Plasma membrane embedding system for spatiotemporal controlled load delivery of biological articles

Abstract

A peptide delivery system for delivering a peptide load to a target cell or a target site and a method of delivering a peptide load to a target site are disclosed. The peptide delivery system may include cells, extracellular vesicles, or lipid-containing particles in which an exogenous polypeptide has been anchored. The exogenous polypeptide has a first transmembrane domain and a second transmembrane domain linked by a loop region, wherein the loop region comprises a first cleavage site, a peptide load, and a second cleavage site; and optionally, a targeting moiety for targeting the peptide delivery system to the target cell or the target site.

Description

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 63 / 467,518, filed May 18, 2023, under 35 USC § 119(e), the entire contents of which are expressly incorporated herein by reference.

[0002] Incorporation declarations regarding the reference to sequence lists This application includes a sequence list, which has been submitted electronically in XML file format, the entire contents of which are incorporated herein by reference. The XML copy was created on May 16, 2024, and is named “P71267_SL.xml”, with a size of 451,938 bytes. Background Technology

[0003] The use of peptide-based therapies to treat living organisms is on the rise. These therapies include hormones, cytokines, chemokines, prodrugs, and various forms of antibodies, such as chimeric antigen receptors (CARs), bispecific killer cell binders (BIKEs), and trispecific killer cell binders (TRIKEs). Any peptide or protein that functions in vivo is a potential therapeutic agent. Despite the promising therapeutic potential of peptides and proteins, several factors limit their bioavailability, thereby affecting their function in vivo.

[0004] One limiting factor is the in vitro production, stability, and multiple-dose delivery of these biological products. These biologics are typically produced in mammalian cells, bacteria, or, more recently, single-celled eukaryotes (such as Pichia pastoris). Pichia pastoris Synthesized in [cell name missing]. Biologics are purified from producing cells, prepared in a delivery system, and administered to patients. Therapeutic levels typically require continuous delivery and / or repeated peptide administration.

[0005] Systemic delivery of biologics can further reduce efficacy, depending on the drug's pharmacokinetic and pharmacodynamic properties, because only a very small percentage of the administered peptides reach the target site. Most biologics may be degraded and metabolized before reaching their intended site of action.

[0006] The third limiting factor is the biotoxicity associated with the mechanism of action (MOA) of these biological products.

[0007] There is an urgent need in this field for improved protein and peptide delivery methods to enhance the safety and efficacy of biopharmaceuticals. Summary of the Invention

[0008] This invention discloses a peptide delivery system for delivering a peptide payload to a target cell or target locus. The peptide delivery system comprises: a cell, extracellular vesicle, or lipid-containing particle in which an exogenous peptide is anchored, the exogenous peptide comprising: a first transmembrane domain and a second transmembrane domain connected by a loop region, wherein the loop region comprises a first cleavage site, a peptide payload, and a second cleavage site; and optionally, a targeting portion for targeting the peptide delivery system to the target cell or the target locus.

[0009] In such peptide delivery systems, the first cleavage site and / or the second cleavage site may contain a proteolytic cleavage substrate, preferably a sheddase cleavage site, an MMP cleavage site, a GrzB cleavage site, or a Casp3 cleavage site.

[0010] Such peptide delivery systems can be configured to release the peptide load under the action of a protease (preferably an exfoliase), which can be endogenous or exogenous (e.g., included as a component of the peptide delivery system).

[0011] Also considered is a peptide delivery system in which the exfoliase is present at the target site and / or released by the target cell.

[0012] In embodiments of the above-described peptide delivery system, the exfoliase may comprise a dedicated exfoliase selected from the following: ADAM protease (metalloproteinase), BACE protease, serine protease granzyme-B, and Site-1 protease. The ADAM protease may include membrane-anchored type 1 proteases, such as ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, ADAMthr20, ADAM21, ADAM28, ADAM30, and ADAM33.

[0013] This paper also considers peptide delivery systems in which the exfoliase is a part-time exfoliase selected from transmembrane peptidase B (Meprin B), MT-MMP (membrane matrix metalloproteinase), proprotein convertase, transmembrane serine protease, matrix metalloproteinase, podocyte protein, cathepsin S, and cathepsin L. The MT-MMP can be membrane-anchored type 1 or GPI-anchored, and can include MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, and MT6-MMP. The proprotein convertase can be selected from PCSK1 / 3, KCSK2, furin, PCSK4, PSCK5 / 6, PACE4, PCSK7, and PCSK9. The transmembrane serine protease may be selected from membrane-anchored type II proteases, including Matriptase, Matriptase-2, Matriptase-3, polymerase-1, Corin, Hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, Spinesin, intestinal peptidase, HAT, DESCL1, TMPRSS11A, HAT-like 4, and HAT-like 5. The matrix metalloproteinase may be selected from soluble proteases, including MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A-B, MMP24, MMP25, MMP26, MMP27, and MMP28. In one embodiment, the matrix metalloproteinase is selected from MMP2, MMP9, and / or MMP25. In one embodiment, MMP2 may be selected from SEQ ID NOS: 2-6. In one embodiment, MMP9 may be selected from SEQ ID NOS: 7-8. In one embodiment, MMP25 may be selected from SEQ ID NOS: 9-17, 18, or 155 and 19-29.

[0014] This article also discloses a peptide delivery system in which the transmembrane domain comprises one or more transmembrane domains from one of the following proteins: CD63, CD9, CD81, CD28, CD4, CD8, CD34, CD69, CD19, CD20, integrin, TNFSF14, TNR1A, aquaporin, NOTCH, NgR1, NRG1, GPi anchor, EGFR, or rhodopsin.

[0015] In some embodiments, the peptide delivery system may further comprise one or more intracellular or extracellular domains selected from or derived from the following proteins: OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1, programmed cell death-I (PD-1), inducible T cell co-stimulatory factor (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CDI1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC class I molecules, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), co-stimulatory ligands (PD-L2, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1, programmed cell death-I (PD-1), inducible T cell co-stimulatory factor (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CD137 / CD137), CD2, CD7, CD27, CD30, B7-1 (CD80), CD7-2 (CD86), PDL-1, programmed cell death-I (CD86), inducible T ... BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecules (ICAM, CD30L, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, IT GAM, CD1 1b, ITGAX, CD1 1e, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a. A ligand that specifically binds to CD83 or any combination thereof.

[0016] In some embodiments, such peptide delivery systems may include a targeting moiety that can bind to BCMA, MUC16 (also known as CA125), EGFR, EGFRvIII, MUCI, Flt-3, WT-1, CD38, CD70, CD90, CD133, MHC-WTI, TSPANI0, MHC-PRAME, MHC-NY-ESOI, HER2 (ERBB2), CA-IX (carbonic anhydrase IX), LIVI, ADAMIO, CHR NA2, LeY, NKG2D, CSI, CD44v6, CD24, LGR5, ALDH, ALDH1, CD54, Sca1, CD271, CD123, CD36, CD109, CD110, CD71-negative, CCA, ABCG2, Claudin-18.2 (Claudin-18A2 or Claudin18 isoform 2), PSCA, DLL3 (Delta-like protein 3, Drosophila Delta homolog 3, Delta3), Mud 7 (Mucinl 7, Muc3, Muc3), FAPα (fibroblast activator protein α), Ly6G6D (lymphocyte antigen 6 complex site protein G6d, c6orf23, G6D, MEGTI, NG25), PSMA, MSLN or RNF43 (E3 ubiquitin ligase RNF43, cyclic finger protein 43), BAFF, C242 antigen, disialotetrahexosylganglioside (GD2), 4-IBB, 5T4, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44v6, CD44, CD51, CD52, CD56, CD74, CEA, CNT0888, CTLA-4, DR5, EpCAM, FAP, fibronectin extra domain-B (fibronectin extra domain-B) extradomain-B), folic acid receptor 1, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scattering factor receptor kinase, IGF-1 receptor, IGF-I, IgGI, LI-CAM, integrin α5β1, integrin αvβ3, podocyte oleanolic acid, MORAb-009, MS4A1, MUC1, mucin CanAg, C-MET, CCR4, CD152, CD10, CD19, CD20, CD200, N-hydroxyacetylneuraminic acid, NPC-IC, PDGF-RA, PDL192, phosphatidylserine, tumor antigen CTAA16.88. VEGF-A, VEGFR-1, VEGFR2, vimentin, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tendinin C, TGF-β, TRAIL-R1, TRAIL-R2, folate receptor, transferrin receptor, or any combination thereof.

[0017] This document also considers the aforementioned peptide delivery system, wherein the second transmembrane domain is connected to an intracellular region comprising one or more intracellular domains from 41BB, ICOS, and CD3ζ. In some embodiments, GrzB can be activated by phosphorylation of the intracellular domains of 41BB, ICOS, or CD3ζ by a kinase, thereby causing GrzB to be transported out of the cell and cleaved at the first and / or second cleavage sites.

[0018] In some embodiments, the peptide delivery system described above may include a third transmembrane domain and a fourth transmembrane domain connected by a second loop region, wherein the third transmembrane domain is connected to an intracellular region containing an intracellular domain from 41BB, ICOS, or CD3ζ. In such embodiments, phosphorylation of the 41BB, ICOS, or CD3ζ intracellular domain by a kinase can activate the release of endogenous GrzB from the cell, causing GrzB to be transported out of the cell and cleaved at GrzB cleavage sites present in the first or second loop region, thereby releasing the peptide load.

[0019] In one embodiment, the second transmembrane domain may be derived from a NOTCH protein having a cleavage site configured to release a functional domain connected to the NOTCH protein transmembrane domain in the intracellular environment.

[0020] Considering the peptide delivery system described above, the payload may include: an antibody, an antibody fragment, a VHH, a cytokine or chemokine composed of a fraction or derivative of sulfated xylan, which is an antagonist of a ligand selected from the following list: IL-1a, IL-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-3, IL-14, IL-15 or IL-17 cytokine family (including IL-25), interferon, G-CSF, M-CSF, GM-CSF, BD NF, CNTF, EGF, EPO, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF12, FGF13, FGF14, FGF15, FGF16, FGF 17. FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, LIF, PDGF, SCF, TGFa, TGFB, TNFa, TNFB, TPO, VEGF, GH, NGF, NT3, NT4, NT5, NT6, NT7, Oncostatin M (OSM), insulin, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-1 family members including MIP-1a, MIP-2, eotaxin (eotaxin-1, eotaxin-2 or eotaxin-3), PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase;Enzymes of the cathepsin family, cell adhesion molecules (e.g., PECAM-1), soluble receptors or cell-binding receptors or virus-binding receptors, cytokine-induced neutrophil chemotactic agents (KC), TNF-α and IFN-γ, and other soluble inflammatory mediators (e.g., reactive oxygen species and nitric oxide), CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, and CCL18 CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, thrombin, urokinase, vaccine, or any combination thereof.

[0021] In several embodiments, the peptide delivery system described above may further include a linker between the first transmembrane domain and the first cleavage site. The linker may be 2-128 amino acids long or 4-20 amino acids long. In some embodiments, the linker may be selected from the linkers listed in Table 4.

[0022] In one embodiment, the peptide delivery system may be configured to be specifically sorted into extracellular vesicles.

[0023] This paper also considers the aforementioned peptide delivery systems configured to release two or more peptide payloads intracellularly or extracellularly.

[0024] This paper also considers peptide delivery systems that contain two or more exfoliase cleavage sites, and each cleavage site may be the same as or different from any other cleavage site.

[0025] This paper also considers peptide delivery systems that include an intracellular domain, wherein the intracellular domain comprises the stem domain of a CD45 phosphatase.

[0026] This paper also considers the aforementioned peptide delivery system, which comprises a single-domain antibody or antibody single chain that binds to an autoassociated antigen (SAA) to bring the SAA into contact, for example, in cis or trans.

[0027] This paper also considers peptide delivery systems comprising a single-domain antibody or antibody single chain that binds to an autoassociated antigen (SAA), and further comprising a CD45 phosphatase-binding moiety or an NKG2A-binding moiety that activates inhibitory signals in the target cells.

[0028] In the above-described peptide delivery system implementation, the peptide load can be any peptide or protein that functions in the human body, including hormones, cytokines, chemokines, prodrugs, and various forms of antibodies, such as chimeric antigen receptors (CARs), bispecific killer cell binders (BIKEs), trispecific killer cell binders (TRIKEs), etc.

[0029] In several embodiments, the peptide delivery system described above may be selected from: CD63-MMP2-a-CD16-a-CD33-Myc-MMP9-CD63-GPi (SEQ ID NO: 124), CD63-MMP2-a-CD16-a-CD33-Myc-MMP9-CytCD63-EV motif (SEQ ID NO: 155), CD63-MMP2-a-CD16-a-CD33-Myc-MMP9-CytCD63 (SEQ ID NO: 36 or 188), TNFSF14-a-CD16-a-CD33-Myc-TNR1A-ADAM17-a-CD38-41BB (SEQ ID NO: 54, 366, or 367), CD63-GrzB-a-CD16-a-CD33-Myc-GrzB-a-CD38-CD28-CD3z (SEQ ID NO: 71 or 252), CD63-MMP9-2L-TNFa-Myc-MMP9-CD63-Cyt (SEQ ID NO: 88 or 295), CD63-MMP9-2L-TNFa-Myc-MMP9-CD63-D-Cyt (lacking EV sorting sequence) (SEQ ID NO: 103 or 311), CD63-DL1-MMP2-2L-TNFa-Myc-MMP2-CD63-Cyt (SEQ ID NO: 104 or 368), CD63-MMP9-2L-TNFa-Myc-MMP9-aCD19CAR (SEQ ID NO: 105 or 369), CD63-MMP2-2L-TNFa-Myc-MMP2-CD63-D-Cyt (SEQ ID NO: 71 or 252), CD63-MMP9 ...9-CD63-D-Cyt (SEQ ID NO: 71 or 252), CD63-MMP9-2L-TNFa-Myc-MMP9-CD63-D-Cyt (SEQ ID NO: 71 or 252), CD63-MMP 106 or 370 or 371), CD63-MMP2-2L-TNFa-Myc-MMP2-aCD19CAR (SEQ ID NO: 107 or 372 or 373), CD69cyt-TMII-TNFa-Myctag-CD28TM-cyt (SEQ ID NO: 119 or 370), CD69cyt-TMII-MMP9-TNFa-Myc-MMP9-CD28TM-cyt (SEQ ID NO: 120 or 377), CD69Cyt-TM-GrzB-TNFa-Myc-GrzB-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 107 or 372 or 373), CD69Cyt-TM-GrzB-nLuc-Myc-GrzB-CD28-CD3z (δ sc-GAGE B) (SEQ ID NO: 106 or 370 or 371), CD63-MMP2-2L-TNFa-Myc-MMP2-aCD19CAR ...9Cyt-TM-GrzB-nLuc-Myc-GrzB-CD28-CD108 or 374), CD69Cyt-TM-nLuc-Myc-a-CD19sc-stem-CD28-CD3z (δ GrzB, GAGE ​​A) (SEQ ID NO: 109 or 375), CD69Cyt-TM-GrzB (primitive)-nLuc-Myc-GrzB (primitive)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 358 or 382), CD69Cyt-TM-GrzB(IEPD)-nLuc-Myc-GrzB(IEPD)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 110 or 383), CD69Cyt-TM-GrzB(SASA)-nLuc-Myc-,GrzB(SASA)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 109 or 374), CD69Cyt-TM-GrzB(primitive)-nLuc-Myc-,GrzB(SASA)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 109 or 375), CD69Cyt-TM-GrzB(primitive ... 111 or 376), or CD69Cyt-TM-GrzB(ADKG)-nLuc-Myc-GrzB(ADKG)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 112 or 384).

[0030] In some embodiments, the peptide delivery system described above may comprise cells to which an exogenous peptide has been anchored. In some embodiments, the cells may be selected from hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs) and their derived cell products, adoptive T cells, dendritic cells (DCs), natural killer (NK) cells, or any therapeutic immune or non-immune cells.

[0031] This article also considers adoptive or engineered immune cells containing peptide loads of such peptide delivery systems, wherein the immune cells are autologous or allogeneic.

[0032] In several implementations, exogenous peptides can be introduced into cells individually via a CRISPR system, or via a viral or non-viral vector, or via one or more mRNAs encoding the exogenous peptide.

[0033] In various embodiments, exogenous peptides of the above peptide delivery systems can be delivered using CPP, micelles, liposomes, nanoparticles, dendritic polymers, nanotubes, electroporation, viral transduction, nuclear transfection, transfection, cell fusion, or microinjection.

[0034] This paper also considers gene constructs encoding exogenous peptides of the peptide delivery system described herein.

[0035] This article also considers methods for treating patients in need, including administering the aforementioned peptide delivery system to the patients.

[0036] This document also discloses a method for delivering peptides to target sites in patients in need, the method comprising: administering a peptide delivery system to a patient, the peptide delivery system comprising a lipid-containing vesicle (e.g., a cell, extracellular vesicle, lipid nanoparticle) in which an exogenous polypeptide is anchored, the exogenous polypeptide comprising: a peptide load; a first transmembrane region and a second transmembrane region; the first transmembrane region comprising a transmembrane domain linked to at least one exfoliase or other proteolytic cleavage site; the second transmembrane region comprising a transmembrane domain linked to at least one exfoliase or other proteolytic cleavage site; and optionally, a targeting portion for targeting the exogenous polypeptide to a target site, wherein when the lipid-containing vesicle reaches the target site, the exfoliase and / or other protease cleave the cleavage sites of the first and second transmembrane regions, releasing the peptide load.

[0037] This paper also considers a method in which the cleavage site is derived from a proteolytic cleavage substrate naturally present in glycosylphosphatidylinositol (GPi) anchored proteins and membrane proteins having one, two, three, four or more transmembrane domains, intracellular domains or extracellular domains in the extracellular matrix or adjacent cells.

