTGTTCCGTTCCAGCCCCCAAT CTCAGAGCGGAGCCGACAGAGAGCAGGGAACCC (SEQ ID NO: 28) wxya GCCCCACCCCCGTCCGCGTTACAACCGGGAGGCCCGCTGGGTCCTGCACCGTCACCCTCCTCCCTGTGACCGCCCACCTGATACCCAAACAACTTTCTCGCCCCTCCAGTCCCCAGCTCGCCGAGCGCTTGCGGGGAGCCACCCAGCCTCAGTTTCCCAGCCCCGGGCGGGCGAGGGGCGATGACGTCATGCCGGCGCGCGGCATTGTGG GGCGGGGCGAGGCGGGGCGCCGGGGGGAGCAACACTGAGACGCCATTTTCGGCGGCGGGAGCGGCGCAGGCGGCCGAGCGGGACTGGCTGGGTCGGCTGGGCTGCTGGTGCGAGGAGCCGCGGGGCTGTGCTCGGCGGCCAAGGGGACAGCGCGTGGGTGGCCGAGGATGCTGCGGGCGGTAGCTCC GGCGCCCCTCGCTGGTGACTGCTGCGCCGTGCCTCACACAGCCGAGGCGGGCTCGGCGCACAGTCGCTGCTCCGCGCTCGCGCCCGGCGGCGCTCCAGGTGCTGACAGCGCGAGAGAGCGCGGCCTCAGGAGCAACAC (SEQ ID NO: 29) wxya TTCCAGAGCTTTCGAGGAAGGTTTCTTCAACTCAAATTCATCCGCCTGATAATTTTCTTATATTTTCCTAAAGAAGGAAGAGAAGCGCATAGAGGAGAAGGGAAATAATTTTTTAGGAGCCTTTTCTTACGGCTATGAGGAATTTGGGGCTCAGTTGAAAAGCCTAAACTGCCTCTCGGGAGGTTGGGCGCGGCGAACTACTTTCAGCGGCGCACGGAGAC GGCGTCTACGTGAGGGGTGATAAGTGACGCAACACTCGTTGCATAAATTTGCGCTCCGCCAGCCCGGAGCATTTAGGGGCGGTTGGCTTTGTTGGGTGAGCTTGTTTGTGTCCCTGTGGGTGGACGTGGTTGGTGATTGGCAGGATCCTGGTATCCGCTAACAGgtactggcccacagccgtaaagacctgcgggggcgtgagaggg gggaatgggtgaggtcaagctggaggcttcttggggttgggtgggccgctgaggggaggggggggcgaggtgacgcgacacccggcctttctgggagagtggggccttgttgacctaaggggggcgagggcagttggcacgcgcacgcgccgacagaaactaacagacattaaccaacagcgattccgtcgcgt ttacttgggaggaaggcggaaaagaggtagtttgtgtggcttctggaaaccctaaatttggaatcccagtatgagaatggtgtcccttcttgtgtttcaatgggatttttacttcgcgagtcttgtgggtttggtttgttttcagtttgcctaacacccgtgcttaggtttgagg cagattggagttcggtcgggggagtttgaatatccggaacagttagtggggaaagctgtggacgcttggtaagagagcgctctggattttccgctgttgacgttgaaaccttgaatgacgaatttcgtattaagtgacttagccttgtaaaattgaggggaggcttgcggaatattaacgtatttaaggcatttt gaaggaatagttgctaattttgaagaatattagggtgtaaaagcaagaaatacaatgatcctgaggtgacacgcttatgttttacttttaaactagGTCACC (SEQ ID NO: 30) CAG gacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtattacgg taaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccct ccccaccccccaattttgtatttattttttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggc ggcggcggccctataaaaagcgaagcgcgcggcgggcg (SEQ ID NO: 173) HLP Tgtttgctgcttgcaatgtttgcccattttagggtggacacaggacgctgtggtttctgagccagggggcgactcagatcccagccagtggacttagcccctgtttgctcctccgataactggggtgaccttggttaatattcaccagcagcctcccccgttgcccctctggatccactgcttaaatacggacgagg acagggccctgtctcctcagcttcaggcaccaccactgacctgggacagtgaat (SEQ ID NO: 174)