[0038] This document also considers methods in which the exfoliase is selected from dedicated or part-time exfoliases. In several embodiments, the dedicated exfoliase may be selected from ADAM protease, BACE protease, serine protease granzyme-B, and Site-1 protease. The ADAM protease may be selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, and ADAM33. The part-time exfoliase may be selected from: transmembrane peptidase B, MT-MMP (membrane matrix metalloproteinase), proprotein convertase, transmembrane serine protease, matrix metalloproteinase, podocyte protein, cathepsin S, and cathepsin L, wherein the transmembrane peptidase B is a membrane-anchored type 1 metalloproteinase, and wherein the MT-MMP is membrane-anchored type 1 or GPI-anchored, and includes MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, and MT6-MMP. The proprotein convertase may be selected from PCSK1 / 3, KCSK2, furin protease, PCSK4, PSCK5 / 6, PACE4, PCSK7, and PCSK9. The transmembrane serine protease may be selected from membrane-anchored type II proteases, including, for example, Matriptase, Matriptase-2, Matriptase-3, polymerase-1, Corin, Hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, Spinesin, intestinal peptidase, HAT, DESCL1, TMPRSS11A, HAT-like 4, and HAT-like 5. The matrix metalloproteinase may be a soluble protease and may include one or more of MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A-B, MMP24, MMP25, MMP26, MMP27, and MMP28. In some embodiments, the matrix metalloproteinase may be selected from MMP2, MMP9, and / or MMP25. For example, MMP2 may be selected from SEQ ID NOS:2-6, MMP9 may be selected from SEQ ID NOS:7-8, and MMP25 may be selected from SEQ ID NOS:9-17, 18, or 155, 19-29.

[0039] This document also considers the method described above, wherein the transmembrane domains of the first and / or second transmembrane regions comprise transmembrane domains derived from one or more of the following: CD63, CD9, CD81, CD28, CD4, CD8, CD34, CD69, CD19, CD20, integrin, TNFSF14, TNR1A, aquaporin, NOTCH, NgR1, NRG1, GPi anchor, EGFR, and rhodopsin.

[0040] This article also considers the method described above, wherein cleavage occurs at one or more of the cleavage sites by endogenous exfoliases.

[0041] This article also considers the method described above, wherein the peptide delivery system further comprises an abscission enzyme, and the abscission enzyme cleaves the peptide delivery system at one or more of the cleavage sites.

[0042] This article also considers the methods described above, wherein the intracellular or extracellular domain of the exogenous polypeptide is selected from one or more of the following intracellular or extracellular domains: OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1, programmed cell death-I (PD-1), inducible T cell co-stimulatory factor (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CDI 1a / CD 18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, lgα (CD79a), DAP-10, Fcγ receptor, MHC class I molecules, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), co-stimulatory ligand (PD-L2, 4-1BB / CD137), 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1, programmed cell death-I (PD-1), PD-2, PD-3, PD-40, PD-1, ... BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecules (ICAM, CD30L, CD70, CD83, HLA-G, MICA, M1 CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, IT GAM, CD1 1b, ITGAX, CD1 1e, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG(CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

[0043] This article also considers the methods described above, wherein the targeted portion binds to BCMA, MUC16 (also known as CA125), EGFR, EGFRvIII, MUCI, Flt-3, WT-1, CD38, CD70, CD90, CD133, MHC-WTI, TSPANI0, MHC-PRAME, MHC-NY-ESOI, HER2 (ERBB2), CA-IX (carbonic anhydrase IX), LIVI, ADAMI0, CHRNA2, Le Y, NKG2D, CSI, CD44v6, CD24, LGR5, ALDH, ALDH1, CD54, Sca1, CD271, CD123, CD36, CD109, CD110, CD71-negative, CCA, ABCG2, Claudin-18.2 (Claudin-18A2 or Claudin18 isoform 2), PSCA, DLL3 (Delta-like protein 3, Drosophila Delta homolog 3, Delta3), Mud 7 (Mucinl) 7. Muc3, FAPα (fibroblast activator protein α), Ly6G6D (lymphocyte antigen 6 complex site protein G6d, c6orf23, G6D, MEGTI, NG25), PSMA, MSLN or RNF43 (E3 ubiquitin protein ligase RNF43, cyclic finger protein 43), BAFF, C242 antigen, disialotetine ganglioside (GD2), 4-IBB, 5T4, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44v6, CD44, CD51, CD52, CD56, C D74, CEA, CNT0888, CTLA-4, DR5, EpCAM, FAP, fibronectin extradomain-B, folate receptor 1, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scattering factor receptor kinase, IGF-1 receptor, IGF-I, IgGI, LI-CAM, integrin α5β1, integrin αvβ3, podin, MORAb-009, MS4A1, MUC1, mucin CanAg, C-MET, CCR4, CD152, CD10, CD19, CD20, CD200, N-hydroxyacetylneuraminic acid, NPC-IC, PDGF-R a. PDL192, phosphatidylserine, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tendinogen C, TGF-β, TRAIL-R1, TRAIL-R2, folate receptor, transferrin receptor or any combination thereof.

[0044] This document also considers a method as described above, wherein the first transmembrane region and / or the second transmembrane region comprises: (i) an intracellular region comprising an intracellular region sequence from 41BB, ICOS, or CD3ζ, and (ii) a GrzB cleavage site; and wherein the action of a kinase on the intracellular region sequence from 41BB, ICOS, or CD3ζ induces the release of the GrzB, causing it to be transported out of the cell and cleaved at the GrzB cleavage site, thereby releasing the peptide payload, the peptide payload optionally comprising a bispecific antibody.

[0045] This paper also considers the method described above, wherein the second transmembrane region comprises a transmembrane region from the NOTCH protein and a cleavage site within the cell membrane, and cleavage of the cleavage site within the cell membrane can release the functional domain in the intracellular environment.

[0046] This article also considers the method described above, wherein the peptide loading comprises: a cytokine or chemokine composed of a portion or derivative of sulfated xylan, which is an antagonist of a ligand selected from the following list: IL-1a, IL-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-3, IL-14, IL-15 or IL-17 cytokine family (including IL-25), interferon, G-CSF, M-CSF, GM-CSF, BDNF, CNTF, EGF , EPO, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FG F18, FGF19, FGF20, FGF21, FGF22, FGF23, LIF, PDGF, SCF, TGFa, TGFB, TNFa, TNFB, TPO, VEGF, GH, NGF, NT3, NT4, NT5, NT6, NT7, Oncostatin M OSM, insulin, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-1 family members including MIP-1a, MIP-2, eosinophil chemokines (eosinophil chemokine-1, eosinophil chemokine-2, or eosinophil chemokine-3), PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase; cathepsins, cell adhesion molecules (e.g., PECAM-1), soluble receptors or cell-binding receptors or virus-binding receptors, cytokine-induced neutrophil chemotactic agents (KC), TNF-α and IFN-γ, and other soluble inflammatory mediators (e.g., reactive oxygen species and nitric oxide), CCL1, CCL2, CCL3, CCL4. CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, thrombin, urokinase, vaccine, or any combination thereof.

[0047] This article also considers methods as described above, wherein the exogenous polypeptide further comprises at least one adapter, wherein the at least one adapter is optionally located between a cleavage site and a first binding moiety (e.g., an anti-CD3VHH domain) present in the first transmembrane region, between a cleavage site and a second binding moiety (e.g., an anti-TAA VHH domain) present in the second transmembrane region, and / or between the first binding moiety and the second binding moiety.

[0048] This article also considers the method described above, wherein the length of the connector is 2-128 amino acids, or preferably 4-20 amino acids.

[0049] This paper also considers the method described above, wherein the connectors are selected from Table 4.

[0050] This article also considers the method described above, wherein the exogenous peptide can also be configured for specific sorting into extracellular vesicles (EVs).

[0051] This article also considers methods as described above, wherein the peptide load comprises two or more bispecific binders, and the bispecific binders are released intracellularly or extracellularly.

[0052] This article also considers methods as described above, wherein the peptide delivery system comprises exfoliase cleavage sites, and each cleavage site may be the same as or different from any other cleavage site.

[0053] This paper also considers a method as described above, wherein the peptide delivery system comprises an intracellular domain, and the intracellular domain comprises the stem domain of CD45 phosphatase.

[0054] This article also considers methods as described above, wherein the peptide delivery system comprises a single-domain antibody or antibody single chain that binds to an autoassociated antigen (SAA) to bring the SAA into contact, for example, in a cis or trans configuration.

[0055] This article also considers methods as described above, which include the use of a peptide delivery system comprising a single-domain antibody or antibody single chain that binds to the self-associated antigen (SAA) and further comprising a CD45 phosphatase-binding moiety or an NKG2A-binding moiety that activates inhibitory signals in the target cells.

[0056] This article also considers the methods described above, wherein the peptide load is any potentially therapeutic peptide that functions in vivo, including hormones, cytokines, chemokines, prodrugs, and various forms of antibodies, such as chimeric antigen receptors (CARs), bispecific killer cell binders (BIKEs), trispecific killer cell binders (TRIKEs), etc.

[0057] This article also considers the methods described above, wherein the peptide delivery system is selected from: CD63-MMP2-a-CD16-a-CD33-Myc-MMP9-CD63-GPi (SEQ ID NO: 124), CD63-MMP2-a-CD16-a-CD33-Myc-MMP9-CytCD63-EV motif (SEQ ID NO: 155), CD63-MMP2-a-CD16-a-CD33-Myc-MMP9-CytCD63 (SEQ ID NO: 36 or 188), TNFSF14-a-CD16-a-CD33-Myc-TNR1A-ADAM17-a-CD38-41BB (SEQ ID NO: 54 or 366 or 367), CD63-GrzB-a-CD16-a-CD33-Myc-GrzB-a-CD38-CD28-CD3z (SEQ ID NO: 71 or 252), CD63-MMP9-2L-TNFa-Myc-MMP9-CD63-Cyt (SEQ ID NO: 88 or 295), CD63-MMP9-2L-TNFa-Myc-MMP9-CD63-D-Cyt (lacking EV sorting sequence) (SEQ ID NO: 103 or 311), CD63-DL1-MMP2-2L-TNFa-Myc-MMP2-CD63-Cyt (SEQ ID NO: 104 or 368), CD63-MMP9-2L-TNFa-Myc-MMP9-aCD19CAR (SEQ ID NO: 105 or 369), CD63-MMP2-2L-TNFa-Myc-MMP2-CD63-D-Cyt (SEQ ID NO: 71 or 252), CD63-MMP9 ... 106 or 370 or 371), CD63-MMP2-2L-TNFa-Myc-MMP2-aCD19CAR (SEQ ID NO: 107 or 372 or 373), CD69cyt-TMII-TNFa-Myctag-CD28TM-cyt (SEQ ID NO: 119 or 370), CD69cyt-TMII-MMP9-TNFa-Myc-MMP9-CD28TM-cyt (SEQ ID NO: 120 or 377), CD69Cyt-TM-GrzB-TNFa-Myc-GrzB-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 107 or 372 or 373), CD69Cyt-TM-GrzB-nLuc-Myc-GrzB-CD28-CD3z (δ sc-GAGE B) (SEQ ID NO: 106 or 370 or 371), CD63-MMP2-2L-TNFa-Myc-MMP2-aCD19CAR ...108 or 374), CD69Cyt-TM-nLuc-Myc-a-CD19sc-stem-CD28-CD3z (δ GrzB, GAGE ​​A) (SEQ ID NO: 109 or 375), CD69Cyt-TM-GrzB (primitive)-nLuc-Myc-GrzB (primitive)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 358 or 382), CD69Cyt-TM-GrzB(IEPD)-nLuc-Myc-GrzB(IEPD)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 110 or 383), CD69Cyt-TM-GrzB(SASA)-nLuc-Myc-,GrzB(SASA)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 109 or 374), CD69Cyt-TM-GrzB(primitive)-nLuc-Myc-,GrzB(SASA)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 109 or 375), CD69Cyt-TM-GrzB(primitive ... 111 or 376), or CD69Cyt-TM-GrzB(ADKG)-nLuc-Myc-GrzB(ADKG)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 112 or 384).

[0058] This document also considers methods as described above, wherein the lipid-containing vesicles comprise cells selected from hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs) and derived cell products, adoptive T cells, dendritic cells (DCs), natural killer (NK) cells, or any therapeutic immune or non-immune cells. In some embodiments, the immune cells may be autologous or allogeneic.

[0059] This paper also considers methods as described above, wherein the peptide delivery system is introduced into the cell individually in the form of a viral or non-viral vector, mRNA, peptide, protein, antibody, nanobody, oligonucleotide, or extracellular vesicle (EV).

[0060] This article also considers methods as described above, wherein the peptide delivery system is delivered using CPP, micelles, liposomes, nanoparticles, dendritic polymers, nanotubes, electroporation, viral transduction, nuclear transfection, transfection, cell fusion, or microinjection.

[0061] One object of the present invention is to deliver peptides.

[0062] One object of the present invention is to target the payload to a specific cell type.

[0063] One object of the present invention is to deliver multiple peptides.

[0064] One object of the present invention is to introduce a peptide delivery system into any cell capable of delivering at least one peptide to a target cell or target site.

[0065] One object of the present invention is to create a single delivery system that can be integrated into any cell. Attached Figure Description

[0066] Figure 1 This is a diagram of a general embodiment of the present invention that includes two transmembrane domains (i.e., "dispanin").

[0067] Figure 2 This is a diagram of an implementation scheme for a "double transmembrane protein" delivery system, which sequentially includes a type II transmembrane domain, a first MMP cleavage sequence, a first adapter ("Adapter-a"), an anti-CD3 VHH domain, a second adapter ("Adapter-b"), an anti-TAA VHH domain, a third adapter ("Adapter-c"), a second MMP cleavage sequence, and a GPI anchor.

[0068] Figure 3 Showing Figure 2 The amino acid sequence and multiple domains / motifs of the illustrated embodiment.

[0069] Figure 4A This is a diagram of a general embodiment of the invention, which uses a modified tetraspanin (CD63) that is linked to a specific ligand (GAGE) (here, TNF-α) and contains a specific motif, GYEVM, for EV-specific delivery.

[0070] Figure 4B Showing Figure 4A The amino acid sequence and multiple domains / motifs of the illustrated embodiment.

[0071] Figure 5A A general embodiment of the invention is shown, which uses a modified tetraspan protein (CD63) without an EV sorting motif.

[0072] Figure 5B Showing Figure 5A The amino acid sequence and multiple domains / motifs of the illustrated embodiment.

[0073] Figure 6 An embodiment of the invention is shown, which, among other things, uses a modified transmembrane C-type lectin protein (CD69).

[0074] Figure 7 The invention is illustrated in a general embodiment that uses a modified tetratransmembrane protein comprising four different types of transmembrane domains (TMI–TMIV).

[0075] Figure 8A general embodiment of the invention is shown, which uses a modified tetraspan membrane protein.

[0076] Figure 9 This is a diagram illustrating an embodiment of the bitransmembrane protein of the present invention.

[0077] Figure 10 This is the amino acid sequence of the CD63-ring-EV sorting implementation scheme, showing... Figure 9 The corresponding structural domains and motifs of the illustrated implementation scheme.

[0078] Figure 11 This is a diagram illustrating an embodiment of the bitransmembrane protein of the present invention.

[0079] Figure 12 yes Figure 11 The amino acid sequences of the illustrated embodiments show the corresponding domains and motifs.

[0080] Figure 13 This is a diagram illustrating an embodiment of the bitransmembrane protein of the present invention.

[0081] Figure 14 yes Figure 13 The amino acid sequences of the illustrated embodiments show the corresponding domains and motifs.

[0082] Figure 15 This is a diagram illustrating an embodiment of the bitransmembrane protein of the present invention.

[0083] Figure 16 The amino acid sequence of the CD63-Grn-Ba-CD16-La-CD33-Grn-Ba-CD38CAR embodiment is shown. Figure 15 The corresponding structural domains and motifs of the illustrated implementation scheme.

[0084] Figure 17A This is a diagram of an embodiment of the present invention, encoded by CD69cyt-TM-II-TNFa-Myc-CD28TM-CYT, for delivering TNFα.

[0085] Figure 17B It is the amino acid sequence of CD69cyt-TM-II-TNFa-Myc-CD28TM-CYT.

[0086] Figure 18A This is a figure showing an embodiment of the present invention encoded by CD69cyt-TMII-9-TNFa-myctag-9-CD28TM-cyt containing two MMP9 cleavage sites.

[0087] Figure 18BIt is the amino acid sequence of CD69cyt-TMII-9-TNFa-Myctag-9-CD28TM-cyt.

[0088] Figure 19 This is a diagram of an embodiment of the present invention, wherein αTAA-αSAA serve as loads that can be delivered after pyrolysis.

[0089] Figure 20 This is a diagram showing the plasmid transmembrane domains, protease motifs, and payloads of several embodiments disclosed in this application.

[0090] Figure 21 This is a diagram showing the experimental setup / model of the embodiment of the invention shown in this application.

[0091] Figure 22A , Figure 22B and Figure 22C Three options for experimental setups / models involving the measurement of TNFα and NF-κB luciferase activity are shown.

[0092] Figure 23A The diagram shows an experimental setup / model of the embodiment disclosed in this application, in which granzyme B causes the release of TNFα load from the surface of NK cells.

[0093] Figure 23B This is a diagram showing the experimental setup / model of the embodiment disclosed in this application, in which granzyme B causes the release of TNFα load from the surface of K562 cells.

[0094] Figure 24 This is a diagram showing the function of TNFα loading in direct intercellular contact with HEK293-NF-κB-Luc reporter cells.

[0095] Figure 25 This is a graph showing the delivery of TNFα load after MMP9 cleavage. Note: GAGE ​​and MMP9 co-transfection. We show EV depletion vs. EV non-depletion to control MMP transfer via EV.

[0096] Figure 26 The graph shows that TNFα loads are primarily delivered to the EV.

[0097] Figure 27 This figure shows the delivery of TNFα load after MMP2 cleavage. In this experiment, supernatant from HEK293 cells expressing the GAGE ​​construct was used.