[0122] The promoter may be a tissue-specific promoter. In general, a tissue-specific promoter directs the transcription of a nucleic acid (e.g., an engineered nucleic acid encoding a chimeric protein, such as a membrane-cleavable chimeric protein having the formula S-C-MT or MT-C-S) , allowing representation to be restricted to specific cell types, organelles, or tissues. Tissue-specific promoters include, but are not limited to, albumin (liver-specific, Pinkert et al., (1987)), lymphoid-specific promoters (Calame and Eaton, 1988), T-cell receptors (Winoto and Baltimore, (1989) ) and immunoglobulin (Banerji et al., (1983); Queen and Baltimore, 1983), neuron-specific promoters (such as the neurofilament promoter; Byrne and Ruddle, 1989), pancreas-specific promoters (Edlund et al., (1985)) or mammary gland-specific promoters (whey promoter, U.S. Patent No. 4,873,316 and European Application Publication No. 264,166) and developmentally regulated promoters such as the murine hox promoter ( Kessel and Gruss, Science 249:374-379 (1990)) or the alpha-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3:537-546 (1989)), the contents of each of these documents All are fully incorporated herein by reference. A promoter may be constitutive in each particular cell type, organelle or tissue. Tissue-specific promoters and / or regulatory elements may also include promoters from: liver fatty acid binding (FAB) protein gene specific for colonic epithelial cells; insulin gene specific for pancreatic cells; transthyroid specific for liver cells Transphyretin, .α1.-antitrypsin, plasminogen activator inhibitor type 1 (PAI-I), lipoprotein AI and LDL receptor genes; myelin specific for oligodendritic cells Basic protein (MBP) gene; glial fibrillary acidic protein (GFAP) gene specific for glial cells; OPSIN specific for targeted eye; and neural specific enolase (NSE) activation specific for nerve cells son. Examples of tissue-specific promoters include, but are not limited to, those for creatine kinase (which has been used to direct expression in muscle and heart tissue) and immunoglobulin heavy or light chains for expression in B cells. chain promoter. Other tissue-specific promoters include the human smooth muscle alpha-actin promoter. Exemplary tissue-specific expression elements for liver include, but are not limited to, HMG-COA reductase promoter, sterol regulatory element 1, phosphoenolpyruvate carboxykinase (PEPCK) promoter, human C-reactive protein (CRP) Promoter, human glucokinase promoter, cholesterol L 7-α hydroxylase (hydroylase) (CYP-7) promoter, β-galactosidase α-2,6 sialylkansferase (sialykansferase) promoter, insulin Like growth factor binding protein (IGFBP-I) promoter, aldolase B promoter, human transferrin promoter and type I collagen promoter. Exemplary tissue-specific expression elements for the prostate include, but are not limited to, the prostatic acid phosphatase (PAP) promoter, the prostate secretory protein 94 (PSP 94) promoter, the prostate-specific antigen complex promoter, and human glandular kallikrein Enzyme gene promoter (hgt-1). Exemplary tissue-specific expression elements for gastric tissue include, but are not limited to, the human H+ / K+-ATPase alpha subunit promoter. Exemplary tissue-specific expression elements for the pancreas include, but are not limited to, pancreatitis-associated protein promoter (PAP), elastase 1 transcriptional enhancer, pancreatic-specific amylase and elastase enhancer promoters, and pancreatic Cholesterol esterase gene promoter. Exemplary tissue-specific expression elements for use in the endometrium include, but are not limited to, the uteroglobin promoter. Exemplary tissue-specific expression elements for adrenal cells include, but are not limited to, the cholesterol side chain cleavage (SCC) promoter. Exemplary tissue-specific expression elements for the nervous system in general include, but are not limited to, the gamma-gamma enolase (neuron-specific enolase, NSE) promoter. Exemplary tissue-specific expression elements for the brain include, but are not limited to, the neurofilament heavy chain (NF-H) promoter. Exemplary tissue-specific expression elements for lymphocytes include, but are not limited to, the human CGL-1 / granzyme B promoter, terminal deoxytransferase (TdT), lambda 5, VpreB, and lck (lymphocyte-specific tyrosine protein kinase p561ck) promoter, human CD2 promoter and its 3' transcriptional enhancer, and 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. A tissue-specific expression element for breast cells is such as, but not limited to, the human alpha-lactalbumin promoter. Exemplary tissue-specific expression elements for lung include, but are not limited to, the cystic fibrosis transmembrane conductance regulator (CFTR) gene promoter.

[0123] In some embodiments, the promoters of the disclosure are regulated by signals within the tumor microenvironment. A tumor microenvironment regulates a promoter if the activity of the promoter in the presence of the tumor microenvironment is increased or decreased by at least 10% relative to the activity of the promoter in the absence of the tumor microenvironment. In some embodiments, the activity of the promoter is increased or decreased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% relative to the activity of the promoter in the absence of the tumor microenvironment , at least 80%, at least at least 90%, at least 100%. For example, the activity of the promoter is increased or decreased by 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, relative to the activity of the promoter in the absence of the tumor microenvironment. 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%.

[0124] In some embodiments, the activity of the promoter is increased or decreased by at least 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold) relative to the activity of the promoter in the absence of the tumor microenvironment , 15 times, 20 times, 25 times, 50 times or 100 times). For example, the activity of the promoter is increased or decreased by at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold relative to the activity of the promoter in the absence of the tumor microenvironment. In some embodiments, the activity of the promoter is increased or decreased by 2-10 times, 2-20 times, 2-30 times, 2-40 times, 2-50 times relative to the activity of the promoter in the absence of tumor microenvironment. times, 2-60 times, 2-70 times, 2-80 times, 2-90 times or 2-100 times.

[0125] In some embodiments, the promoters of the disclosure are activated under hypoxic conditions. A "hypoxic condition" is a condition in which the body or body region lacks an adequate supply of oxygen at the tissue level. Hypoxic conditions can lead to inflammation (eg, levels of inflammatory cytokines increase under hypoxic conditions). In some embodiments, a promoter active under hypoxic conditions is operably linked to nucleotides encoding a chimeric protein that reduces the expression of inflammatory cytokine activity, thereby reducing hypoxic-induced damage. inflamed. In some embodiments, the promoter active under hypoxic conditions comprises a hypoxia responsive element (HRE). A "hypoxia responsive element (HRE)" is a responsive element that responds to hypoxia inducible factor (HIF). In some embodiments, the HRE comprises the consensus motif NCGTG (where N is A or G). Activation Condition Controlled Polypeptide (ACP) Promoter System

[0126] In some embodiments, the synthetic promoter is a promoter system comprising an Activation Condition Controlled Polypeptide- (ACP-) binding domain sequence and a promoter sequence. This system is also referred to herein as an "ACP-responsive promoter". Generally, the ACP promoter system includes a first expression cassette encoding an activation condition control polypeptide (ACP) and a second expression cassette encoding an ACP-responsive promoter operably linked to an exogenous multikaryotic A nucleotide sequence, such as an exogenous polynucleotide sequence encoding a membrane cleavable chimeric protein described herein or any other protein of interest (eg, a protease). In some embodiments, the first expression cassette and the second expression cassette are each encoded by separate engineered nucleic acids. In other embodiments, the first expression cassette and the second expression cassette are encoded by the same engineered nucleic acid. The ACP-responsive promoter can be operably linked to a nucleotide sequence encoding a single protein of interest or multiple proteins of interest.

[0127] The promoter of the ACP promoter system (e.g., the promoter sequence driving the expression of ACP or the promoter sequence of the ACP-responsive promoter) can include any promoter sequence described herein (see "promoter" above) . ACP responsive promoters can be derived from minP, NFkB response element, CREB response element, NFAT response element, SRF response element 1, SRF response element 2, AP1 response element, TCF-LEF response element promoter fusion, hypoxia responsive element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducer molecule responsive promoter and its tandem repeats. In some embodiments, the ACP responsive promoter comprises a minimal promoter.