[0098] Figure 28 This graph shows that the TNFα load is primarily delivered to the EV. Cleavage occurs again via MMP2. Note: EV depletion vs. EV not depleted.

[0099] Figure 29 This is a graph showing the differential expression of CD63 and CD69 transmembrane GAGE ​​in HEK293 cells.

[0100] Figure 30 This figure shows the results of in vitro reactivity analysis of TNFα loading delivery after MMP9 cleavage and NF-B activation.

[0101] Figure 31 This is a graph showing the percentage of K562 cells that are positive for TNFα expression on their surface 3 days after transduction.

[0102] Figure 32 This is a diagram showing the delivery of TNFα loads from K562 cells cleaved by granzyme B (GrzB) secreted by NK92 cells.

[0103] Figure 33 The percentage of K562 cells positive for surface TNFα and αCD19 / CAR expression 6 days after cell sorting is shown. (Surface expression of GAGE ​​nanoluciferase in K562 transduced cells (MOI 6)).

[0104] Figure 34A and Figure 34B This figure shows the release of nanoluciferase (nLuc) loads from K562 cells via cleavage of granzyme B (GrzB) secreted by NK92 cells. E:T ratio was 3:1, and incubation time was 4 hours.

[0105] Figure 35 The percentage of KHYG-1 cells positive for surface nanoluciferase (nLuc) expression was shown 3 days after transduction.

[0106] Figure 36 This is a graph showing data on the release of nanoluciferase (nLuc) loads from KHYG-1 cells via granzyme B (GrzB) cleavage after 1–3 hours of co-culturing with Nalm-6 cells. Data are normalized to MFI at different time points.

[0107] Figure 37A and Figure 37B This is a graph showing the percentage of KHYG-1 cells positive for surface nanoluciferase and MFI 6 days after cell sorting.

[0108] Figure 38 This is a graph showing the release of nanoluciferase (nLuc) loads from KHYG-1 cells via granzyme B (GrzB) cleavage after 4 and 6 hours of co-culturing with K562 cells.

[0109] Figure 39A and Figure 39B This is a graph showing the percentage of NK92 cells with surface nanoluciferase positivity and MFI 10 days after transduction of NK92 cells.

[0110] Figure 40 This is a diagram showing the release of nanoluciferase (nLuc) loads from NK92 cells via granzyme B (GrzB) cleavage after 4 hours of co-culturing with Raji cells.

[0111] Figure 41 This is a graph showing the percentage of NK92 cells that are positive for surface nanoluciferase after transduction (unsorted cells) and 3 days after cell sorting.

[0112] Figure 42 The figure shows the release of nanoluciferase (nLuc) loads from NK92 cells after Caspase 3 cleavage 6 hours and 24 hours after co-culturing with K562 cells. Detailed Implementation

[0113] The details shown herein are merely illustrative examples and are intended for a discussion of various embodiments. Their purpose is to provide what is considered the most useful and easily understood description of the principles and concepts of the methods and compositions described herein. In this regard, no further details are attempted beyond what is necessary for a basic understanding, and the description allows those skilled in the art to clearly see how several forms can be embodied in practice.

[0114] The invention will now be described with reference to more detailed embodiments. However, the invention may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to make this disclosure sufficient and complete, and to fully convey its scope to those skilled in the art.

[0115] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be restrictive. As used in the specification and appended claims, the singular forms “a,” “an,” and “described” are intended to include the plural forms as well, unless the context clearly indicates otherwise. All publications, patent applications, patents, and other references mentioned herein are expressly and entirely incorporated by reference.

[0116] Unless otherwise stated, the numerical parameters listed in the following description and appended claims are approximate values ​​and may vary depending on the desired characteristics sought, and are therefore modified by the term "about". At least, and not in an attempt to limit the application of the equivalent doctrine to the scope of the claims, each numerical parameter should be interpreted according to the number of significant figures and ordinary rounding.

[0117] Although the numerical ranges and parameters listed are approximate, the values ​​listed in the specific embodiments are reported as precisely as possible. However, any value contains some inherent error due to the standard deviation present in the respective test measurements. Every numerical range given throughout this specification will include every narrower numerical range falling within such a wider range, as such narrower numerical ranges are explicitly stated herein. The applicant also considers the ranges derived from data points and the explicit ranges disclosed herein.

[0118] Every reference cited in this article is incorporated into the article in its entirety through citation.

[0119] This document considers peptide or protein cargo delivery systems with directed delivery and / or site-specific release and activation of the cargo. The cargo is targeted to its predetermined site and / or activated upon arrival at the target. This approach can increase the dose at the local site or target site while limiting the number of off-target side effects. The peptide or protein delivery systems of the present invention may include certain safety and specificity mechanisms so that the biologic is released only at a high dose at the target site, without release in other tissues (e.g., tissues that may also express antigens of specific antibody-derived biologics). In addition to making these peptide-based drugs safer and more widely applicable, our systems can also reduce treatment costs and be tailored for more personalized or patient-specific uses. Furthermore, the delivery systems and methods disclosed herein can achieve effects equivalent to more frequent dosing without the need for repeated peptide administration. For example, delivery via cargo cells storing the peptide or protein to be delivered can provide an in vivo reservoir of inactive peptides or proteins (e.g., prodrugs).

[0120] Mammalian cells possess the hardware necessary to shed many cell membrane-bound proteins. Shedding is a controlled, irreversible post-translational process carried out by a family of proteolytic enzymes, including exfoliases, which are described further below. Cell biology allows cells to control the expression levels and functions of substrate proteins, as well as their localization on the cell surface.

[0121] Proteolytic cleavage substrates include glycosylphosphatidylinositol (GPi)-anchored proteins, membrane proteins having one, two, three, four, or more transmembrane domains, intracellular domains, extracellular domains in the extracellular matrix, or even extracellular domains in adjacent cells. Membrane proteins with two transmembrane domains considered in this paper are referred to herein as "dispanins." Membrane proteins with four transmembrane domains considered in this paper are referred to herein as "tetraspanins." Cleavage can target the proximal or distal regions of membrane-bound proteins and can lead to the release of intracellular and / or extracellular protein domains (extracellular and / or intracellular domain release).

[0122] We have designed a cell-based delivery system that allows the direct delivery and release of at least one peptide or protein payload to a desired site of action. The system is characterized by the payload being anchored to the cell via a transmembrane domain (TMD). A TMD is a portion of a protein molecule that spans the cell membrane, typically consisting of one or more α-helices or β-sheets that cross the lipid bilayer of the membrane, anchoring the protein in place and allowing it to interact with molecules on both sides of the membrane. Many important proteins possess transmembrane domains, including receptors, transporters, and enzymes. These domains are commonly involved in transmembrane signal transduction or the transport of molecules into and out of the cell.

[0123] The delivery system considered in this paper consists of or contains single- or multiple-transmembrane (TM) proteins embedded in the membrane of a carrier (e.g., cells, extracellular vesicles, or other lipid-containing particles or compositions). Additional motifs may be present, which regulate the delivery / release of the payload in the desired environment. Therefore, our goal is not direct systemic injection of biologics, but rather infusion of engineered cells or EVs configured to deliver the cargo directly to the desired site and release it at the appropriate time.

[0124] The delivery systems considered in this article are applicable to any cell type and can be used to deliver any peptide of interest. Suitable cell types include, but are not limited to, hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs) and their derived cell products, adoptive T cells, dendritic cells (DCs), natural killer cells (NKs), NKT cells, or any therapeutic immune or non-immune cells. Such cells can be used for autologous or allogeneic therapies.

[0125] In one embodiment, we engineered NK cells to generate and deliver a bispecific conjugate (GAGE) within the tumor microenvironment (TME). The TME comprises cellular and non-cellular components (partners) that modulate multiple aspects of tumorigenesis, including tumorigenesis, invasion, and metastasis (The updated landscape of tumor microenvironment and drug repurposing. Ming-Zhu Jin & Wei-Lin Jin. Signal Transduction and Targeted Therapy, volume 5, Article number: 166 (2020)). This delivery is further modulated and can be induced by either NK cell activation (through activation-induced cleavage) or the tumor environment (through cleavage upon encountering proteases specifically enriched in the TME). Furthermore, the release of this activation stimulus may lead to NK cell expansion and activation, depending on the availability of tumor antigens. In embodiments, the delivery system described herein comprises a next-generation cancer immunotherapy system with enhanced capabilities to generate and release payloads under desired conditions and at specific sites.

[0126] There are no particular limitations on the exfoliases that can be used in this payload delivery system, and they will be described further below. For example, the exfoliase can be selected from matrix metalloproteinases (MMPs) and granzyme-B (GrzB), which allow for environment-specific cargo release. Furthermore, models based on the ADAM family of MMPs and the serine protease granzyme B have been proposed. In these models, additional functions, such as tumor targeting, amplification, and / or activation, are regulated by adding extracellular and / or intracellular domains. These additional domains can include extracellular cytokines, drugs or prodrugs, chimeric antigen receptor (CAR)-like or dual antigen receptor (DAR)-like domains, or intracellular regulators, inhibitors, proteins containing signaling domains, etc.

[0127] Furthermore, this system has been designed for the passive release of specific MMPs dependent on cancer cell release within the tumor environment, or for the active release induced by receptor-mediated activation of producing cells.

[0128] These systems can also be designed to sort into specific subcellular compartments. We designed a protein molecule that can actively sort into extracellular vesicles (EVs) such as exosomes. Cells release these exosomes in large quantities, and the payload can be released after being cleaved by proteases within the TME or other target sites. These EVs can also be obtained from in vitro culture and given to patients as off-the-shelf therapeutics.

[0129] The simplest form of the invention comprises a prodrug form of a peptide, comprising: a first transmembrane region and a second transmembrane region; the first transmembrane region comprising at least one transmembrane unit connected to at least one cleavage site; the second transmembrane region comprising a transmembrane unit connected to at least one cleavage site; optionally, the delivery system may include a targeting portion for targeting the prodrug to target cells; wherein when the prodrug contacts the target cells, an exfoliase cleaves the cleavage site to release the peptide.

[0130] We primarily use a TMD ensemble in this multi-transmembrane load delivery system. The N-terminus of each TMD consists of a type II TMD carrying a adapter / load, and a type I TMD is attached to its C-terminus. The TMD is preferably at least one or more of the following: CD63, CD9, CD81, CD28, CD4, CD8, CD34, CD69, CD19, CD20, integrin, TNFSF14, TNR1A, aquaporin, NOTCH, NgR1, NRG1, GPi anchor, EGFR, rhodopsin, or any related TMD that provides similar functionality to the expression system usable in this invention.

[0131] Optionally, the second transmembrane region has an intracellular region containing 41BB, ICOS, and CD3ζ, and MMP / GrzB cleavage sites that cleave the extracellular MMP / GrzB, wherein the action of phosphatases on 41BB, ICOS, and CD3ζ cleaves and activates MMP / GrzB, enabling it to be transported out of the cell and cleave the MMP / GrzB cleavage sites, thereby releasing the bispecific antibody / load. Optionally, the first and second transmembrane regions are embedded with a NOTCH protein-dependent traction system, which has secretase cleavage sites within the cell membrane, and multiple cargo / loads are attached to several cleavage sites extracellularly or intracellularly. Optionally, this system can be embedded by recruiting self-associated antigens (α-SAA) through the addition of single-chain antibodies.

[0132] This engineered system can be introduced into cells individually as a viral or non-viral vector, mRNA, peptide, protein, antibody, nanobody, oligonucleotide, or extracellular vesicle (EV). Delivery can be achieved using CPP, micelles, liposomes, nanoparticles, dendritic polymers, nanotubes, electroporation, viral transduction, nuclear transfection, transfection, cell fusion, or microinjection. Viral vectors may contain one or more genes encoding GAGE, and the resulting viral particles may carry these genes. When the virus infects cells, one or more genes are stably integrated and the encoded protein is expressed. Non-viral vectors may be in the form of plasmids carrying one or more GAGE ​​genes. After transfection, cells express one or more genes encoding one or more GAGE ​​proteins. Cells with stable expression can be selected. It is anticipated that one of the gene delivery strategies that can be employed is a site-specific integration strategy, such as CRISPR (e.g., Cas9, Cpf1, or other Cas effector protein complexes) or a transposon-transposase strategy. CRISPR Cas systems can be introduced via electroporation or LNP and can integrate genes encoding GAGE.

[0133] This invention relates to a peptide delivery system with site-directed release and peptide activation. Site-directed release means that the peptide-producing cells or EVs containing this peptide delivery system are targeted to their predetermined sites. The cells or EVs are then activated to release the peptide at the target site. This approach can increase the delivered dose while limiting the number of off-target side effects. The peptide delivery system of this invention includes certain safety and specificity mechanisms that ensure high-dose release of the biologic / load only at the target site, without release in other tissues that may also express antigens of the specific biologic (e.g., antibody-derived biologics). In addition to making these peptide-based drugs safer, more suitable, and more specific, our system can also reduce treatment costs. Furthermore, this invention achieves the equivalent of more frequent dosing without repeated administration of the peptide because delivery via cargo cells (which will produce and store the peptide) provides an in vivo reservoir of inactive peptides.

[0134] Mammalian cells possess the hardware necessary to shed a variety of cell membrane-bound proteins. Lysis is a controlled, irreversible post-translational modification carried out by a family of proteases, or exfoliases. Cell biology allows cells to control the expression levels and functions of substrate proteins, as well as their localization on the cell surface. Exfoliases are known as a class, including dedicated and part-time exfoliases. (Lichtenthaler SF, Lemberg MK, Fluhrer R) Proteolytic ectodomain shedding of membrane proteins in mammals-hardware, concepts, and recent developments. EMBO J . 2018;37(15):e99456. doi:10.15252 / embj.201899456.

[0135] Specialized exfoliases include ADAM proteases, BACE proteases, serine proteases granzyme B, and Site-1 proteases. ADAM proteases (metalloproteinases) are membrane-anchored type 1 proteases, including ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, and ADAM33. BACE (aspartic acid) proteases are also membrane-anchored type 1 proteases, including BACE1 and BACE2. Site-1 (serine) proteases are membrane-anchored type 1 proteases, including SKI-a or S1P.

[0136] Partial exfoliases include transmembrane peptidase B, MT-MMP (membrane matrix metalloproteinase), proprotein convertase, transmembrane serine protease, matrix metalloproteinase, podocyte protein, cathepsin S, and cathepsin L. Transmembrane peptidase B is a membrane-anchored type 1 metalloproteinase. MT-MMPs are membrane-anchored type 1 or GPI-anchored proteases, including MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, and MT6-MMP. Proprotein convertases are membrane-anchored type 1 or soluble proteases, including PCSK1 / 3, PCSK2, furin, PCSK4, PCSK5 / 6, PACE4, PCSK7, and PCSK9. Transmembrane serine proteases are membrane-anchored type II proteases, including Matriptase, Matriptase-2, Matriptase-3, polymerase-1, Corin, Hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, Spinesin, intestinal peptidase, HAT, DESCL1, TMPRSS11A, HAT-like 4, and HAT-like 5. Matrix metalloproteinases are soluble proteases, including MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A-B, MMP24, MMP25, MMP26, MMP27, and MMP28.

[0137] For evaluation, substrate targets exhibiting the highest cleavage efficiency against a specific MMP but with cleavage values ​​below 4000 k (obs) against other MMPs were considered "specific" substrate sequences for that particular MMP. The substrate hexapeptide sequence was sandwiched between constant regions of the M13 phage gene III protein containing the FLAG epitope. .

[0138] Table 1-3 below shows the selected MMP substrates and their specific binding affinity sequences for MMP2, MMP9 and MMP25.

[0139] Table 1: MMP2-specific target substrate* k(obs) (M -1 sec -1 )

[0140] Table 2: MMP9-specific target substrate* k(obs) (M -1 sec -1 )

[0141] Table 3: MMP25 specifically targets substrates* k(obs) (M -1 sec -1 )

[0142]

[0143] Proteolytic cleavage substrates include glycosylphosphatidylinositol (GPi)-anchored proteins, membrane proteins having one, two, three, four, or more transmembrane domains, intracellular domains, extracellular domains in the extracellular matrix, or even extracellular domains in adjacent cells. Cleavage can target the proximal or distal regions of membrane-bound proteins and can lead to the release of intracellular and extracellular protein domains (extracellular and / or intracellular domain release). Figure 1-20 ).

[0144] Membrane-bound proteins that cross the plasma membrane once are called “single-pass transmembrane” molecules, and are composed of type I, II, III, and IV members. Type I transmembrane proteins have an N-terminal signal peptide domain that targets the type I transmembrane protein to the endoplasmic reticulum (ER) lumen during synthesis, resulting in expression in the extracellular space. A termination transfer anchoring sequence anchors the type I transmembrane protein to the lipid membrane. Although both type II and type III use signal anchoring sequences, type II targets the ER lumen with its C-terminal domain, while type III targets the ER lumen with its N-terminal domain. Type IV can be IV-A, whose N-terminal domain targets the cytosol, or it can be IV-B, whose N-terminal domain targets the lumen. The delivery system of the present invention can have one, two, three, four, or more transmembrane proteins (see, for example, Figure 4A , Figure 4B , Figure 5A , Figure 5B , Figure 6 , Figure 7 , Figure 8 , Figure 9 wait).

[0145] Optionally, the transmembrane region following the first or second extracellular loop (ECL) has an intracellular region containing 41BB, ICOS, and CD3ζ, wherein the action of the kinase on 41BB, ICOS, CD28, and CD3ζ activates engineered cells (NK cells and T cells) to release their endogenous GrzB, causing it to cleave GrzB cleavage sites, thereby releasing the bispecific antibody / loador. Figure 6 , Figure 7 , Figure 8 and Figure 15 For example, in NK cells, CD69™ is embedded in a membrane with at least two protease cleavage site motifs that regulate the release of payloads from the desired environment. In this model, single-chain CAR proteins are also expressed at the N-terminus for specific delivery. Optionally, the first and second transmembrane regions are embedded with a NOTCH protein-dependent traction system having secretase cleavage sites within the cell membrane, and multiple cargo / loads are attached to several cleavage sites outside or inside the cell. Figure 7 and Figure 8 This system incorporates more than two protease cleavage site motifs, enabling several potential cargo delivery / release mechanisms. The system also possesses the potential to release intracellular payloads.

[0146] Apart from Figure 7 The model described herein can optionally be embedded into the system by adding a single-chain (α-SAA) to recruit its own related antigens. Figure 8 The system is Figure 7 The improvement features an additional single-chain antibody against an autoassociated antigen (α-SAA) linked to an intracellular domain of CD45. "α-SAA" represents a single-chain antibody or antibody domain targeting or binding to an autoassociated antigen, preferably scFv or camel antibody (VHH domain or VHH domain antibody) or a single-domain antibody. Autoassociated antigens include CD45, CD148, or CD43.

[0147] Linkers are peptide regions that separate functional units in fusion proteins. (Chen X, Zaro JL, Shen WC. Fusionprotein linkers: property, design, and functionality.) Adv Drug Deliv Rev.2013;65(10):1357-1369. doi:10.1016 / j.addr.2012.09.039 discloses several considerations for selecting linkers. Those skilled in the art will be able to follow the teachings of this reference and select linkers based on load. In some cases, when peptides composed of multiple functional domains need to be kept at a certain distance, non-cleavable rigid linkers are used. Human muscle aldolase (HMA) is an example of a rigid linker used to keep protein domains separated at a set distance. Similarly, flexible, non-cleavable linkers are often used in peptides containing multiple functional domains that can move freely. These include polyglycine / serine linkers widely used in chimeric proteins. Linkers can be of any size required to perform their function, but are preferably 2-128 amino acids in length, with a most preferred length of 4-20 amino acids. Other linker lengths considered include 3–100 amino acids, 5–50 amino acids, 6–45 amino acids, 7–40 amino acids, 8–30 amino acids, 10–20 amino acids, and 5–15 amino acids. Table 4 below provides examples of linkers and some constructs containing linkers. Linkers disclosed in iGEM (http: / / parts.igem.org / Protein_domains / Linker) are also considered.

[0148] Table 4: Connectors and certain components containing connectors

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157] Table 5 lists all the relevant sequences disclosed in the following embodiments.

[0158] Table 5: Exemplary protein sequence

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167] In the context of this invention, a payload is any amino acid sequence intended for delivery into living organisms. Suitable payloads include full-length proteins and peptides, enzymes, cytokines, chemokines, signaling proteins, and many other proteins with multiple functions. In this invention, payloads are typically therapeutic antibodies, bispecific binders, and cytokines. The payload size is preferably less than 2000 amino acids.

[0168] The payload delivery system of this invention is intended to be part of cell therapy. The payload delivery system can deliver the payload to cells via electroporation, extracellular vesicles, nanoparticles, and transient or stable genetic modifications. The protein sequences disclosed herein can be extracellular and / or intracellular. Localization on the cell membrane depends on the type of transmembrane system used.

[0169] In some implementations, exfoliases are an actual part of the peptide delivery system. When cells are under conditions intended for the release of exfoliases, the exfoliases are released, thereby enabling their delivery to treat conditions unrelated to the exfoliases. Inactive or cleaved exfoliases can be expressed on the cell surface and can be activated under suitable conditions, where triggers can lead to the activation and / or recombination and assembly of the exfoliase into an activated exfoliase. Furthermore, exfoliases can also be expressed through systems based on artificial promoters that are activated under suitable conditions, including receptor connection and activation or cleavage of receptor-linked membrane-bound transcription factors. Target cells may release certain exfoliases due to apoptosis caused by exogenous or endogenous pathways, including Caspase 3. Caspase-3 is a key protein in mammalian cells involved in apoptosis, or programmed cell death. It belongs to the cysteine ​​protease family called caspases and plays a crucial role in initiating and executing apoptosis. Caspase-3 is considered an effector caspase because it functions downstream of the apoptotic signaling pathway and is responsible for executing the final stage of cell death. Once dead cells release Caspase-3, it is released into the TME and releases peptides.

[0170] Several exfoliated enzymes have been differentially expressed in the tumor microenvironment (TME) compared to normal tissues. For example, MMP, GrzB, and ADAM, ADAMTS are commonly overexpressed in many different types of cancer, including breast, lung, and pancreatic cancer. Differential expression of exfoliated enzymes in the TME is significant for tumor progression and metastasis. For instance, increased expression of MMP9, MMP2, ADAM10, and ADAM17 in the TME is associated with increased release of pro-tumor growth factors and cytokines such as epidermal growth factor (EGF) and tumor necrosis factor-α (TNF-α). This leads to increased tumor cell proliferation, survival, and invasion. Overall, differential expression of exfoliated enzymes in the TME compared to normal tissues highlights the potential of targeting these enzymes as a therapeutic strategy for cancer treatment. Therefore, site-specific delivery of payloads can be leveraged using differential expression of exfoliated enzymes.

[0171] We have designed a cell-based peptide delivery system capable of directly delivering and releasing payloads / cargo to the desired site of action. This system comprises single-transmembrane or multi-transmembrane (TM) proteins, or C-type lectin proteins embedded in the carrier cell membrane, and carries additional motifs that regulate payload delivery / release within the desired microenvironment. Therefore, our goal is not direct systemic injection of biologics, but rather the infusion of engineered cells or their derived extracellular vesicles, which can precisely deliver payloads to the desired site and release them at the appropriate time.

[0172] Although this delivery system is cell type independent, we engineered NK cells to generate and deliver bispecific conjugates within the tumor microenvironment. This delivery is further regulated and can be triggered by either NK cell activation (activation-induced cleavage) or the tumor microenvironment (cleavage upon encountering specifically enriched proteases in the TME). Figure 19 NK cell activation can be mediated by their germline-encoded activation receptors or by specifically engineered receptors, such as chimeric antigen receptors targeting tumor antigens. NK cell activation-induced lysis can be achieved through the release of granzyme B after degranulation or through physical traction (e.g., in Notch activation). Furthermore, this activation-stimulated release will lead to NK cell proliferation and activation depending on the availability of tumor antigens. This is a next-generation cancer immunotherapy system with enhanced capabilities to generate and release payloads under desired conditions and at specific sites.

[0173] Although various exfoliases can be used in this payload delivery system, current research primarily uses matrix metalloproteinases (MMPs) as a universal example, as these enzymes allow for environment-specific cargo release. Models based on the ADAM family of MMPs and serine protease granzyme B have also been proposed. In these models, the addition of extracellular and / or intracellular domains enables the regulation of auxiliary functions such as tumor targeting, amplification, and / or activation. These additional domains can include various components: such as extracellular cytokines, drugs or prodrugs, chimeric antigen receptor (CAR) or diantigen receptor (DAR) domains, or intracellular regulators, inhibitors, proteins containing signaling domains, transcription factors, transcription activators or inhibitors, DNA-modifying enzymes (such as HDAC), proteins containing localization and dimerization domains, proteins containing degradation and dimerization domains, proteins containing hypoxia-responsive domains, proteins that activate immune cells, proteins that enhance immune cell persistence and proliferation, proteins that induce immune cell migration, and splitting receptors (such as splitting CARs), etc.

[0174] Furthermore, the system is designed for the passive release of specific MMPs dependent on cancer cell release in a tumor environment, or for the active release induced by receptor-mediated activation of producing cells.

[0175] Membrane embedding systems can also be designed in such a way that they are sorted into specific subcellular compartments. In some embodiments, we engineer the payload to be sorted into extracellular vesicles (EVs) such as exosomes. The cell releases these exosomes in large quantities, and the payload can be released after being cleaved by proteases within the TME or other target sites. Figures 4A-4B , Figures 5A-5B , Figure 9 , Figure 10 and Figures 24-28 In theory, these EVs can also be obtained from in vitro culture and given to patients as ready-made therapeutic agents.

[0176] In some embodiments, in NK cells or T cells, CD69™ is embedded in a membrane having at least two protease cleavage site motifs, which regulate the release of the desired environmental load. Figures 17A-17B and Figures 18A-18B ).Apart from Figures 17A-17B and Figures 18A-18B In addition to the model described herein, the system may optionally be equipped with a single-chain CAR protein at the N-terminus for specific delivery. Figure 6 ).

[0177] In some implementations, a modified tetraspan membrane protein may be used, in which more than two protease cleavage site motifs are inserted, possessing several potential cargo delivery / release capabilities. This system also has the potential to release intracellular payloads. Figure 7 ).

[0178] In some implementations, the further modified tetraspan membrane protein can be used with an additional single strand of an anti-self-associated antigen (a-SAA) linked to an intracellular domain of CD45. Figure 8 ).

[0179] In some embodiments, the intracellular or extracellular domains include OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1, programmed death receptor-I (PD-1), inducible T cell co-stimulatory factor (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CDI 1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Feγ receptor, MHC class I molecules, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), and co-stimulatory ligands (PD-L2, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1, programmed death receptor-I (PD-1), inducible T cell co-stimulatory factor (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CD137)), inducible T cell co-stimulatory factor (CD79a), DAP-10, Feγ receptor, MHC class I molecules, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), and co-stimulatory ligands (PD-L2, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1, programmed death receptor-I (CD80), inducible T cell co-stimulatory factor (CD79a), inducible T cell co-stimulatory factor (CD7 BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecules (ICAM, CD30L, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, IT GAM, CD1 1b, ITGAX, CD1 1e, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD 19a. A ligand that specifically binds to CD83 or any combination thereof.

[0180] In some implementations, the antigen-binding domain specifically binds to BCMA, MUC16 (also known as CA125), EGFR, EGFRvIII, MUCI, Flt-3, WT-1, CD38, CD70, CD90, CD133, MHC-WTI, TSPANI0, MHC-PRAME, MHC-NY-ESOI, HER2 (ERBB2), CA-IX (carbonic anhydrase IX), LIVI, ADAMI0, CHRNA2, and LeY. NKG2D, CSI, CD44v6, CD24, LGR5, ALDH, ALDH1, CD54, Sca1, CD271, CD123, CD36, CD109, CD110, CD71-negative, CCA, ABCG2, Claudin-18.2 (Claudin-18A2 or Claudin18 isoform 2), PSCA, DLL3 (Delta-like protein 3, Drosophila Delta homolog 3, Delta3), Mud 7 (Mucinl) 7. Muc3, FAPα (fibroblast activator protein α), Ly6G6D (lymphocyte antigen 6 complex site protein G6d, c6orf23, G6D, MEGTI, NG25), PSMA, MSLN or RNF43 (E3 ubiquitin protein ligase RNF43, cyclic finger protein 43), BAFF, C242 antigen, disialotetine ganglioside (GD2), 4-IBB, 5T4, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44v6, CD44, CD51, CD52, CD56, C D74, CEA, CNT0888, CTLA-4, DR5, EpCAM, FAP, fibronectin extradomain-B, folate receptor 1, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scattering factor receptor kinase, IGF-1 receptor, IGF-I, IgGI, LI-CAM, integrin α5β1, integrin αvβ3, podin, MORAb-009, MS4A1, MUC1, mucin CanAg, C-MET, CCR4, CD152, CD10, CD19, CD20, CD200, N-hydroxyacetylneuraminic acid, NPC-IC, PDGF-R a. PDL192, phosphatidylserine, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tendinogen C, TGF-β, TRAIL-R1, TRAIL-R2, folate receptor, transferrin receptor or any combination thereof.These antigen-binding domains can be used in CARs and DARs targeting these antigens. CARs and / or antibodies targeting these antigens are disclosed in, for example, the following literature: BCMA-WO201616630, WO2020150339, WO2019196713, WO2016014565, WO2017025038; MUC16: US9,169,328, WO2016149368, WO2020023888; EGFRvIII: WO2017125830, WO2016016341; Flt3: WO2018222935, WO20200102 84. WO2017173410; CD20: WO2018145649, WO2020010235, WO2020123691; CD38: WO2017025323; CD70: WO2019152742, WO201 8152181; CD33: WO2016014576; CD133: WO2018072025; CSI: WO2019030240; RORI: WO2016115559; CD19: WO2002077029, USI 1,077,144; Claudin: WO2018006882, WO2021008463; DLL3: WO2020180591; WTI: US20160152725A1, US7622119B2; CD23: US6011138A, C N1568198A; CD30: US10815301B2, US10808035B2; PRAME: US20180148503A1, WO2020186204A1; LIVI: US20200231699A1; NKG2D: WO2021 l 79353A1, US20210269501A1; FAP α: US20200246383A1, US20210115102A1; PSMA: US20210277141A1, WO2020108646A1; MSLN: CN109680002A, CN109628492A.

[0181] In some embodiments, the exfoliase cleavage site comprises one or more of a plurality of protease cleavage sites and can be recognized by one or more of the following: thrombin, trypsin, plasmin, prostate-specific antigen (PSA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase (MMP), matriptase (MT-SP1), podin, integrin metalloproteinase (ADAM), and transmembrane serine protease (TMPRSS).

[0182] In some embodiments, the exfoliase that recognizes a protease cleavage site or one or more of a plurality of protease cleavage sites is one or more of the following: thrombin, trypsin, plasmin, prostate-specific antigen (PSA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uP). AR), matrix metalloproteinase (MMP), matriptase (MT-SP1), pod protein, integrin metalloproteinase (ADAM), transmembrane serine protease (TMPRSS), granzyme B, activated protein C, caspase, cathepsin, chymotrypsin, elastase, guanidinobenzoatase, HtrA1, human neutrophil elastase, lactoferrin, Marapsin, NS3 / 4A, PACE4, tissue plasminogen activator (tPA), DESC1, DPP-4, hepsin, matriptase-2, secretase, kallikrein-related peptidase (KLK) and trypsin, or serine proteases, cysteine-type lysosomal proteases, metalloproteinases, coagulation factor proteases or aspartic-type lysosomal proteases.

[0183] In some embodiments, the payload comprises a cytokine or chemokine composed of a portion or derivative of sulfated xylan, which is an antagonist selected from ligands included in the following list: IL-1a, IL-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-3, IL-14, IL-15 or IL-17 cytokine family (including IL-25), interferon, G-CSF, M-CSF, GM-CSF, BDNF, CNTF, EGF, EPO, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18 , FGF19, FGF20, FGF21, FGF22, FGF23, LIF, PDGF, SCF, TGFa, TGFB, TNFa, TNFB, TPO, VEGF, GH, NGF, NT3, NT4, NT5, NT6, NT7, Oncostatin M OSM, insulin, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-1 family members including MIP-1a, MIP-2, eosinophil chemokines (eosinophil chemokine-1, eosinophil chemokine-2, or eosinophil chemokine-3), PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase; cathepsins, cell adhesion molecules (e.g., PECAM-1), soluble receptors or cell-binding receptors or virus-binding receptors, cytokine-induced neutrophil chemotactic agents (KC), TNF-α and IFN-γ, and other soluble inflammatory mediators (e.g., reactive oxygen species and nitric oxide), CCL1, CCL2, CCL3, etc. CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, or any combination thereof.

[0184] In some embodiments, the payload may contain thrombin. In other embodiments, the payload may be a vaccine. In yet another embodiment, the payload may be urokinase delivery. The construct of the present invention enables payload delivery across the blood-brain barrier.

[0185] As used herein, “α-TAA” refers to a single-chain antibody or antibody domain that targets or binds to a tumor-associated antigen, preferably scFv, camel antibody (VHH domain antibody or VHH domain), single-domain antibody, or antibody-binding domain. Exemplary tumor-associated antigens that bind to the initiating peptide used in this invention include, for example, pan-B cell antigens (such as CD20 found on the surface of malignant B cells such as non-Hodgkin's lymphoma and non-malignant B cells) and pan-T cell antigens (such as CD2, CD3, CD5, CD6, CD7). Other exemplary tumor-associated antigens include, but are not limited to, MAGE-1, MAGE-3, MUC-1, HPV 16, HPV E6 & E7, TAG-72, CEA, α-Lewisy, L6-antigen, CD19, CD22, CD25, CD30, CD33, CD37, CD44, CD52, CD56, mesothelin, PSMA, HLA-DR, EGF receptor, VEGF receptor, and HER2 receptor. Carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) are two examples of such tumor-associated antigens. Other targets include the MICA / B ligand of NKG2D. These molecules are expressed on a variety of tumor types but are not typically expressed on healthy cells.Other specific examples of tumor-associated antigens include epithelial cell adhesion molecules (Ep-CAM / TACSTD1), mesothelin, tumor-associated glycoprotein 72 (TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1, MDA7, cancer-associated viral vaccines (such as human papillomavirus antigen), prostate-specific antigens (PSA, PSMA), RAGE (renal antigen), CAMEL (melanoma CTL recognition antigen), and CT antigens (such as MAGE-B5, MAGE-B6, M...). AGE-C2, MAGE-C3 and MAGE-D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE and SAGE), mucin antigens (such as MUC1, mucin-CA125, etc.), cancer-associated ganglioside antigens, tyrosinases, gp75, C-myc, Mart1, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7, Ep-CAM, tumor-derived heat shock proteins, etc. (see also Acres, etc.) et al., Curr Opin Mol Ther 2004 February, 6:40-7; Taylor-Papadimitriou et al., Biochim Biophys Acta. 1999 Oct. 8; 1455(2-3):301-13; Emens et al., Cancer Biol Ther. 2003 July-August; 2(4 Suppl 1):S161-8; and Ohshima et al., Int J Cancer. 2001 Jul. 1; 93(1):91-6). Other exemplary tumor-associated antigen targets include CA 195 tumor-associated antigen-like antigen (see, for example, U.S. Patent No. 5,324,822) and female urine squamous cell carcinoma-like antigen (see, for example, U.S. Patent No. 5,306,811), as well as breast cell tumor-associated antigen as described in U.S. Patent No. 4,960,716.