[0128] In some embodiments, the ACP binding domain includes one or more zinc finger binding sites. In some embodiments, the ACP responsive promoter comprises a minimal promoter and the ACP binding domain comprises one or more zinc finger binding sites. The ACP binding domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more zinc finger binding sites. In some embodiments, the transcription factor is a zinc finger-containing transcription factor. In some embodiments, the zinc finger-containing transcription factor is a synthetic transcription factor. In some embodiments, the ACP binding domain includes one or more zinc finger binding sites and the ACP has a DNA binding zinc finger protein domain (ZF protein domain). In some embodiments, the ACP has a DNA-binding zinc finger protein domain (ZF protein domain) and an effector domain. In some embodiments, the ACP binding domain includes one or more zinc finger binding sites and the ACP has a DNA binding zinc finger protein domain (ZF protein domain) and an effector domain. In some embodiments, ZF protein domains are modular in design and consist of zinc finger arrays (ZFAs). Zinc finger arrays contain 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 for any desired nucleic acid sequence, eg, a ZFA with desired specificity for an ACP binding domain with a particular zinc finger binding site composition and / or configuration. ZF motifs can be directly adjacent to each other, or separated by flexible linker sequences. In some embodiments, the ZFA is an array, string or chain of ZF motifs arranged in series. ZFA can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 zinc fingers body. ZFA can have 1-10, 1-15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1- 9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3- 5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4- 9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10 or 5-15 zinc finger phantoms. ZF protein domains can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more ZFAs. ZF domains can have 1-10, 1-15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10 or 5-15 ZFAs. In some embodiments, the ZF protein domain comprises 1 to 10 ZFAs. In some embodiments, the ZF protein domain comprises at least one ZFA. In some embodiments, the ZF protein domain comprises at least 2 ZFAs. In some embodiments, the ZF protein domain comprises at least 3 ZFAs. In some embodiments, the ZF protein domain comprises at least 4 ZFAs. In some embodiments, the ZF protein domain comprises at least 5 ZFAs. In some embodiments, the ZF protein domain comprises at least 10 ZFAs.

[0129] In some embodiments, the ACP is a transcriptional regulator. In some embodiments, ACP is a transcriptional repressor. In some embodiments, ACP is a transcriptional activator. In some embodiments, ACP is a transcription factor. In some embodiments, the ACP comprises a DNA binding domain and a transcriptional effector domain. In some embodiments, the DNA binding domain comprises a tetracycline (or derivative thereof) repressor (TetR) domain. In some embodiments, ACP is an antigen recognition receptor of the disclosure.

[0130] The ACP may also further comprise an effector domain, such as a transcriptional effector domain. For example, a transcriptional effector domain can be an effector or activator domain of a transcription factor. Transcription factor activation domains are also known as transactivation domains and serve as scaffolding domains for proteins such as transcriptional co-regulators for activating or repressing gene transcription. Any suitable transcriptional effector domain may be used in the ACP, including but not limited to the herpes simplex virus protein 16 (VP16) activation domain; the activation domain consisting of four tandem copies of VP16, the VP64 activation domain; the p65 of NFκB Activation domain; Estin-Barr virus R transactivator (Rta) activation domain; triple activator comprising VP64, p65 and Rta activation domain, called VPR activation domain; human E1A related The histone acetyltransferase (HAT) core domain of protein p300, known as the p300 HAT core activation domain; the Krüppel-associated box (KRAB) repression domain; the repressor element silencing transcription factor (REST) ​​repression domain; The WRPW motif of the shape-associated basic helix-loop-helix repressor protein, known as the WRPW repression domain; the DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and the HP1α chroma repression domain or any combination thereof.

[0131] In some embodiments, the effector domain is a transcriptional effector domain selected from: a herpes simplex virus protein 16 (VP16) activation domain; an activation domain consisting of four tandem copies of VP16, i.e. Activation domain of VP64; activation domain of p65 of NFκB; activation domain of R transactivator (Rta) of Estin-Barr virus; triple activator comprising VP64, p65 and Rta activation domain, the triple activator is called VPR activation domain; histone acetyltransferase (HAT) core domain of human E1A-associated protein p300, known as p300 HAT core activation domain; Krüppel-associated box (KRAB) repression domain; repressor element silences transcription factors (REST) ​​repression domain; the WRPW motif of the hairy-associated basic helix-loop-helix repressor protein, known as the WRPW repression domain; DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and HP1 alpha shading repression domain.

[0132] In some embodiments, the ACP is a small molecule (eg, drug) inducible polypeptide. For example, in some embodiments, ACP is inducible by tetracycline (or a derivative thereof) and comprises a TetR domain and a VP16 effector domain. In some embodiments, the ACP includes an estrogen receptor variant, such as ERT2, and can be produced by tamoxifen or its metabolites (such as 4-hydroxy-tamoxifen [4-OHT], N-desmethyltamoxifen moxifen, tamoxifen-N-oxide, or endoxifene), regulated by tamoxifen-controlled nuclear localization.

[0133] In some embodiments, the ACP is a small molecule (eg, drug) inducible polypeptide that includes a repressor protease and one or more cognate cleavage sites for the repressor protease. In some embodiments, the repressor protease is active (cleaves the cognate cleavage site) in the absence of the specified agent and is inactive (not cleaves the cognate cleavage site) in the presence of the specified agent. In some embodiments, the specific agent is a protease inhibitor. In some embodiments, a protease inhibitor specifically inhibits a given repressible protease of the disclosure. A repressor protease can be any protease described herein that is capable of being inactivated by the presence or absence of a particular agent (for exemplary repressor proteases, cognate cleavage sites, and protease inhibitors, see "Protease Cleavage Sites" above. ").