[0186] Preferably, such tumor-associated antigens are located on or within the cell membrane of tumor cells. Examples of tumor-associated antigens are described, for example, by DeVita et al. (Eds., “Biological Therapy of Cancer”, 2nd Edition, Chapter 3: Biology of Tumor Antigens, Lippincott Company, ISBN 0-397-51416-6 (1995)). Non-limiting examples of such tumor antigens include CD19, CD20, CD30, CD33, CD38, CD133, BCMA, TEM8, EpCAM, ROR1, folate receptor, CD70, CEA, BAGE, CA-125, CDK-1, MART-1, MUC-1, MUM-1, PSA, PSMA, HER-2, IL13Rα, IL13Rα2, AIM-2, AIM-3, BRAP, RTN4, GLEA2, T NKS2, KIAA0376, RBPSUH, NKTR, EGFRvIII, LICAM, Livin, Livinβ, Nestin, OLIG2, ART1, ART4, Gli1, Cav-1, CD74, E-cadherin, GAGE-1, Ganglioside / GD2, PROX1, PSCA, βhCG, WT1, Mesothelin, melan-A, SSX-2, PLK1, VEGF-A, VEGFR2, and Tie-2.

[0187] Time-based air conditioning control and delivery The present invention envisions the use of embodiments to achieve targeted payload release in tumor microenvironments or tissue-specific environments. This allows for optimal timing and anatomical localization of cargo release and activation, such as the release and demasking of pro-inflammatory cytokines (TNF, IL2, IL15, IL18) in the tumor microenvironment, or the conditional release of certain growth factors in non-malignant diseases (e.g., GMCSF in bone marrow transplantation, IL2 in certain immunodeficiency disorders, and insulin in diabetes). Furthermore, the temporal and conditional cargo release according to the invention is envisioned to limit currently observed systemic off-target effects and / or limit persistent exposure and response. Conditioned induction of responses is beneficial in various diseases with pronounced diurnal rhythm-like patterns in symptom development, such as inflammatory bowel disease and diabetes.

[0188] It is also crucial to utilize bispecific conjugates and other proteins with short half-lives and / or a high risk of off-target activity in other tissues within the correct microenvironment and at the right time. Embodiments of the present invention are applicable to situations where spatiotemporal release is critical. One such example is the generation of a bitransmembrane protein encoding a bispecific CD38-CD3 conjugate in an extracellular loop, which is cleaved only in the bone marrow microenvironment and used in conjunction with an anti-BCMA CAR, thereby reducing the released conjugate and the off-target activity of the CAR. In this model system, the very short half-life of the bispecific CD38-CD3 conjugate advantageously makes systemic distribution and effects less likely. Furthermore, with embodiments of the present invention, due to the continuous protein renewal and release by the transporting cells, a single administration of cells is expected to be more potent than multiple administration regimens of other delivery systems, and in some cases achieve equivalent therapeutic effects, while incurring limited side effects.

[0189] Example 1: General Structure In one embodiment, the present invention includes a delivery system for a protein payload, wherein an exfoliating enzyme present in vivo releases the payload. (Reference) Figures 1-2 A payload delivery system for delivering a single payload includes a first transmembrane anchoring region, a cytoplasmic region, a first site that can be cleaved by an exfoliase, a first adapter, a payload sequence, a second adapter, a second site that can be cleaved by an exfoliase, and an optional single-stranded specific CAR.

[0190] The transmembrane anchoring region can be selected from CD63, CD9, CD81, CD28, CD4, CD8, CD34, CD69, CD19, CD20, integrin, TNFSF14, TNR1A, aquaporin, NOTCH, NgR1, NRG1, GPi anchor, EGFR, rhodopsin, or any relevant TMD that provides a similar function such as membrane anchoring, and signal transduction regulation can be used for expression in this invention.

[0191] The cytoplasmic domain may be selected from CD63, CD69, CD9, CD28, CD81, CD34, CD3, CD4, CD8 or any related cytoplasmic domain that can be used to provide similar functions to this system (e.g., in terms of anchoring, intracellular fluid mobility and / or sorting to EVs and signal transduction).In some embodiments, the intracellular or extracellular domains include OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1, programmed death receptor-I (PD-1), inducible T cell co-stimulatory factor (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CDI 1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Feγ receptor, MHC class I molecules, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), and co-stimulatory ligands (PD-L2, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1, programmed death receptor-I (PD-1), inducible T cell co-stimulatory factor (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CD137) / CD137), CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1, programmed death receptor-I (CD80), inducible T cell co-stimulatory factor ... BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecules (ICAM, CD30L, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 la, LFA-1, IT GAM, CD1 1b, ITGAX, CD11e, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD 19a. A ligand that specifically binds to CD83 or any combination thereof.

[0192] The connector can be selected from any suitable connector disclosed herein, such as those listed in Table 4.

[0193] The exfoliase and its cleavage site can be selected from part-time or dedicated exfoliases. Dedicated exfoliases can be selected from ADAM proteases, BACE proteases, serine proteases granzyme-B, and Site-1 proteases. Adam proteases (metalloproteinases) are membrane-anchored type I proteases, including ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, and ADAM33. BACE (aspartic acid) proteases are also membrane-anchored type I proteases, including BACE1 and BACE2. Site-1 (serine) proteases are membrane-anchored type I proteases, including SKI-a or S1P.

[0194] Partial exfoliases and their cleavage sites include transmembrane peptidase B, MT-MMP (membrane matrix metalloproteinase), proprotein convertase, transmembrane serine protease, matrix metalloproteinase, podocyte protein, cathepsin S, and cathepsin L. Transmembrane peptidase B is a membrane-anchored type 1 metalloproteinase. MT-MMPs are membrane-anchored type 1 or GPI-anchored proteases, including MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, and MT6-MMP. Proprotein convertases are membrane-anchored type 1 or soluble proteases, including PCSK1 / 3, PCSK2, furin, PCSK4, PCSK5 / 6, PACE4, PCSK7, and PCSK9. Transmembrane serine proteases are membrane-anchored type II proteases, including Matriptase, Matriptase-2, Matriptase-3, polymerase-1, Corin, Hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, Spinesin, intestinal peptidase, HAT, DESCL1, TMPRSS11A, HAT-like 4, and HAT-like 5. Matrix metalloproteinases are soluble proteases, including MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A-B, MMP24, MMP25, MMP26, MMP27, and MMP28.

[0195] In some embodiments, the payload comprises a cytokine or chemokine composed of a portion or derivative of sulfated xylan, which is an antagonist selected from ligands comprising the following list: IL-1a, IL-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-3, IL-14, IL-15, or the IL-17 cytokine family (including IL-25), interferon, G-CSF, M-CSF, GM-CSF, BDNF, CNTF, EGF, EPO. , FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF1 8. FGF19, FGF20, FGF21, FGF22, FGF23, LIF, PDGF, SCF, TGFa, TGFB, TNFa, TNFB, TPO, VEGF, GH, NGF, NT3, NT4, NT5, NT6, NT7, Oncostatin M OSM, insulin, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-1 family members including MIP-1a, MIP-2, eosinophil chemokines (eosinophil chemokine-1, eosinophil chemokine-2, or eosinophil chemokine-3), PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase; cathepsins, cell adhesion molecules (e.g., PECAM-1), soluble receptors or cell-binding receptors or virus-binding receptors, cytokine-induced neutrophil chemotactic agents (KC), TNF-α and IFN-γ, and other soluble inflammatory mediators (e.g., reactive oxygen species and nitric oxide), CCL1, CCL2, CCL3, etc. CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, or any combination thereof.

[0196] In other embodiments, the system can be further modified by adding more cleavable exfoliases and more payload, either extracellularly or internally. The exfoliases cleave the peptides or proteins to release them. The peptides or proteins may be active upon cleavage, or may still require additional activation through mechanisms present in the cell. These activation mechanisms can be naturally occurring or genetically engineered. Figures 4A-4B and Figures 5A-5B An embodiment of the invention comprising four transmembrane domains is shown. To generate a fine delivery system capable of accommodating different payloads in intracellular and extracellular environments, multiple transmembrane crossings are required. In this embodiment, we use a tetratransmembrane protein (TMD) linked by payloads spanning exfoliation sites to form the delivery system. This includes a first protease site cleavable by MMP9, a TNF-α payload, and a second protease site cleavable by MMP9. Figures 4A-4B and Figures 5A-5B The difference lies in whether or not an EV sorting sequence exists.

[0197] Example 2: BIKE delivery In one embodiment, the delivery system of the present invention is configured to deliver bikes. (Reference) Figure 2 One embodiment of the invention comprises a BIKE flanked by a protease cleavage sequence embedded in an extracellular loop between a type II transmembrane protein and a GPI anchor. This particular embodiment is encoded by the amino acid sequence shown below in SEQ ID NO: 123 or SEQ ID NO: 1. The construct consists of an N-terminal intracellular domain, followed by a type II transmembrane domain, a cleavage sequence, and a payload, followed by another cleavage sequence. The C-terminus of the protein is bound to the plasma membrane via a GPi anchor. Exfoliation causes cleavage, thereby releasing the payload into the extracellular environment.

[0198]

[0199]

[0200] This sequence is also Figure 3 The diagram is shown in the figure, accompanied by a legend to illustrate the structural domains and motifs.

[0201] The corresponding cDNA is shown below:

[0202] like Figure 3 As shown, SEQ ID NO: 123 or SEQ ID NO: 1 is divided into the following functional motifs: CD63-cytoplasmic MCGACKENYC (SEQ ID NO: 125), encoded by the following DNA: ATGTGCGGCGCATGCAAGGAAAACTACTGT (SEQ ID NO: 126) CD63-TM-HELICAL (from inside to outside) LMITFAIFLSLIMLVEVAAAI (SEQ ID NO: 127), encoded by the following DNA:

[0203] The adapter-a: GGSGPVRRYQ (SEQ ID NO: 129) is encoded by the following DNA:

[0204] MMP2: GGPLGVRGG (SEQ ID NO:131), which is encoded by the following DNA:

[0205] VL-a-CD16-118AA

[0206] It is encoded by the following DNA:

[0207] Linker-b and linker-c: GGGGSGGGGSGGGGS 15AA (SEQ ID NO:135), encoded by the following DNA: (SEQ ID NO: 136).

[0208] VH-a-CD16-107AA It is encoded by the following DNA:

[0209]

[0210] Flanking 20AA: PSGQAGAAASESLFVSNHAY (SEQ ID NO:139), encoded by the following DNA:

[0211] 7AA flanking peptide: EASGGPE (SEQ ID NO:141), which is encoded by the following DNA:

[0212] VL-a-CD33-116AA (SEQ ID NO: 143), which is encoded by the following DNA:

[0213] VH-a-CD33

[0214]

[0215] Myc-tag EQKLISEEDL (SEQ ID NO: 147), which is encoded by the following DNA:

[0216] MMP9: GGPLGMTS (SEQ ID NO:149), encoded by the following DNA:

[0217] CD63-extracellular

[0218]

[0219] GPi-anchor: VLRDKLVKCEGISLLAQNTSWLLLLLLSLSLLQATDFMSL (SEQ ID NO: 153), encoded by the following DNA:

[0220] Example 3: Loading delivered to extracellular vesicles refer to Figures 4A-4B This embodiment comprises a payload containing a cleavable MMP sequence embedded in an extracellular loop between the third and fourth transmembrane domains of a tetramembrane protein. Due to the presence of the GYEVM motif (SEQ ID NO: 337), this specific molecule will be sorted into extracellular vesicles.

[0221] The amino acid sequence in SEQ ID NO 155 below encodes this construct.

[0222]

[0223] The corresponding cDNA is shown below:

[0224]

[0225] SEQ ID NO: 155 is divided into the following functional motifs: CD63-cytoplasm: MCGACKENYC (SEQ ID NO: 157), encoded by the following DNA:

[0226] CD63-TM-HELICAL (from inside to outside) LMITFAIFLSLIMLVEVAAAI (SEQ ID NO:159), encoded by the following DNA:

[0227] The adapter-a: GGSGPVRRYQ (SEQ ID NO:161), is encoded by the following DNA:

[0228] MMP2: GGPLGVRGG (SEQ ID NO:163), encoded by the following DNA:

[0229] VL-a-CD16-118AA

[0230]

[0231] Linker-b and linker-c: GGGGSGGGGSGGGGS 15AA (SEQ ID NO:167), encoded by the following DNA:

[0232] (SEQ ID NO: 168).

[0233] VH-a-CD16-107AA

[0234] It is encoded by the following DNA:

[0235] Flanking 20AA: PSGQAGAAASESLFVSNHAY (SEQ ID NO: 171), encoded by the following DNA:

[0236] 7AA flanking peptide: EASGGPE (SEQ ID NO: 173), encoded by the following DNA:

[0237] VL-a-CD33-116AA

[0238]

[0239] VH-a-CD33

[0240]

[0241] Connector and Myc-TAG GGSGEQKLISEEDLGG (SEQ ID NO: 179)

[0242] CD63-TM-HELICAL (outside in) LVVAAAALGIAFVEVLGIVFA (SEQ ID NO: 185)

[0243] CD63-cytoplasmic CCLVKSIRSGYEVM (SEQ ID NO: 187)

[0244] Example 4: BIKE expressed on cell surface refer to Figures 5A-5B One embodiment of the present invention is a BIKE containing an MMP / GrzB cleavage sequence embedded in an extracellular loop between the third and fourth transmembrane regions of a tetramembrane protein. Because of the lack of the signaling motif GYEVM (SEQ ID NO: 337) required for sorting to extracellular vesicles, this molecule will be expressed on the cell surface.

[0245] The construct has the amino acid sequence listed in SEQ ID NO: 188 below:

[0246] The corresponding cDNA is shown below (SEQ ID No: 189):

[0247]

[0248] The following SEQ ID NO 188 is divided into the following categories: Figure 11 The functional structural domains and motifs shown below are: CD63-cytoplasmic: MCGACKENYC (SEQ ID NO: 190), encoded by the following cDNA:

[0249] CD63-TM-HELICAL (from inside to outside) LMITFAIFLSLIMLVEVAAAI (SEQ ID NO: 192), encoded by the following cDNA:

[0250] The adapter-a: GGSGPVRRYQ (SEQ ID NO: 194), is encoded by the following cDNA:

[0251] MMP2: GGPLGVRGG (SEQ ID NO: 196), encoded by the following cDNA:

[0252] VL-a-CD16-118AA

[0253] It is encoded by the following cDNA:

[0254] Linker-b and linker-c: GGGGSGGGGSGGGGS 15AA (SEQ ID NO:200), encoded by the following cDNA:

[0255] VH-a-CD16-107AA

[0256] It is encoded by the following cDNA:

[0257] Flanking 20AA: PSGQAGAAASESLFVSNHAY (SEQ ID NO:204), encoded by the following cDNA:

[0258] 7AA flanking peptide: EASGGPE (SEQ ID NO:206), encoded by the following cDNA:

[0259] VL-a-CD33-116AA

[0260] It is encoded by the following cDNA:

[0261] VH-a-CD33

[0262] It is encoded by the following cDNA:

[0263] The Myc-tag ggsgeqkliseedlgg (SEQ ID NO: 380) is encoded by the following cDNA:

[0264] MMP9: ggPLGMTS (SEQ ID NO: 149), encoded by the following cDNA:

[0265] CD63-extracellular

[0266] It is encoded by the following cDNA:

[0267] CD63-TM-HELICAL (outermost to innermost) LVVAAAALGIAFVEVLGIVFA (SEQ ID NO: 185), encoded by the following cDNA:

[0268] CD63-cytoplasmic CCLVKS (SEQ ID NO: 325) is encoded by the following cDNA:

[0269] Example 5: Delivery of TRIKE containing ADAM 17 refer to Figures 13-14 In one embodiment, the present invention comprises a TRIKE containing an ADAM17 target sequence embedded in both type II TM (TNFSF14) and type I TM (TNR1A) domains. This molecule is expressed on the cell surface and cleaves upon NK cell activation.

[0270] The construct is listed below in SEQ ID NO: 212:

[0271] The corresponding cDNA is shown below:

[0272]

[0273] Domains and motifs: SEQ ID NO: 212 is divided into the following and as follows Figure 14 The functional structural domains and motifs are shown.

[0274] TNFSF14 cytoplasmic MSCSVAR (SEQ ID NO:214) is encoded by the following cDNA:

[0275] TNFSF14 transmembrane (from inside to outside) VGLGLLLLLMGAGLAVQGWFL (SEQ ID NO: 216), encoded by the following cDNA:

[0276] Extracellular TNFSF14 containing cleavage sequences:

[0277] It is encoded by the following cDNA:

[0278] Linker-a: GGSGPVRRYQ (SEQ ID NO:220), encoded by the following cDNA:

[0279] VL-a-CD16-118AA

[0280]

[0281] TRIKE-connector: GGGGSGGGGSGGGGS (SEQ ID NO: 224), encoded by the following cDNA:

[0282] VH-a-CD16-107AA

[0283] It is encoded by the following cDNA:

[0284] Flanking 20AA: PSGQAGAAASESLFVSNHAY (SEQ ID NO: 228), encoded by the following cDNA:

[0285] 7AA flanking peptide: EASGGPE (SEQ ID NO: 230), encoded by the following cDNA:

[0286] VL-a-CD33-116AA

[0287] It is encoded by the following cDNA:

[0288] TRIKE-connector: GGGGSGGGGSGGGGS (SEQ ID NO: 234), encoded by the following cDNA: TCTGGAGGCGGCGGATCTGGCGGAGGAGGCAGCGGCGGCGGCGGTTCA (SEQ ID NO: 235).

[0289] TRIKEVH-a-CD33

[0290] It is encoded by the following cDNA:

[0291] Myc-tag GGSG EQKLISEEDLS GGSG (SEQ ID NO: 238), which is encoded by the following cDNA:

[0292] TNR1A is extracellular and contains the ADAM17 cleavage sequence SLECTKLCLPQIENVKGTEDS (SEQ ID NO: 240), encoded by the following cDNA:

[0293] Vh-a-CD38

[0294] It is encoded by the following cDNA:

[0295] The linker GGGGSGGGGSGGGGS (SEQ ID NO: 244) is encoded by the following cDNA:

[0296] Vl-a-CD38

[0297] It is encoded by the following cDNA:

[0298]

[0299] TNR1A-transmembrane SGTTVLLPLVIFFGLCLLSLLFIGLMYRY (SEQ ID NO: 248) is encoded by the following cDNA:

[0300] 41BB-intracellular: KRGRKKLLYIFKQPFMRPVQTTQEEDG (SEQ ID NO: 250), encoded by the following cDNA:

[0301] Example 6: BIKE with anti-CD38 containing granzyme B cleavage sequence refer to Figure 15 and Figure 16 In some embodiments, the present invention comprises BIKE+ antiCD38 containing a granzyme-B cleavage sequence having an activation domain of SEQ ID NO 71 or 252. The molecule will be membrane-bound and released after granzyme-B release following NK cell degranulation. AntiCD38 will remain on NK cells after the release of the remaining cargo and will have a function similar to a chimeric antigen receptor (CAR).

[0302]

[0303] The corresponding cDNA is shown below:

[0304]

[0305]

[0306] SEQ ID NO: 252 is divided into as follows Figure 15 And the functional structural domains and motifs shown below.

[0307] CD63-cytoplasmic MCGACKENYC (SEQ ID NO: 254) is encoded by the following cDNA:

[0308] CD63-TM-HELICAL (from inside to outside) LMITFAIFLSLIMLVEVAAAI (SEQ ID NO: 256), encoded by the following cDNA:

[0309] Granzyme B cleavage site: IEPD (SEQ ID NO: 258), encoded by the following cDNA: ATCGAGCCTGAC (SEQ ID NO: 259). Alternatively, granzyme B cleavage sites: EEEEVEADSEEEEEEE (SEQ ID NO: 260), AQGVISADASNLDDFY (SEQ ID NO: 261), or LEADKGKLEYD (SEQ ID NO: 262) can be used.

[0310] Connector-a: GGSG (SEQ ID NO: 263) GGCGGCAGCGGC (SEQ ID NO: 264).

[0311] VL-a-CD16-118AA

[0312] It is encoded by the following cDNA:

[0313] VH-a-CD16-107AA

[0314] It is encoded by the following cDNA:

[0315] Flanking 20AA: PSGQAGAAASESLFVSNHAY (SEQ ID NO: 269), encoded by the following cDNA:

[0316] 7AA flanking peptide: EASGGPE (SEQ ID NO: 271), encoded by the following cDNA:

[0317] VL-a-CD33-116AA

[0318] It is encoded by the following cDNA:

[0319] TRIKE VH-a-CD33

[0320] It is encoded by the following cDNA:

[0321] Myc-tag: GGSGEQKLISEEDLGG (SEQ ID NO: 277), encoded by the following cDNA:

[0322] Granulase B cleavage site. IEPD (SEQ ID NO: 279) ATCGAGCCTGAT (SEQ ID NO: 280).

[0323] CD38Vh:

[0324] It is encoded by the following cDNA:

[0325] Linker: GGGGSGGGGSGGGGS (SEQ ID NO: 283), encoded by the following cDNA:

[0326] CD38V1:

[0327] It is encoded by the following cDNA:

[0328]

[0329] Hinge stem region:

[0330] CD28 transmembrane: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 289), encoded by the following cDNA:

[0331] CD28 intracellular: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:291), encoded by the following cDNA:

[0332] CD3z intracellular:

[0333] Example 7: HEK293 cells with TNF-α and MMP2 or MMP9 cleavage sites refer to Figures 4A-4B , Figures 5A-5B and Figures 9-12 In some implementation schemes, based on the above teachings, we have created Figures 4A-4B , Figure 9 and Figure 10 The constructed CD63-9-2L-TNFa-Myc-9-CD63-cyt shown (containing the EV sorting motif), and Figures 5A-5B , Figure 11 and Figure 12 The construct shown is CD63-9-2L-TNFa-Myc-9-CD63-D-Cyt (lacking the EV sorting motif). These constructs differ only in the extracellular vesicle sorting motif.

[0334] The construct CD63-9-2L-TNFa-Myc-9-CD63-cyt (EV sorting motif) LeGo-CD63-9-2L-TNFa-Myc-9-CD63-cyt is shown in SEQ ID NO: 295 below.

[0335]

[0336] refer to Figures 4A-4B , Figure 9 and Figure 10 The domains and motifs are encoded by the following amino acid sequences.

[0337] CD63-cytoplasm: MAVEGGMKCVK (SEQ ID NO: 297).

[0338] CD63-TM-HELICAL (from inside to outside) FLLYVLLLAFCACAVGLIAVG (SEQ ID NO: 298).

[0339] CD63-Extracellular VGAQLVLSQTIIQGATPGS (SEQ ID NO: 299).

[0340] CD63-TM-HELICAL (outermost from the inside) LLPVVIIAVGVFLFLVAFVGC (SEQ ID NO: 300).

[0341] CD63-cytoplasmic CGACKENYC (SEQ ID NO: 301).

[0342] CD63-TM-HELICAL (from inside to outside) LMITFAIFLSLIMLVEVAAAI ((SEQ ID NO: 302).

[0343] Connector: GGSGPVRRYQ (SEQ ID NO: 303).

[0344] MMP9: GGPLGMTS (SEQ ID NO: 304).

[0345] TNFa:

[0346] Connector + Myc-tag: GGSGEQKLISEEDL (SEQ ID NO: 306).

[0347] MMP9: GGPLGMTS (SEQ ID NO: 307).

[0348] CD63-extracellular

[0349] CD63-TM-HELICAL (outer to inner) LVVAAAALGIAFVEVLGIVFA (SEQ ID NO: 309).

[0350] CD63-cytoplasmic LVKSIRSGYEVM (SEQ ID NO: 310).

[0351] The construct CD63-9-2L-TNFa-Myc-9-CD63-D-Cyt (lacking the EV sorting motif) is described below in SEQ ID NO: 311.

[0352]

[0353] refer to Figures 5A-5B , Figure 11 and Figure 12 The domains and motifs are encoded by the following amino acid sequences.

[0354] CD63-cytoplasm:

[0355] CD63-TM-HELICAL (from inside to outside) FLLYVLLLAFCACAVGLIAVG (SEQ ID NO: 314).

[0356] CD63-Extracellular VGAQLVLSQTIIQGATPGS (SEQ ID NO: 315).

[0357] CD63-TM-HELICAL (outermost) LLPVVIIAVGVFLFLVAFVGC (SEQ ID NO: 316).

[0358] CD63-cytoplasmic CGACKENYC (SEQ ID NO: 317).

[0359] CD63-TM-HELICAL (from inside to outside) LMITFAIFLSLIMLVEVAAAI (SEQ ID NO: 318).

[0360] Connector: GGSGPVRRYQ (SEQ ID NO: 319).

[0361] MMP9: GGPLGMTS (SEQ ID NO: 320).

[0362] TNFa-

[0363] Connector + Myc-tag: GGSGEQKLISEEDL (SEQ ID NO: 322).

[0364] MMP9: GGPLGMTS (SEQ ID NO: 323).

[0365] CD63-extracellular

[0366] CD63-TM-HELICAL (outermost) LVVAAAALGIAFVEVLGIVFA (SEQ ID NO: 185).

[0367] CD63-cytoplasmic CCLVKS (SEQ ID NO: 325).

[0368] Functional testing Example 8: TNFα assay using HEK 293 cells refer to Figure 21 As shown in Figure 22, we use HEK293 NF- A functional assay was developed for β-luciferase reporter cells to measure the presence of TNFα.

[0369] Refer to Figure 22 and Figure 24 To test the functionality of the GAGE ​​payload in this system, HEK293 cells were transfected with the GAGE ​​plasmid using polyethyleneimine (PEI) in the following transient transfection: - Day 0: 400,000 cells were seeded in 6-well plates.

[0370] - Day 1: Transfect 2.5 μg GAGE ​​plasmid into HEK293 wt cells.

[0371] -Day 2: Combine HEK293+GAGE and HEK293 NF- B-luciferase reporter cells were co-cultured for 24 hours or cultured separately.

[0372] - Day 3: Collect and lyse reporter cells, and use NF-125- The functional activity of TNFα in HEK293 cells was determined using a β-luciferase reporter gene assay. (Reference) Figure 26 It is easy to observe that TNFα expressed on HEK293 cells is functional regardless of the presence of EV sorting motifs.

[0373] Example 9: Determination of load release via MMP 9 or MMP2 Next, refer to Figure 25 and Figure 27 The test was conducted to determine whether cells expressing GAGE ​​were released in the presence of MMP9 or MMP2. HEK293 cells were transfected with GAGE ​​and either MMP9 or MMP2 plasmids in the following transient transfections using polyethyleneimine (PEI): - Day 0: 400,000 cells were seeded in 6-well plates.

[0374] - Day 1: Transfect 2.5 μg GAGE ​​and MMP9 or MMP2 plasmids into HEK293 wt cells.

[0375] - Day 2: Co-culture HEK293+GAGE and HEK293+MMP9 or HEK293+MMP2 cells for 24 hours.

[0376] -Day 3: Collect 200 μL of supernatant and administer HEK293 NF- B reporter cells.

[0377] After 6 hours of incubation, reporter cells were collected, lysed with 1% Triton, and then NF-κB was used. The functional activity of TNFα released from HEK293 cells was determined using a β-luciferase reporter gene assay. The substrate (D-luciferin) was automatically added to the sample, and luminescence was measured.

[0378] refer to Figure 25 and Figure 27 It is easy to observe that TNFα is expressed higher in HEK293 cells for EV-sorted plasmids, indicating that most GAGE ​​loads are enriched in EVs more than non-EV-sorted plasmids.

[0379] Example 10: Load delivery to extracellular vesicles To verify that the GAGE ​​load was primarily delivered to the EV, the experiment of Example 9 was repeated using an Amicon® Ultra 0.5 mL centrifuge filter to deplete the supernatant of the EV. HEK293 NF- Luciferase reporter cells were used for luciferase reporter assays. Supernatant from the co-culture, either depleted of EVs or added unchanged, was added to HEK293 reporter cells, which respond to TNFα-loaded NF-κB expression. Figure 26 and Figure 28 The results showed a significant decrease in luciferase activity after EV depletion, indicating that GAGE ​​was present in the supernatant containing extracellular vesicles.

[0380] Example 11: Selection of TMDs with Better Cell Surface Expression To evaluate selective transmembrane (TM) proteins that are primarily expressed on the cell surface, we exchanged CD63 TM with C-type lectin transmembrane CD69. Figure 31 The study showed that the percentage of TNFα-positive cells using CD69 as the TM protein was higher than that using CD63 TM.

[0381] Next, to assess whether GAGE ​​TNFα expressed on HEK293 cells could be released in the presence of MMP9, MMP9 lysis was performed by directly co-culturing GAGE ​​TNFα-expressing cells with MMP-expressing cells, MMP9-transfected cells, or supernatants providing recombinant MMP9 protein. HEK293 NF- β-luciferase reporter cells were used to verify the successful MMP9 cleavage of the MMP9 protease substrate linked to TNFα GAGE ​​using luciferase assays. Figures 17A-17B , Figures 18A-18B , Figure 29 and Figure 30As shown, luciferase activity was observed only in clones containing MMP9 cleavage substrates, indicating that TNFα can be released via cleavage by specific proteases.

[0382] Example 12: Releasing the load using granzyme B We further evaluated the invention using another exfoliase cleavage substrate of granzyme B (GrzB) protein linked to TNFα GAGE ​​as a payload. First, two clones, CD69-GrzB-TNFα-Myc-GrzB-aCD19-CAR and CD69-TNFα-Myc-aCD19-CAR, were generated and transduced into K562 cells. Figure 31 ).

[0383] Next, we evaluated whether GAGE ​​TNFa containing GrzB cleavage substrate expressed on K562 cells could be released upon co-culture with NK92 cells. NK92 cell encounter with target cells typically leads to induced degranulation of NK92 cells containing granzyme B. GrzB cleavage accompanied by TNFa release was performed by co-culturing K562 cells expressing GAGE ​​TNFa with NK92 cells. After 4 hours of incubation, cells were centrifuged, and the supernatant was collected. HEK293 NF-... B-luciferase reporter cells were used to confirm the successful cleavage of GrzB protease substrates linked to TNFα GAGE ​​using luciferase assays. Figure 32 As shown, luciferase activity was high in clones containing GrzB cleavage substrates, indicating that TNFα is specifically released by cleavage of specific proteases.

[0384] Example 13: Determination of the function of delivering nano-luciferase as evidence of principle To increase readout sensitivity, we repeated the experiment of Example 12 with another GAGE ​​payload, in which we used nanoluciferase as the payload.

[0385] We generated the following three clones: -CD69-GrzB-nLuc-Myc-GrzB-aCD19-CAR (GAGE 1 or original GAGE) -CD69-nLuc-Myc-aCD19-CAR(GAGE A) -CD69-GrzB-nLuc-Myc-GrzB-CAR(GAGE B) These plasmids were transduced into K562 cells using MOI6, and then assayed using a luciferase assay. (Reference) Figure 33 We observed the surface expression of GAGE ​​nLuc in K562 cells.

[0386] Next, we evaluated whether GAGE ​​nLuc, expressing a GrzB cleavage substrate, could be released during co-culture with NK92 cells. GrzB cleavage accompanying nLuc release was achieved by co-culturing K562 cells expressing GAGE ​​nLuc with NK92 cells. After 4 hours of incubation, cells were centrifuged, and the supernatant was collected. Successful cleavage of the GrzB protease substrate linked to nLuc GAGE ​​was verified by direct luciferase assay. Figure 34A As shown, luciferase activity was higher in clones containing GrzB cleavage substrates, indicating that nLuc is specifically released by cleavage of the specific protease. Further activation of NK92 cells with PAM / Ion had no significant effect on additional substrate cleavage, a finding supported by... Figure 34B The lack of difference in luciferase activity assays reflects this.

[0387] Example 14: Additional clones to evaluate load fragmentation and release We generated the following clones in KHYG-1: CD69-GrzB-nLuc-Myc-GrzB-aCD19-CAR (GAGE1 or original GAGE), CD69-nLuc-Myc-aCD19-CAR (GAGE A), and CD69-GrzB-nLuc-Myc-GrzB-CAR (GAGE B) to further evaluate specific lysis and load release. We transduced these three clones into KHYG-1 at MOI30. Reference Figure 35 We observed the surface expression of GAGE ​​nLuc in KHYG1 cells.

[0388] KHYG-1 cells expressing GAGE ​​nLuc were co-cultured with Nalm-6 cells (highly expressing CD19). Figure 36 After the three different time points shown, luciferase activity was higher in clones containing GrzB cleavage substrate and nLuc linked to sc-aCD19CAR, indicating that nLuc was specifically released by cleavage of specific proteases during co-culture and had the highest cleavage at three hours of incubation.

[0389] Next, we sort the cells to reduce the background activity of the nanoluciferase. For example... Figure 37A As shown, the KHYG-1 cell positivity rate of the three clones exceeded 99%. Interestingly, compared with the other two clones, the MFI of nLuc GAGE-positive cells was higher in the clone without cleavage substrate sites. Figure 37B ).

[0390] We replicated the experiment on sorted cells and selected two different time points (4 hours and 6 hours) for co-culturing sorted KHYG-1 cells expressing GAGE ​​nLuc with K562 cells. Figure 38 As shown, luciferase activity was higher in clones containing GrzB cleavage substrate and nLuc, indicating that nLuc was specifically released by cleavage of the specific protease during co-culture, with the highest cleavage occurring at 6 hours of incubation.

[0391] To evaluate the specificity and sensitivity of granzyme B cleavage, we generated three clones of CD69-GrzB-nLuc-Myc-GrzB-aCD19-CAR in NK92 cells using different GrzB cleavage substrates: (GrzB(IEPD (SEQ IDNOS: 258)), GrzB(AQGVISADASNLDDFY (SEQ ID NOS: 261)), and GrzB(LEADKGKLEYD (SEQ IDNOS: 262)). We transduced these clones into NK92 cells at MOI30. (Reference) Figure 39A and 39B We observed the surface expression of GAGE ​​nLuc in NK92 cells.

[0392] After co-culturing NK92 cells expressing GAGE ​​nLuc with Raji cells for 4 hours, luciferase activity was significantly higher. Compared to the control clone (GAGE A), the luciferase activity in the clone containing GrzB (LEADKGKLEYD (SEQ ID NO: 262)) linked to nLuc as a lysis substrate increased by 9-fold, indicating that this specific substrate has a higher affinity for GrzB lysis during co-culture. Figure 40 ).

[0393] Example 15: Releasing the load via Caspase 3: We generated the following clones in NK92: CD69-Casp3-nLuc-Myc-Casp3-aCD19-CAR (GAGECasp3) and CD69-nLuc-Myc-aCD19-CAR (GAGE A) to further evaluate specific cleavage and load release. We transduced these two clones into NK92 at MOI30. Reference Figure 41 We observed the surface expression of GAGE ​​nLuc in NK92 cells.

[0394] NK92 cells expressing GAGE ​​Casp3-nLuc were co-cultured with K562 cells Figure 42After the two different time points shown, the luciferase activity was higher in clones containing the Casp3 cleavage substrate of nLuc, indicating that nLuc was specifically released by cleavage of specific proteases during co-culture.

[0395] CD69cyt-TMII-TNFa-Myctag-CD28TM-cyt The cDNA of CD69Cyt-TM-II-TNFa-Myc-CD28TM-Cyt is shown in SEQ ID NO: 326 below.

[0396]

[0397] The corresponding amino acid sequence is shown in SEQ ID NO: 327.

[0398]

[0399] SEQ ID NO: 327 is also shown in Figures 17A-17B The diagram includes illustrations of structural domains and motifs.

[0400] CD69-cytoplasmic MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVP (SEQ ID NO: 328).

[0401] CD69-TM-II type (from inside to outside) VLCAVMNVVFITILIIALIAL (SEQ ID NO 329).