[0134] In some embodiments, the ACP has a degron domain (see "Degron Systems and Domains" above for exemplary degron sequences). The degron domains can be in any order or position relative to the individual domains of the ACP. For example, a degron domain can be N-terminal to a repressor protease, C-terminal to a repressor protease, N-terminal to a ZF protein domain, C-terminal to a ZF protein domain, N-terminal to an effector domain, or an effector domain. C-terminus of the object domain. Multi-cistronic and multi-promoter systems

[0135] In some embodiments, engineered nucleic acids (eg, engineered nucleic acids comprising expression cassettes) are configured to produce chimeric proteins. For example, nucleic acids can be configured to produce 2-20 different chimeric proteins. In some embodiments, the nucleic acid is configured to produce 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 Kinds, 3-20 Kinds, 3-19 Kinds, 3-18 Kinds, 3-17 Kinds, 3-16 Kinds, 3-15 Kinds, 3-14 Kinds, 3-13 Kinds, 3-12 Kinds, 3-11 Kinds Kinds, 3-10 Kinds, 3-9 Kinds, 3-8 Kinds, 3-7 Kinds, 3-6 Kinds, 3-5 Kinds, 3-4 Kinds, 4-20 Kinds, 4-19 Kinds, 4-18 Kinds kinds, 4-17 kinds, 4-16 kinds, 4-15 kinds, 4-14 kinds, 4-13 kinds, 4-12 kinds, 4-11 kinds, 4-10 kinds, 4-9 kinds, 4-8 kinds Kinds, 4-7 Kinds, 4-6 Kinds, 4-5 Kinds, 5-20 Kinds, 5-19 Kinds, 5-18 Kinds, 5-17 Kinds, 5-16 Kinds, 5-15 Kinds, 5-14 Kinds Kinds, 5-13 Kinds, 5-12 Kinds, 5-11 Kinds, 5-10 Kinds, 5-9 Kinds, 5-8 Kinds, 5-7 Kinds, 5-6 Kinds, 6-20 Kinds, 6-19 Kinds kinds, 6-18 kinds, 6-17 kinds, 6-16 kinds, 6-15 kinds, 6-14 kinds, 6-13 kinds, 6-12 kinds, 6-11 kinds, 6-10 kinds, 6-9 kinds kinds, 6-8 kinds, 6-7 kinds, 7-20 kinds, 7-19 kinds, 7-18 kinds, 7-17 kinds, 7-16 kinds, 7-15 kinds, 7-14 kinds, 7-13 kinds kinds, 7-12 kinds, 7-11 kinds, 7-10 kinds, 7-9 kinds, 7-8 kinds, 8-20 kinds, 8-19 kinds, 8-18 kinds, 8-17 kinds, 8-16 kinds kinds, 8-15 kinds, 8-14 kinds, 8-13 kinds, 8-12 kinds, 8-11 kinds, 8-10 kinds, 8-9 kinds, 9-20 kinds, 9-19 kinds, 9-18 kinds species, 9-17 species, 9-16 species, 9-15 species, 9-14 species, 9-13 species, 9-12 species, 9-11 species, 9-10 species, 10-20 species, 10-19 species kinds, 10-18 kinds, 10-17 kinds, 10-16 kinds, 10-15 kinds, 10-14 kinds, 10-13 kinds, 10-12 kinds, 10-11 kinds, 11-20 kinds, 11-19 kinds kinds, 11-18 kinds, 11-17 kinds, 11-16 kinds, 11-15 kinds, 11-14 kinds, 11-13 kinds, 11-12 kinds, 12-20 kinds, 12-19 kinds, 12-18 kinds kinds, 12-17 kinds, 12-16 kinds, 12-15 kinds, 12-14 kinds, 12-13 kinds, 13-20 kinds, 13-19 kinds, 13-18 kinds, 13-17 kinds, 13-16 kinds species, 13-15 species, 13-14 species, 14-20 species, 14-19 species, 14-18 species, 14-17 species, 14-16 species, 14-15 species, 15-20 species, 15-19 species species, 15-18 species, 15-17 species, 15-16 species, 16-20 species, 16-19 species, 16-18 species, 16-17 species, 17-20 species, 17-19 species, 17-18 species species, 18-20 species, 18-19 species or 19-20 chimeric proteins. In some embodiments, the nucleic acid is configured to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19 or 20 chimeric proteins.

[0136] In some embodiments, the engineered nucleic acid can be polycistronic, that is, more than one individual polypeptide can be produced from a single mRNA transcript (eg, multiple chimeric proteins). The engineered nucleic acid can be polycistronic through the use of various linkers, for example, a polynucleotide sequence encoding a first chimeric protein can be linked to a nucleotide sequence encoding a second chimeric protein, such as in One gene:linker:second gene is ligated in 5' to 3' orientation. The linker may encode a 2A ribosomal skipping element, such as T2A. Other 2A ribosome skipping elements include, but are not limited to, E2A, P2A, and F2A. The 2A ribosome skipping element allows for the production of individual polypeptides encoded by the first and second genes during translation. The linker may encode a cleavable linker polypeptide sequence, such as a furin cleavage site or a TEV cleavage site, wherein upon expression, the cleavable linker polypeptide is cleaved such that separate polypeptides encoded by the first and second genes are produced. A cleavable linker can include a polypeptide sequence that further facilitates cleavage, such as the flexible linker (eg, a Gly-Ser-Gly sequence).

[0137] The linker may encode an internal ribosome entry site (IRES) that allows the production of separate polypeptides encoded by the first and second genes during translation. A linker may encode a splice acceptor, such as a viral splice acceptor.

[0138] A linker can be a combination of linkers, such as a furin-2A linker, which can generate a single polypeptide by means of 2A ribosomal hopping followed by further cleavage of the furin site to allow complete removal of the 2A residues. In some embodiments, a combination of linkers can include a furin sequence, a flexible linker, and a 2A linker. Thus, in some embodiments, the linker is a Furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, the linkage system of the present disclosure is a Furin-Gly-Ser-Gly-T2A fusion polypeptide.

[0139] In general, polycistronic systems can use any number or combination of linkers to express any number of genes or portions thereof (e.g., engineered nucleic acids can encode first, second, and third chimeric proteins, each separated by a linker, such that separate polypeptides encoded by the first, second and third chimeric proteins are produced).

[0140] The engineered nucleic acid can express genes from multiple ORFs using multiple promoters, ie, more than one individual mRNA transcript can be produced from a single engineered nucleic acid. For example, a first promoter can be operably linked to a polynucleotide sequence encoding a first chimeric protein, and a second promoter can be operably linked to a polynucleotide sequence encoding a second chimeric protein. In general, any number of promoters can be used to express any number of chimeric proteins. In some embodiments, at least one of the ORFs expressed from multiple promoters can be polycistronic.

[0141] A "linker" as used herein may refer to a polypeptide linking the first polypeptide sequence and the second polypeptide sequence, the polycistronic linker described above, or an additional promoter operably linked to the additional ORF described above. engineered cells

[0142] Provided herein are engineered cells, and methods of producing engineered cells that produce membrane-cleavable chimeric proteins. In general, engineered cells of the disclosure can be engineered to express a chimeric protein provided herein, such as a membrane cleavable chimeric protein having the formula S-C-MT or MT-C-S described herein protein. These cells are referred to herein as "engineered cells". These cells, which normally contain the engineered nucleic acid, do not occur in nature. In some embodiments, the cell is engineered to include a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding a chimeric protein (eg, a membrane cleavable chimeric protein). An engineered cell can comprise the engineered nucleic acid integrated into the genome of the cell. Engineered cells may comprise engineered nucleic acids capable of expression without integration into the cellular genome (eg, engineered with a transient expression system such as plastids or mRNA).

[0143] The present disclosure also contemplates additive and synergistic interactions between one or more chimeric proteins and the engineered cells that produce them. In some embodiments, cells are engineered to produce at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) embedded A fusion protein, such as at least two membrane cleavable chimeric proteins. In other embodiments, the cell is engineered to produce at least one chimeric protein with an effector molecule that is not naturally produced by the cell. The effector molecule may, for example, complement the function of an effector molecule naturally produced by the cell.

[0144] In some embodiments, cells are engineered to express membrane-tethered anti-CD3 and / or anti-CD28 agonist extracellular domains.

[0145] In some embodiments, cells (eg, immune cells or stem cells) are engineered to produce chimeric proteins. For example, cells can be engineered to produce 2-20 different chimeric proteins, such as 2-20 different membrane-cleavable chimeric proteins. In some embodiments, the cells are engineered to produce 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 Kinds, 3-20 Kinds, 3-19 Kinds, 3-18 Kinds, 3-17 Kinds, 3-16 Kinds, 3-15 Kinds, 3-14 Kinds, 3-13 Kinds, 3-12 Kinds, 3-11 Kinds Kinds, 3-10 Kinds, 3-9 Kinds, 3-8 Kinds, 3-7 Kinds, 3-6 Kinds, 3-5 Kinds, 3-4 Kinds, 4-20 Kinds, 4-19 Kinds, 4-18 Kinds kinds, 4-17 kinds, 4-16 kinds, 4-15 kinds, 4-14 kinds, 4-13 kinds, 4-12 kinds, 4-11 kinds, 4-10 kinds, 4-9 kinds, 4-8 kinds Kinds, 4-7 Kinds, 4-6 Kinds, 4-5 Kinds, 5-20 Kinds, 5-19 Kinds, 5-18 Kinds, 5-17 Kinds, 5-16 Kinds, 5-15 Kinds, 5-14 Kinds Kinds, 5-13 Kinds, 5-12 Kinds, 5-11 Kinds, 5-10 Kinds, 5-9 Kinds, 5-8 Kinds, 5-7 Kinds, 5-6 Kinds, 6-20 Kinds, 6-19 Kinds kinds, 6-18 kinds, 6-17 kinds, 6-16 kinds, 6-15 kinds, 6-14 kinds, 6-13 kinds, 6-12 kinds, 6-11 kinds, 6-10 kinds, 6-9 kinds kinds, 6-8 kinds, 6-7 kinds, 7-20 kinds, 7-19 kinds, 7-18 kinds, 7-17 kinds, 7-16 kinds, 7-15 kinds, 7-14 kinds, 7-13 kinds kinds, 7-12 kinds, 7-11 kinds, 7-10 kinds, 7-9 kinds, 7-8 kinds, 8-20 kinds, 8-19 kinds, 8-18 kinds, 8-17 kinds, 8-16 kinds kinds, 8-15 kinds, 8-14 kinds, 8-13 kinds, 8-12 kinds, 8-11 kinds, 8-10 kinds, 8-9 kinds, 9-20 kinds, 9-19 kinds, 9-18 kinds species, 9-17 species, 9-16 species, 9-15 species, 9-14 species, 9-13 species, 9-12 species, 9-11 species, 9-10 species, 10-20 species, 10-19 species kinds, 10-18 kinds, 10-17 kinds, 10-16 kinds, 10-15 kinds, 10-14 kinds, 10-13 kinds, 10-12 kinds, 10-11 kinds, 11-20 kinds, 11-19 kinds kinds, 11-18 kinds, 11-17 kinds, 11-16 kinds, 11-15 kinds, 11-14 kinds, 11-13 kinds, 11-12 kinds, 12-20 kinds, 12-19 kinds, 12-18 kinds kinds, 12-17 kinds, 12-16 kinds, 12-15 kinds, 12-14 kinds, 12-13 kinds, 13-20 kinds, 13-19 kinds, 13-18 kinds, 13-17 kinds, 13-16 kinds species, 13-15 species, 13-14 species, 14-20 species, 14-19 species, 14-18 species, 14-17 species, 14-16 species, 14-15 species, 15-20 species, 15-19 species species, 15-18 species, 15-17 species, 15-16 species, 16-20 species, 16-19 species, 16-18 species, 16-17 species, 17-20 species, 17-19 species, 17-18 species species, 18-20 species, 18-19 species or 19-20 chimeric proteins. In some embodiments, the cells are engineered to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19 or 20 chimeric proteins.