[0402] CD69-extracellular SVGQYNGGSGPVRRYQ (SEQ ID NO 330).

[0403] TNFa

[0404] Connector GGSG (SEQ ID NO 332).

[0405] Myc-label EQKLISEEDL (SEQ ID NO: 333).

[0406] CD28TM FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO 334).

[0407] CD69cyt-TMII-9-TNFa-Myctag-9-CD28TM-cyt The cDNA of CD69cyt-TMII-9-TNFa-Myctag-9-CD28TM-cyt is shown in SEQ ID NO: 335.

[0408]

[0409]

[0410] The corresponding amino acid sequence is shown in SEQ ID NO: 336 below, and this sequence is also displayed in Figures 18A-18B The diagram includes illustrations of structural domains and motifs.

[0411]

[0412] Domains and motifs CD69-cytoplasmic MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVP (SEQ ID NO: 338).

[0413] CD69-TM-II type (from inside to outside) VLCAVMNVVFITILIIALIAL (SEQ ID NO: 339).

[0414] CD69-extracellular SVGQYNGGSGPVRRYQ (SEQ ID NO: 340).

[0415] Connector GGSG (SEQ ID NO: 341).

[0416] MMP9 GGPLGMTSG (SEQ ID NO: 342).

[0417] Connector GSGSSS (SEQ ID NO: 343).

[0418] TNFa

[0419] Myc-label EQKLISEEDL (SEQ ID NO 345).

[0420] CD28TM FWVLVVVGGVLACYSLLVTVAFIIFW (SEQ ID NO: 346).

[0421] refer to Figure 20 Some GAGE ​​DNA sequences are shown below: LeGo-CD63-9-2L-TNFa-Myc-9-CD63-cyt

[0422] It is encoded by the following cDNA:

[0423] LeGo-CD63-9-2L-TNFa-Myc-9-CD63-D-Cyt

[0424] It is encoded by the following cDNA:

[0425] LeGo-CD63-DL1-2-2L-TNFa-Myc-2-CD63-Cyt

[0426] It is encoded by the following cDNA:

[0427]

[0428] LeGo-CD63-9-2L-TNFa-Myc-9-CD19CAR

[0429] It is encoded by the following cDNA:

[0430]

[0431] LeGo-CD63-2-2L-TNFa-Myc-2-CD63-D-Cyt

[0432] It is encoded by the following cDNA:

[0433]

[0434] LeGo_CD63-2-2L-TNFa-Myc-2-CD19CAR

[0435] It is encoded by the following cDNA:

[0436]

[0437] LeGo-CD63-2L-TNFa-Myc-CD63-Cyt

[0438] It is encoded by the following cDNA:

[0439]

[0440] LeGo-CD63-DL1-2L-TNFa-Myc-CD63-Cyt

[0441] It is encoded by the following cDNA:

[0442] LeGO-CD69Cyt-TM-GrnzB-TNFa-Myc-GrnzB-a-CD19sc-stem-CD28-CD3Z

[0443]

[0444] It is encoded by the following cDNA:

[0445]

[0446] LeGO-CD69Cyt-TM-II-9-TNFa-Myc-9-CD28TM-Cyt

[0447] It is encoded by the following cDNA:

[0448] LeGO-CD69Cyt-TM-II-TNFa-Myc-CD28TM-Cyt

[0449] It is encoded by the following cDNA:

[0450]

[0451] LeGO-CD69Cyt-TM-GrzB-TNFa-Myc-GrzB-a-CD19sc-stem-CD28-CD3z

[0452] It is encoded by the following cDNA:

[0453]

[0454] LeGO-CD69Cyt-TM-GrzB-nLuc-Myc-GrzB-a-CD19sc-stemCD28-CD3z

[0455] It is encoded by the following cDNA:

[0456]

[0457] LeGO-CD69Cyt-TM-GrzB-nLuc-Myc-GrzB-CD28-CD3z, (δ-sc, GAGE ​​B)

[0458]

[0459] It is encoded by the following cDNA:

[0460] CD69Cyt-TM-nLuc-Myc-a-CD19sc-stem-CD28-CD3z, (δ-GrzB, GAGE ​​A)

[0461]

[0462] It is encoded by the following cDNA:

[0463]

[0464] CD69Cyt-TM-GrzB(IEPD)-nLuc-Myc-GrzB(IEPD)-a-CD19sc-stem-CD28-CD3z

[0465] It is encoded by the following cDNA:

[0466]

[0467] CD69Cyt-TM-GrzB(SASA)-nLuc-Myc-GrzB(SASA)-a-CD19sc-stem-CD28-CD3z

[0468] It is encoded by the following cDNA:

[0469]

[0470] CD69Cyt-TM-GrzB(ADKG)-TNFa-Myc-GrzB(ADKG)-a-CD19sc-stem-CD28-CD3z

[0471]

[0472] It is encoded by the following cDNA:

[0473]

[0474] Example 19: Generation of Lentiviral Vectors The transfer vector containing the target gene was transfected into 293T cells along with lentiviral packaging vectors (pMDLg / pRRE and pRSV-Rev) and envelope vector (PHC mv-VSV-G), and the viral supernatant was harvested.

[0475] Small-scale production of VSV-G pseudotyped lentiviral vector This method describes the production of VSV-G pseudotyped lentiviral vectors in 6-well plates using a calcium phosphate transfection kit. The volume of viral supernatant per well is 4 ml, and the viral concentration depends on the vector used.

[0476] Materials Table 6.

[0477]

[0478] Common materials: T75 culture flasks, cell culture pipettes, micropipettes and tips, 1.5 ml microcentrifuge tubes, 5 ml syringes, trypan blue, Virkon, and Mytdes.

[0479] plasmid Carrier: depends on the choice Gag-pol plasmid: pMDLg / pRRE Rev plasmid: pRSV-Rev Encapsulation plasmid: phCMV-VSV-G step Maintaining HEK293FT cells Cells are maintained by separating them every other day at a ratio of 1:5 to 1:4 and keeping them in T75 or T150 culture flasks. After thawing, cells should be passaged at least three times before being used for virus production to ensure cell recovery and entry into the exponential growth phase. Using cells with high passage counts is not recommended, as this can negatively impact virus yield.

[0480] Day 1: Laying out the substrate & transdyeing - Plate the cells at a rate of 500,000 cells / well in complete growth medium.

[0481] - Discard the culture medium in the culture flask.

[0482] - Wash cells with 10 ml of room temperature PBS.

[0483] Add 1 ml (T75) or 2 ml (T150) of TrypLE Express and incubate at 37°C for 5 minutes.

[0484] - After incubation, add 10-20 ml of complete culture medium to the bottle, resuspend thoroughly, and disperse the clumps by pipetting several times.

[0485] - Use trypan blue counting to prepare a cell suspension of 250,000 cells / ml.

[0486] - Add 2 ml of suspension to each well (one well per carrier) of a 6-well plate (coated with poly-L-lysine).

[0487] - Place the plate in an incubator for at least 7-8 hours.

[0488] After incubation, perform cell transfection. Confirm that the cells have adhered and reached the optimal confluence of 80%. If the cells are in good condition and at the appropriate density, return the plate to the incubator and prepare the transfection mixture. If the confluence is below 60%, terminate the transfection process.

[0489] - Prepare 1 ml of complete growth medium containing chloroquine at a final concentration of 25 μM. Preheat it in an incubator. Importantly, allow the components of the calcium phosphate precipitation kit to return to room temperature before initiating transfection.

[0490] - Prepare the plasmid mixture (total 4 µg DNA) in a microcentrifuge tube as follows: - 2 μg LeGO-iG2 vector (including target gene) - 1 μg pMDLg / pRRE(Gag / Pol) - 0.75 μg pRSV-REV(Rev) - 0.25 μg phCMV-VSV-G (encapsulated) - Mix the plasmids and add ddH2O to bring the volume to 54 μl. Add 6 μl of 2.5 M CaCl2 solution to the DNA mixture. - In a separate microtube, add 60 µl of 2X HeBS buffer. Add the CaCl2 / DNA mixture and vortex. - Let the mixture stand at room temperature for 15 minutes. Do not exceed 30 minutes to avoid reducing transfection efficiency.

[0491] During this 15-minute period, remove the petri dish, discard the culture medium, and add 1 ml of preheated complete growth medium containing 25 μM chloroquine.

[0492] - After 15 minutes of incubation, add 120 µl of the mixture dropwise into the well while gently rotating the petri dish in a circular motion.

[0493] - Cover the petri dish and place it in an incubator for 10-12 hours.

[0494] Day 2: Change the culture medium - 10-12 hours after transfection: - Aspirate the culture medium containing the transfection mixture and chloroquine from the well and discard it.

[0495] Add 2 ml of complete growth medium to each well. Ensure the medium is preheated to at least room temperature, preferably 37°C. Place the cells in an incubator.

[0496] Day 3: Collect the supernatant 1 - 24 hours after changing the culture medium: - Examine GFP expression in cells under a UV microscope. Transfection efficiency should be higher than 90%.

[0497] - Prepare a 0.45 μm filter, a 5 ml syringe, a 5 ml microtubule, and a 1.5 mm tube for each well.

[0498] - Collect the culture medium from the petri dish using a 5 ml syringe. Filter the supernatant into a 5 ml microtube using a filter. Filter gently, avoiding air bubbles and excessive force. Once finished, place the syringe and filter into Virkon solution.

[0499] - Take 100 μl of the sample from the filtered supernatant and place it into a 1.5 ml microcentrifuge tube (virus titration). Divide the remaining supernatant into equal portions as needed and store at -80°C for long-term preservation.

[0500] Add 2 ml of complete growth medium to each culture dish. Ensure the medium is preheated to at least room temperature, preferably 37°C. Place the cells in an incubator.

[0501] Day 4: Collect supernatant 2 - 48 hours after changing the culture medium: - Collect the viral supernatant in the same manner as the previous day.

[0502] - Discard board.

[0503] Generative cell lines The lentiviral particles harvested in the above steps were transformed into K562 and KHYG-1 cells, sorted, and amplified. The expression of target genes in the resulting cells was tested using appropriate antibodies or fluorescently labeled proteins.

[0504] 1. Prepare the culture medium: RPMI 10% FBS = 400 μL / well 2. Add lentiviral vector and transduction medium.

[0505] 3. Label 24-well plates with the date, name, cell type, and virus used for transduction.

[0506] 4. Set the centrifuge temperature to 32℃.

[0507] 5. Count the cells using trypan blue and prepare 10⁻¹⁰ cells in medium + 10% FBS. 6 Cell suspension of cells per ml.

[0508] 6. Take 250 50,000 cells were distributed into 24-well plates.

[0509] 7. Take the required amount of protamine sulfate stock solution and adjust it to a final concentration of 8%. g / ml.

[0510] 8. Avoid repeated freezing / thawing of the stock solution.

[0511] 9. Add culture medium according to the calculation results.

[0512] 10. Store the virus on dry ice before use. Quickly thaw the required amount of virus.

[0513] 11. Be careful to avoid mixing viruses and minimize contact with air.

[0514] 12. Add the calculated amount of virus to each well.

[0515] 13. For KHYG-1 cells, protamine sulfate (8... IL-2 (100 IU / ml) and IL-2 (100 IU / ml) were added to each well using a pipette.

[0516] 14. Carefully mix the cells by pipetting up and down.

[0517] 15. Centrifuge the plate at 32°C at 1000 × g for 1 hour.

[0518] 16. Remove the plate and incubate it in an incubator for 4 hours to overnight (depending on the construct and virus titer; testing should be performed).

[0519] 17. At the end of the incubation, centrifuge again at 32°C for 1 hour using a 1000 × g centrifuge plate.

[0520] 18. Carefully remove 80% of the culture medium from all wells and replenish with 500 μL of fresh, preheated serum-containing culture medium.

[0521] 19. Place the petri dish back into the incubator.

[0522] Day 1 & Day 2: Examine cells under a microscope and look for cell colonies.

[0523] Day 3: Analyze the cells using flow cytometry.

[0524] HEK293 NF-κB luciferase assay procedure 1. One day prior to the assay, harvest NF-κB reporter (Luc)-HEK293 cells and seed them at a density of 20,000 cells per well into 100 μl of assay medium (DMEM + 10% FBS + 1% P / S) in 96-well plates. Incubate the cells overnight at 37°C and 5% CO2 for approximately 24 hours.

[0525] 2. On the second day, aspirate the culture medium from the 96-well microplate and add 100 μL of supernatant from GAGE-expressing cells (cultured alone or co-cultured with target cells) to the corresponding wells, and incubate at 37°C and 5% CO2 for 6 hours.

[0526] 3. After 6 hours, aspirate the culture medium from the cells and add 100 μL of 0.1% Triton buffer to each sample well.

[0527] 4. Gently shake the plate several times to ensure that the lysis buffer completely covers the cells.

[0528] 5. Incubate at room temperature for 10 minutes to allow lysis to occur.

[0529] 6. Transfer 30 μL of the lysate to a new white opaque 96-well plate for use with a luciferase assay system. Set the GLOMAX machine to automatically add 50 μL of D-luciferin or nanoGLOW assay system (Promega) per well and measure luminescence using a photometer according to the manufacturer's instructions. The luciferase activity data presented in the results were normalized to the percentage of GAGE ​​expression in cells and MFI.

[0530] Flow cytometry Staining and washing were performed in flow cytometry collection buffer. Single-cell suspensions of cells were incubated on ice for 10 minutes with blocking reagent, followed by antibody staining and viability staining on ice for 30 minutes. Samples were analyzed on a Cytoflex flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (TreeStar, Ashland, OR). Cell sorting was performed using an AriaFusion (BD Biosciences) machine. Sorted cells were cultured in antibiotic-containing medium for two weeks. Subsequently, cells were cultured in antibiotic-free conditions.

[0531] Example 20 Experiments were conducted related to the following Caspase 3 sequences and constructs: CD69Cyt-TM-Cas3(DEVD)-TNFa-Myc-Cas3(DEVD)-a-CD19sc-stem-CD28-CD3z Cas3 (DEVDG) sequences are given in bold, underline, and uppercase. DNA sequence

[0532]

[0533] CD69Cyt-TM-Cas3(DEVD)-TNFa-Myc-Cas3(DEVD)-a-CD19sc-stem-CD28-CD3z protein sequence

[0534] CD69Cyt-TM-Cas3(EAALVDMVNDG)-TNFa-Myc-Cas3(EAALVDMVNDG)-a-CD19sc-stem-CD28-CD3z The sequence of Cas3 (EAALVDMVNDG(SEQ ID NO: 388)) is given in bold, underline, and uppercase.

[0535] DNA sequence

[0536]

[0537] >CD69Cyt-TM-Cas3(EAALVDMVNDG)-TNFa-Myc-Cas3(EAALVDMVNDG)-a-CD19sc-stem-CD28-CD3z protein sequence

[0538]

[0539] CD69Cyt-TM-Cas3(EDYGRDSGPP)-TNFa-Myc-Cas3(EDYGRDSGPP)-a-CD19sc-stem-CD28-CD3z Cas3 ( EDYGRDSGPP (SEQ ID NO:391) The sequence is given in bold, underline, and uppercase.

[0540] DNA sequence

[0541]

[0542] CD69Cyt-TM-Cas3(EDYGRDSGPP)-TNFa-Myc-Cas3(EDYGRDSGPP)-a-CD19sc-stem-CD28-CD3z protein sequence

[0543]

[0544] CD69Cyt-TM-Cas3(TPSEPDSGQGPPQ)-TNFa-Myc-Cas3(TPSEPDSGQGPPQ)-a-CD19sc-stem-CD28-CD3z The sequence of Cas3 (TPSEPDSGQGPPQ (SEQ ID NO:394)) is given in bold, underline, and uppercase.

[0545] DNA sequence

[0546]

[0547] CD69Cyt-TM-Cas3(TPSEPDSGQGPPQ)-TNFa-Myc-Cas3(TPSEPDSGQGPPQ)-a-CD19sc-stem-CD28-CD3z The sequence of Cas3 (TPSEPDSGQGPPQ (SEQ ID NO:394)) is given in bold, underline, and uppercase.

[0548] protein sequence

Claims

1. A peptide delivery system for delivering a peptide payload to a target cell or target site, the peptide delivery system comprising: An exogenous polypeptide is anchored therein to a cell, extracellular vesicle, or lipid-containing particle, wherein the exogenous polypeptide comprises: A first transmembrane domain and a second transmembrane domain are connected by a loop region, wherein the loop region includes a first cleavage site, a peptide load, and a second cleavage site; And optionally, for targeting the peptide delivery system to the target cell or the target portion of the target site.

2. The peptide delivery system of claim 1, wherein the first cleavage site and / or the second cleavage site comprises a proteolytic cleavage substrate, preferably an exfoliase cleavage site, an MMP cleavage site, a GrzB cleavage site, and a Casp3 cleavage site.

3. The peptide delivery system of claim 2, configured to release the peptide load upon cleavage by a protease, preferably an exfoliase, the protease being endogenous or exogenous (e.g., included as a component of the peptide delivery system).

4. The peptide delivery system of claim 3, wherein the exfoliase is present at the target site and / or released by the target cell.

5. The peptide delivery system of claim 3 or 4, wherein the exfoliase is selected from the following dedicated exfoliases: ADAM protease (metalloproteinase), BACE protease, serine protease granzyme-B, and Site-1 protease.

6. The peptide delivery system of claim 5, wherein the ADAM protease comprises membrane-anchored type 1 proteases, such as ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, ADAMthr20, ADAM21, ADAM28, ADAM30, and ADAM33.

7. The peptide delivery system of claim 3 or 4, wherein the exfoliase is selected from the following part-time exfoliases: transmembrane peptidase B, MT-MMP (membrane matrix metalloproteinase), proprotein convertase, transmembrane serine protease, matrix metalloproteinase, podocyte protein, cathepsin S, and cathepsin L.

8. The peptide delivery system of claim 7, wherein the MT-MMP is membrane-anchored type 1 or GPI-anchored, and includes MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP and MT6-MMP.