[0146] In some embodiments, the engineered cell comprises one or more engineered nucleic acids encoding a promoter operably linked to a nucleotide sequence encoding a chimeric protein. In some embodiments, the cell is engineered to include a plurality of engineered nucleic acids, e.g., at least two engineered nucleic acids, each encoding a protein operably linked to at least one (e.g., 1, 2) or 3) the promoter of the nucleotide sequence of the chimeric protein. For example, cells can be engineered to contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 The engineered nucleic acids each encode a promoter operably linked to a nucleotide sequence encoding at least one (eg, 1, 2, or 3) chimeric proteins. In some embodiments, the cell is engineered to contain 2, 3, 4, 5, 6, 7, 8, 9, 10 or more engineered nucleic acids, each encoding A promoter operably linked to a nucleotide sequence encoding at least one (eg, 1, 2, or 3) chimeric proteins. The engineered cell may comprise an engineered nucleic acid encoding at least one of the aforementioned linkers, such as a polypeptide linking the first and second polypeptide sequences, one or more of the aforementioned polycistronic A linker, one or more additional promoters operably linked to the additional ORF, or a combination thereof.

[0147] In some embodiments, cells (eg, immune cells or stem cells) are engineered to express proteases. In some embodiments, the cell is engineered to express a protease that is heterologous to the cell. In some embodiments, the cell is engineered to express a protease that is heterologous to the cell expressing the chimeric protein, such as a heterologous protease that cleaves a protease cleavage site of a membrane-cleavable chimeric protein. In some embodiments, the engineered cell comprises one or more engineered nucleic acids encoding a promoter operably linked to a nucleotide sequence encoding a protease, such as a heterologous protease. Proteases and protease cleavage sites are described in more detail herein in the section entitled "Protease Cleavage Sites".

[0148] In some embodiments, cells (eg, immune cells or stem cells) are engineered to produce at least one homing molecule. "Homing" refers to the active navigation (migration) of cells to a target site (eg, cell, tissue (eg, tumor) or organ). "Homing molecule" refers to a molecule that directs a cell to a target site. In some embodiments, the function of the homing molecule is to recognize and / or initiate the interaction of the engineered cell with the target site. Non-limiting examples of homing molecules include CXCR1, CCR9, CXCR2, CXCR3, CXCR4, CCR2, CCR4, FPR2, VEGFR, IL6R, CXCR1, CSCR7, and PDGFR.

[0149] 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 can be produced by engineered cells of the disclosure include: CXC chemokine receptors (e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7), CC chemokine receptors (CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, and CCR11), CX3C chemokine receptors (e.g., CX3CR1, which binds to CX3CL1), and XC Chemokine receptors (eg, 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 (promote T cell recruitment), CCL3 and / or CXCL5, CCL21 (Th1 recruitment and polarization). In some embodiments, the cells are engineered to produce CXCR4.

[0150] 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).

[0151] In some embodiments, cells (eg, immune cells or stem cells) are engineered to produce receptors that detect interleukins, including but not limited to IL6R.

[0152] 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 Receptors of the VEGF family, including but not limited to VEGF-C and VEGF-D).

[0153] In some embodiments, cells (eg, immune cells or stem cells) are engineered to produce one or more integrins. Cells of the disclosure can be engineered to produce any combination of integrin alpha and beta subunits. The alpha subunit of an integrin can be, but is not limited to: ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, IGTA7, ITGA8, ITGA9, IGTA10, IGTA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGA2B, ITGAX. The β subunit of integrin can be, but not limited to: ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7 and ITGB8.

[0154] In some embodiments, cells (eg, 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 MMPs. In some embodiments, cells are engineered to produce inhibitors of molecules (eg, proteins) that inhibit MMPs. For example, cells can be engineered to express an inhibitor of membrane type 1 MMP (MT1-MMP) (eg, an RNAi molecule) or TIMP metallopeptidase inhibitor 1 (TIMP-1).

[0155] In some embodiments, cells (e.g., immune cells or stem cells) are engineered to produce ligands (e.g., hematopoietic E- / L-selectin Ligands (HCELL), Dykstran et al., Stem Cells. 2016 Oct;34(10):2501-2511).

[0156] The term "homing molecule" also encompasses transcription factors that regulate the production of molecules that improve / enhance cell homing.

[0157] Also provided herein are engineered cells engineered to produce chimeric proteins, at least two of which include effector molecules that modulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or more) of the chimeric proteins includes stimulating at least one immunostimulatory mechanism in the tumor microenvironment or suppressing tumor Effector molecules of at least one immunosuppressive mechanism in the microenvironment. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or more) chimeric protein includes an effector molecule that inhibits at least one immunosuppressive mechanism in the tumor microenvironment , and at least one chimeric protein (eg, 1, 2, 3, 4, 5 or more) inhibits at least one immunosuppressive mechanism in the tumor microenvironment. In other embodiments, at least two (eg, 2, 3, 4, 5 or more) chimeric proteins include effector molecules that stimulate at least one immunostimulatory mechanism in the tumor microenvironment. In other embodiments, at least two (e.g., 1, 2, 3, 4, 5, or more) chimeric proteins include effector molecules that inhibit at least one immunosuppressive mechanism in the tumor microenvironment .

[0158] In some embodiments, cells (eg, immune cells or stem cells) are engineered to produce at least one chimeric protein comprising an effector molecule that stimulates T cell signaling, activity and / or recruitment. In some embodiments, cells are engineered to produce at least one chimeric protein comprising an effector molecule that stimulates antigen presentation and / or processing. In some embodiments, the cells are engineered to produce at least one chimeric protein comprising an effector molecule that stimulates natural killer cell-mediated cytotoxic signaling, activity and / or recruitment. In some embodiments, the cells are engineered to produce at least one chimeric protein comprising an effector molecule that stimulates dendritic cell differentiation and / or maturation. In some embodiments, the cells are engineered to produce at least one chimeric protein comprising an effector molecule that stimulates immune cell recruitment. In some embodiments, the cells are engineered to produce at least one chimeric protein comprising an effector molecule that stimulates M1 macrophage signaling, activity and / or recruitment. In some embodiments, the cell is engineered to produce at least one chimeric protein comprising an effector molecule that stimulates Th1 polarization. In some embodiments, cells are engineered to produce at least one chimeric protein comprising an effector molecule that stimulates matrix degradation. In some embodiments, the cells are engineered to produce at least one chimeric protein comprising an effector molecule that stimulates the production of an immunostimulatory metabolite. In some embodiments, cells are engineered to produce at least one chimeric protein comprising an effector molecule that stimulates type I interferon signaling. In some embodiments, cells are engineered to produce at least one chimeric protein comprising an effector molecule that inhibits negative co-stimulatory signaling. In some embodiments, cells are engineered to produce at least one chimeric protein comprising an effector molecule that inhibits pro-apoptotic signaling (eg, via TRAIL) of anti-tumor immune cells. In some embodiments, cells are engineered to produce at least one chimeric protein comprising an effector molecule that inhibits T regulatory (Treg) cell signaling, activity and / or recruitment. In some embodiments, the cells are engineered to produce at least one chimeric protein comprising an effector molecule that inhibits a tumor checkpoint molecule. In some embodiments, cells are engineered to produce at least one chimeric protein comprising an effector molecule that activates Stimulator of Interferon Genes (STING) signaling. In some embodiments, the cells are engineered to produce at least one chimeric protein comprising an effector molecule that inhibits myeloid-derived suppressor cell signaling, activity and / or recruitment. In some embodiments, cells are engineered to produce at least one chimeric protein that includes an effector molecule that degrades an immunosuppressive factor / metabolite. In some embodiments, the cells are engineered to produce at least one chimeric protein comprising an effector molecule that inhibits vascular endothelial growth factor signaling. In some embodiments, cells are engineered to produce at least one chimeric protein comprising effector molecules that directly kill tumor cells (e.g., granzymes, perforins, oncolytic viruses, cytolytic peptides and enzymes, Anti-tumor antibodies that e.g. trigger ADCC).

[0159] In some embodiments, at least one chimeric protein comprising an effector molecule that stimulates T cell signaling, activity and / or recruitment, stimulates antigen presentation and / or processing, stimulates natural killer cell-mediated Cytotoxic signaling, activation and / or recruitment, stimulation of dendritic cell differentiation and / or maturation, stimulation of immune cell recruitment, stimulation of macrophage signaling, stimulation of matrix degradation, stimulation of production of immunostimulatory metabolites, or stimulation of type I interferon signaling; and at least one chimeric protein comprising an effector molecule that inhibits negative co-stimulatory signaling, inhibits pro-apoptotic signaling of anti-tumor immune cells, inhibits T regulatory (Treg) cell signaling, activity and / or recruitment, inhibits Tumor checkpoint molecules, activate Stimulator of Interferon Genes (STING) signaling, inhibit myeloid-derived suppressor cell signaling, activity and / or recruitment, degrade immunosuppressive factors / metabolites, inhibit vascular endothelial growth factor signaling, or directly kill tumors cell.

[0160] In some embodiments, cells (e.g., immune cells or stem cells) are engineered to produce at least one chimeric protein comprising an effector molecule selected from the group consisting of: 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. Illustrative immune checkpoint molecules that can be targeted for block 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 (belongs to the CD2 family of molecules and is expressed on all NK, γδT, and memory CD8+ (αβ) T cells), CD160 (also known as BY55 ) and CGEN-15049. Immune checkpoint inhibitors include antibodies or antigen-binding fragments thereof or other binding proteins that bind to and block or inhibit the activity of 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. 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 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. Illustrative 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), BMS -936559 (anti-PD-L1 - BMS), tremelimumab (anti-CTLA-4; Medimmune / AstraZeneca), ipilimumab (anti-CTLA-4; Yervoy® - BMS), Leary Lirilumab (anti-KIR; BMS), monalizumab (anti-NKG2A; Innate Pharma / AstraZeneca).

[0161] In some embodiments, cells (e.g., immune cells or stem cells) are engineered to produce at least one chimeric protein comprising an effector molecule selected from the group consisting of: 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, the chimeric protein comprising MIP1α (CCL3), MIP1β (CCL5 ) and an effector molecule of CCL21; and / or at least one chimeric protein comprising an effector molecule selected from CpG oligodeoxynucleotides; and / or at least one chimeric protein comprising Effector molecules selected from microbial peptides.

[0162] In some embodiments, cells (e.g., immune cells or stem cells) are engineered to produce IFN-β and at least one chimeric protein comprising an effector molecule selected from the group consisting of: interleukins, Antibodies, Chemoki...

Claims

1. A membrane-cleavable chimeric protein, oriented from N-terminus to C-terminus, having the following formula: S-C-MT or MT-C-S, wherein S comprises a secretible effector molecule, C comprises a protease cleavage site, and MT comprises a cell membrane chain domain, wherein S-C-MT or MT-C-S is conformated to represent a single polypeptide, and wherein the protease cleavage site comprises an amino acid sequence of PRAEYSKGG (SEQ ID NO: 181); or an amino acid sequence of PRAEPIKGG (SEQ ID NO: 182); or an amino acid sequence of PRAEAYKGG (SEQ ID NO: 183); or an amino acid sequence of PRAESSKGG (SEQ ID NO: 184); or an amino acid sequence of PRAEFTKGG (SEQ ID NO: 185); or an amino acid sequence of DEPHYSQRR (SEQ ID NO: 187); or an amino acid sequence of PPLGPIFNPG (SEQ ID NO: 187). The amino acid sequence of 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).

2. The membrane-cleavable chimeric protein of claim 1, wherein the secretible effector molecule comprises a signal peptide or a signal anchor sequence, wherein the signal peptide comprises a natural signal peptide native to the secretible effector molecule, or the signal peptide comprises a non-natural signal peptide, or the signal anchor sequence comprises a non-natural signal anchor sequence of the secretible effector molecule.

3. The membrane-cleavable chimeric protein of claim 2, 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 luciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, serum albumin precursor, azuril precursor, osteobindingin, CD33, IL-6, IL-8, CCL2, TIMP2, VEGFB, osteoprotegerin, serine protease inhibitor E1, GROα, CXCL12, IL-21, CD8, NKG2D, TNFR2, and GMCSF.

4. The membrane-cleavable chimeric protein of claim 1, wherein the secretible effector molecule is selected from the therapeutic class, wherein the therapeutic class is selected from the group consisting of: cytokines, chemokines, homing molecules, growth factors, coactivators, tumor microenvironment modulators, ligands, antibodies, peptides and enzymes.

5. If the membrane in request item 4 can cleave the chimeric protein, wherein: a. The cytokine line was selected from the following groups: IL-1-β, 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-γ, and TNF-α; b. The chemokine line was selected from the following groups: CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1; c. The homing molecule line was selected from the following groups: anti-integrin α4, β7; anti-MAdCAM; SDF1; and MMP-2; d. The growth factor line was selected from the following groups: FLT3L and GM-CSF; e. The co-activating molecule line was selected from the following groups: 4-1BBL and CD40L. f. The tumor microenvironment modifier is selected from the group consisting of: adenosine deaminase, TGFβ inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2; g. The TGFβ inhibitor is selected from the group consisting of: anti-TGFβ peptides, anti-TGFβ antibodies, TGFβ-TRAP, and combinations thereof; h. The immune checkpoint inhibitor is selected from the group consisting of: 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; or i. The VEGF inhibitor comprises anti-VEGF antibodies, anti-VEGF peptides, or combinations thereof.

6. The membrane-cleavable chimeric protein of claim 1, wherein the secretible effector molecule comprises an IL-15, IL-12, or IL-12p70 fusion protein.

7. The membrane of claim 1 is capable of cleaving chimeric proteins, wherein the protease cleavage site contains the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191).

8. The membrane-cleavable chimeric protein of claim 1, wherein the cell membrane chain domain comprises a transmembrane-intracellular domain or a transmembrane domain.

9. The membrane-cleavable chimeric protein of claim 8, wherein the transmembrane-intracellular domain and / or transmembrane domain is derived from PDGFR-β, CD8, CD28, CD3ζ chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, or BTLA, wherein the cell membrane chain domain comprises a cell surface receptor or its cell membrane binding portion.

10. The membrane-cleavable chimeric protein of claim 1, wherein the cell membrane chain domain includes a post-translational modification tag, or is capable of post-translational modification to modify the chimeric protein to include a motif of the post-translational modification tag, wherein the post-translational modification tag is capable of associating with the cell membrane, wherein the post-translational modification tag includes a lipid anchoring domain, wherein the lipid anchoring domain is selected from the group consisting of: GPI lipid anchors, myristylation tags, and palmitylation tags.

11. If the membrane of claim 1 is capable of cleaving chimeric proteins, wherein: a. When expressed in a cell, the secretible effector molecule is chained to the cell membrane of that cell; and / or b. When expressed in a cell that expresses a protease capable of cleaving the protease's cleavage site, the secretible effector molecule is released from the cell membrane; and / or c. wherein the protease expressed on the cell membrane is endogenous to that cell; and / or d. the protease is the ADAM17 protease.

12. An engineered nucleic acid comprising a cassette including a promoter and an exogenous polynucleotide sequence encoding a membrane-cleavable chimeric protein as claimed in any of claims 1 to 11, wherein the promoter is selected from the group consisting of constitutive promoters, inducible promoters, tissue-specific promoters, and synthetic promoters.

13. A representation carrier comprising an engineered nucleic acid as claimed in claim 12.

14. An isolated cell comprising a membrane-cleavable chimeric protein as claimed in any one of claims 1 to 11, an engineered nucleic acid as claimed in claim 12, or an expression vector as claimed in claim 13.

15. The isolated cells as requested in item 14, wherein the cell line is selected from the following groups: T cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), congenital 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.

16. The isolated cells as requested in item 15, wherein the T cell line is selected from the following groups: CD8+ T cells, CD4+ T cells, γ-δ T cells, virus-specific T cells, and natural killer T (NKT) cells.

17. The isolated cells as claimed in claim 15, wherein the T cells are regulatory T cells.

18. The isolated cell of any one of claims 14 to 17, wherein the cell further comprises a protease capable of cleaving the protease cleavage site, wherein the protease is ADAM17 protease.

19. The isolated cells of any one of claims 14 to 17, wherein the cells further comprise an antigen recognition receptor, wherein the antigen recognition receptor is a CAR system.

20. A pharmaceutical composition comprising a membrane-cleavable chimeric protein as claimed in any one of claims 1 to 11, an engineered nucleic acid as claimed in claim 12, an expression vector as claimed in claim 13, or a cell-isolated and pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or combination thereof as claimed in any one of claims 14 to 19.

21. Use of any of claims 14 to 19, or of any of claims 20, of an isolated cell or a pharmaceutical composition, for the preparation of a medicament for treating cancer.

22. A method for inducing the release of membrane chain effector molecules, comprising: a) Provide cells as requested in any of items 14 to 19; and b) the cell is cultured under conditions suitable for expressing the membrane-bound protease and the membrane-cleavable chimeric protein, wherein, after expression, the membrane-cleavable chimeric protein chain is extended to the cell membrane of the cell, and wherein, after expression, the membrane-bound protease cleaves the homologous membrane-bound protease cleavage site of the membrane-cleavable chimeric protein, thereby releasing the secretible effector molecule from the cell membrane.

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