9. The peptide delivery system of claim 7, wherein the proprotein convertase is selected from PCSK1 / 3, KCSK2, furin protease, PCSK4, PCSK5 / 6, PACE4, PCSK7 and PCSK9.

10. The peptide delivery system of claim 7, wherein the transmembrane serine protease is selected from membrane-anchored type II proteases, including Matriptase, Matriptase-2, Matriptase-3, polymerase-1, Corin, Hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, Spinesin, intestinal peptidase, HAT, DESCL1, TMPRSS11A, HAT-like 4, and HAT-like 5.

11. The peptide delivery system of claim 7, wherein the matrix metalloproteinase is selected from soluble proteases, including MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A-B, MMP24, MMP25, MMP26, MMP27 and MMP28.

12. The peptide delivery system of claim 11, wherein the matrix metalloproteinase is selected from MMP2, MMP9 and / or MMP25.

13. The peptide delivery system of claim 12, wherein the MMP2 is selected from SEQ ID NO: 2-6.

14. The peptide delivery system of claim 12, wherein the MMP9 is selected from SEQ ID NO: 7-8.

15. The peptide delivery system of claim 12, wherein the MMP25 is selected from SEQ ID NO: 9-17, 18 or 155, 19-29.

16. The peptide delivery system of any of the preceding claims, wherein the transmembrane domain comprises one or more transmembrane domains from the group consisting of: CD63, CD9, CD81, CD28, CD4, CD8, CD34, CD69, CD19, CD20, integrin, TNFSF14, TNR1A, aquaporin, NOTCH, NgR1, NRG1, GPI anchor, EGFR, or rhodopsin.

17. The peptide delivery system of claim 1, further comprising one or more intracellular or extracellular domains selected from or derived from the following proteins: OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1, programmed cell death-I (PD-1), inducible T cell co-stimulatory factor (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CDI 1a / CD 18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, lgα (CD79a), DAP-10, Fcγ receptor, MHC class I molecules, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), co-stimulatory ligands (PD-L2, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1, programmed cell death-I (PD-1), co-stimulatory ligands (PD-L2, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD7-1 (CD80), CD7-2 (CD86), PDL-1, programmed cell death-I ... BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecules (ICAM, CD30L, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD11a, LFA-1, IT GAM, CD1 1b, ITGAX, CD1 1e, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a. A ligand that specifically binds to CD83 or any combination thereof.

18. The peptide delivery system of claim 1, comprising a targeting portion, wherein the targeting portion binds to BCMA, MUC16 (also known as CA125), EGFR, EGFRvIII, MUCI, Flt-3, WT-1, CD38, CD70, CD90, CD133, MHC-WTI, TSPANI0, MHC-PRAME, MHC-NY-ESOI, HER2 (ERBB2), CA-IX (carbonic anhydrase IX), LIVI, ADAMI0, CHRNA2, LeY, NKG2D, CSI, CD44v6, CD24, LGR5, ALDH, ALDH1, CD54, Sca1, CD271, CD123, CD36, CD109, CD110, CD71-negative, CCA, ABCG2, Claudin-18.2 (Claudin-18A2 or Claudin18 isoform 2), PSCA, DLL3 (Delta-like protein 3, Drosophila Delta homolog 3, Delta3), Mud 7 (Mucinl 7, Muc3, Muc3), FAPα (fibroblast activator protein α), Ly6G6D (lymphocyte antigen 6 complex site protein G6d, c6orf23, G6D, MEGTI, NG25), PSMA, MSLN or RNF43 (E3 ubiquitin protein ligase RNF43, cyclic finger protein 43), BAFF, C242 antigen, disialotetrahexosylganglioside (GD2), 4-IBB, 5T4, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44v6, CD44, CD51, CD52, CD56, CD74, CEA, CNT0888, CTLA-4, DR5, EpCAM, FAP, fibronectin extradomain-B, folate receptor 1, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scattering factor receptor kinase, IGF-1 receptor, IGF-I, IgGI, LI-CAM, integrin α5β1, integrin αvβ3, podocyte oleanolic acid, MORAb-009, MS4A1, MUC1, mucin CanAg, C-MET, CCR4, CD152, CD10, CD19, CD20, CD200, N-hydroxyacetylneuraminic acid, NPC-IC, PDGF-RA, PDL192, phosphatidylserine, tumor antigen CTAA16.

88. VEGF-A, VEGFR-1, VEGFR2, vimentin, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tendinin C, TGF-β, TRAIL-R1, TRAIL-R2, folate receptor, transferrin receptor, or any combination thereof.

19. The peptide delivery system of claim 1, wherein the second transmembrane domain is connected to an intracellular region comprising one or more intracellular domains from 41BB, ICOS, and CD3ζ.

20. The peptide delivery system of claim 19, wherein GrzB is activated by phosphorylation of an intracellular domain kinase of the 41BB, ICOS, or CD3ζ, and GrzB is transported out of the cell and cleaved at the first cleavage site and / or the second cleavage site.

21. The peptide delivery system of claim 1, comprising a third transmembrane domain and a fourth transmembrane domain connected by a second loop region, wherein the third transmembrane domain is connected to an intracellular region comprising an intracellular domain from 41BB, ICOS, or CD3z.

22. The peptide delivery system of claim 21, wherein the cell is activated by phosphorylation of an intracellular domain kinase of the 41BB, ICOS, or CD3ζ to release endogenous GrzB, such that the GrzB is transported out of the cell and cleaved at GrzB cleavage sites present in the first or second loop region, thereby releasing the peptide load.

23. The peptide delivery system of claim 22, wherein the second transmembrane domain is derived from a NOTCH protein having a cleavage site, the cleavage site being configured to release a functional domain connected to the transmembrane domain of the NOTCH protein in the intracellular environment.

24. The peptide delivery system of any one of the preceding claims, wherein the payload comprises: an antibody, an antibody fragment, VHH, a cytokine or chemokine composed of a portion or derivative of sulfated xylan, which is an antagonist selected from the list of ligands comprising: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-3, IL-14, IL-15, or IL-17. The cytokine family includes IL-25, interferon, G-CSF, M-CSF, GM-CSF, and BDNF. , CNTF, EGF, EPO, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF12, FGF13, FGF14, FGF15, FGF16, FGF1 7. FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, LIF, PDGF, SCF, TGFa, TGFB, TNFa, TNFB, TPO, VEGF, GH, NGF, NT3, NT4, NT5, NT6, NT7, Oncostatin M (OSM), insulin, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-1 family members including MIP-1a, MIP-2, eosinophil chemokines (eosinophil chemokine-1, eosinophil chemokine-2 or eosinophil chemokine-3), PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase;Enzymes of the cathepsin family, cell adhesion molecules such as PECAM-1, soluble receptors or cell-binding receptors or virus-binding receptors, cytokine-induced neutrophil chemokines (KC), TNF-α and IFN-γ, and other soluble inflammatory mediators such as reactive oxygen species and nitric oxide, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, C CL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, thrombin, urokinase, vaccine, or any combination thereof.

25. The peptide delivery system of any one of the preceding claims, further comprising a connector between the first transmembrane domain and the first cleavage site.

26. The peptide delivery system of claim 25, wherein the linker length is 2-128 amino acids or 4-20 amino acids.

27. The peptide delivery system of claim 25, wherein the adapter is selected from the adapters listed in Table 4.

28. The peptide delivery system of claim 1, configured to be specifically sorted into extracellular vesicles.

29. The peptide delivery system of claim 1, configured to release two or more peptide payloads intracellularly or extracellularly.

30. The peptide delivery system of claim 1, comprising two or more exfoliase cleavage sites, wherein each cleavage site may be the same as or different from any other cleavage site.

31. The peptide delivery system of claim 1, comprising an intracellular domain, wherein the intracellular domain comprises the stem domain of CD45 phosphatase.

32. The peptide delivery system of claim 1, comprising a single-domain antibody or antibody single chain that binds to an autoassociated antigen (SAA) to bring the SAA into contact, for example, in a cis or trans configuration.

33. The peptide delivery system of claim 1 or 11, comprising a single-domain antibody or antibody single chain that binds to an autoassociated antigen (SAA), and further comprising a CD45 phosphatase-binding moiety or an NKG2A-binding moiety that activates an inhibitory signal in the target cell.

34. The peptide delivery system of claim 1, wherein the peptide load is any peptide or protein that functions in the human body, including hormones, cytokines, chemokines, prodrugs, and various forms of antibodies, such as chimeric antigen receptors (CARs), bispecific killer cell binders (BIKEs), trispecific killer cell binders (TRIKEs), etc.

35. The peptide delivery system of claim 1, wherein the peptide delivery system is selected from: CD63-MMP2-a-CD16-a-CD33-Myc-MMP9-CD63-GPi (SEQ ID NO: 124), CD63-MMP2-a-CD16-a-CD33-Myc-MMP9-CytCD63-EV motif (SEQ ID NO: 155), CD63-MMP2-a-CD16-a-CD33-Myc-MMP9-CytCD63 (SEQ ID NO: 36 or 188), TNFSF14-a-CD16-a-CD33-Myc-TNR1A-ADAM17-a-CD38-41BB (SEQ ID NO: 124). 54 or 366 or 367), CD63-GrzB-a-CD16-a-CD33-Myc-GrzB-a-CD38-CD28-CD3z (SEQ ID NO: 71 or 252), CD63-MMP9-2L-TNFa-Myc-MMP9-CD63-Cyt (SEQ ID NO: 88 or 295), CD63-MMP9-2L-TNFa-Myc-MMP9-CD63-D-Cyt (lacking EV sorting sequence) (SEQ ID NO: 103 or 311), CD63-DL1-MMP2-2L-TNFa-Myc-MMP2-CD63-Cyt (SEQ ID NO: 104 or 368), CD63-MMP9-2L-TNFa-Myc-MMP9-aCD19CAR (SEQ ID NO: ...104 or 368), CD63-MMP9-2L-TNFa-My 105 or 369), CD63-MMP2-2L-TNFa-Myc-MMP2-CD63-D-Cyt (SEQ ID NO: 106 or 370 or 371), CD63-MMP2-2L-TNFa-Myc-MMP2-aCD19CAR (SEQ ID NO: 107 or 372 or 373), CD69cyt-TMII-TNFa-Myctag-CD28TM-cyt (SEQ ID NO: 119 or 370), CD69cyt-TMII-MMP9-TNFa-Myc-MMP9-CD28TM-cyt (SEQ ID NO: 120 or 377), CD69Cyt-TM-GrzB-TNFa-Myc-GrzB-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 105 or 369 ...CD63-D-Cyt (SEQ ID NO: 106 or 370 or 371), CD63-MMP2-2L-TNFa-Myc-MMP2-aCD19CAR (SEQ ID 107 or 372 or 373), CD69Cyt-TM-GrzB-nLuc-Myc-GrzB-CD28-CD3z (δ sc-GAGE B) (SEQ ID NO:108 or 374), CD69Cyt-TM-nLuc-Myc-a-CD19sc-stem-CD28-CD3z (δ GrzB, GAGE ​​A) (SEQ ID NO: 109 or 375), CD69Cyt-TM-GrzB (primitive)-nLuc-Myc-GrzB (primitive)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 358 or 382), CD69Cyt-TM-GrzB(IEPD)-nLuc-Myc-GrzB(IEPD)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 110 or 383), CD69Cyt-TM-GrzB(SASA)-nLuc-Myc-,GrzB(SASA)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 109 or 374), CD69Cyt-TM-GrzB(primitive)-nLuc-Myc-,GrzB(SASA)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 109 or 375), CD69Cyt-TM-GrzB(primitive ... 111 or 376), or CD69Cyt-TM-GrzB(ADKG)-nLuc-Myc-GrzB(ADKG)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 112 or 384).

36. The peptide delivery system of any one of the preceding claims, comprising cells in which the exogenous polypeptide has been anchored.

37. The peptide delivery system of claim 36, wherein the cells are selected from hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs) and their derived cell products, adoptive T cells, dendritic cells (DCs), natural killer (NK) cells, or any therapeutic immune / non-immune cells.

38. An adoptive cell or engineered immune cell comprising the peptide load of claim 1, wherein the immune cell is autologous or allogeneic.

39. The peptide delivery system of claim 1, wherein the exogenous polypeptide is introduced into the cell individually via a CRISPR system, or in the form of a viral or non-viral vector or one or more mRNAs.

40. The peptide delivery system of claim 39, wherein the exogenous polypeptide is delivered using CPP, micelles, liposomes, nanoparticles, dendritic polymers, nanotubes, electroporation, viral transduction, nuclear transfection, transfection, cell fusion, or microinjection.

41. A gene construct encoding the exogenous polypeptide of claim 1.

42. A method of treating a patient in need, comprising administering to the patient the peptide delivery system of any one of claims 1-37.

43. A method for delivering a peptide to a target site in a patient in need, the method comprising: Administering a peptide delivery system to a patient, the peptide delivery system comprising lipid-containing vesicles (e.g., cells, extracellular vesicles, lipid nanoparticles) in which an exogenous peptide is anchored, the exogenous peptide comprising: Peptide loading; First transmembrane region and second transmembrane region; The first transmembrane region includes a transmembrane domain connected to at least one exfoliase or other protein hydrolysis cleavage site; The second transmembrane region includes a transmembrane domain linked to at least one exfoliase or other proteolytic cleavage site; and Optionally, it is used to target the exogenous polypeptide to the target site of the target portion; When the lipid-containing vesicles reach the target site, exfoliase and / or other proteases cleave the cleavage sites of the first and second transmembrane regions, releasing the peptide load.

44. The method of claim 43, wherein the cleavage site is derived from a proteolytic cleavage substrate naturally present in glycosylphosphatidylinositol (GPi) anchored proteins and membrane proteins, wherein the membrane proteins have one, two, three, four or more transmembrane domains, intracellular domains or extracellular domains in the extracellular matrix or adjacent cells.

45. The method of claim 43, wherein the abscisic acid is selected from dedicated abscisic acid or part-time abscisic acid.

46. ​​The method of claim 45, wherein the dedicated exfoliase is selected from ADAM protease, BACE protease, serine protease granzyme B, and Site-1 protease.

47. The method of claim 46, wherein the ADAM protease is selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30 and ADAM33.

48. The method of claim 45, wherein the part-time exfoliating enzyme is selected from: transmembrane peptidase B, MT-MMP (membrane matrix metalloproteinase), proprotein convertase, transmembrane serine protease, matrix metalloproteinase, podocyte protein, cathepsin S and cathepsin L, wherein the transmembrane peptidase B is a membrane-anchored type 1 metalloproteinase, and wherein the MT-MMP is a membrane-anchored type 1 or GPI-anchored enzyme, and includes MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP and MT6-MMP.

49. The method of claim 48, wherein the proprotein convertase is selected from PCSK1 / 3, KCSK2, furin protease, PCSK4, PCSK5 / 6, PACE4, PCSK7, and PCSK9.

50. The method of claim 48, wherein the transmembrane serine protease is selected from membrane-anchored type II proteases, including, for example, Matriptase, Matriptase-2, Matriptase-3, polymerase-1, Corin, Hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, Spinesin, intestinal peptidase, HAT, DESCL1, TMPRSS11A, HAT-like 4, and HAT-like.

51. The method of claim 48, wherein the matrix metalloproteinase is a soluble protease and comprises one or more of MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A-B, MMP24, MMP25, MMP26, MMP27 and MMP28.

52. The method of claim 51, wherein the matrix metalloproteinase is selected from MMP2, MMP9 and / or MMP25.

53. The method of claim 52, wherein the MMP2 is selected from SEQ ID NOS: 2-6.

54. The method of claim 52, wherein the MMP9 is selected from SEQ ID NOS: 7-8.

55. The method of claim 52, wherein the MMP25 is selected from SEQ ID NOS: 9-17, 18 or 155, 19-29.

56. The method of claim 43, wherein the transmembrane domains of the first transmembrane region and / or the second transmembrane region comprise transmembrane domains derived from one or more of the following: CD63, CD9, CD81, CD28, CD4, CD8, CD34, CD69, CD19, CD20, integrin, TNFSF14, TNR1A, aquaporin, NOTCH, NgR1, NRG1, GPi anchor, EGFR, and rhodopsin.

57. The method of claim 43, wherein cleavage occurs at one or more of the cleavage sites by an endogenous exfoliase.

58. The method of claim 43, wherein the peptide delivery system further comprises an abscission enzyme, and the abscission enzyme cleaves the peptide delivery system at one or more of the cleavage sites.

59. The method of claim 43, wherein the intracellular or extracellular domain of the exogenous polypeptide is selected from one or more of the following intracellular or extracellular domains: OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1, programmed cell death-I (PD-1), inducible T cell co-stimulatory factor (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CDI 1a / CD 18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, lgα (CD79a), DAP-10, Fcγ receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), co-stimulatory ligand (PD-L2, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1, programmed cell death-I (PD-1), co-stimulatory ligand (PD-L2, 4-1BB / CD137, CD2, CD7, CD27, CD30), LFA-1 (CD137, CD70, CD30), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, IgG1 (CD79a), DAP-10, Fcγ receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), co-stimulatory ligand (PD-L2, 4-1BB BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecules (ICAM, CD30L, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, IT GAM, CD1 1b, ITGAX, CD1 1e, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGLl, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

60. The method of claim 43, wherein the targeting portion may bind to BCMA, MUC16 (also known as CA125), EGFR, EGFRvIII, MUCI, Flt-3, WT-1, CD38, CD70, CD90, CD133, MHC-WTI, TSPANI0, MHC-PRAME, MHC-NY-ESOI, HER2 (ERBB2), CA-IX (carbonic anhydrase IX), LIVI, ADAMI0, CHRNA2, LeY, NKG2D, CSI, CD44v6, CD24, LGR5, ALDH, ALDH1, CD54, Sca1, CD271, CD123, CD36, CD109, CD110, CD71-negative, CCA, ABCG2, Claudin-18.2 (Claudin-18A2 or Claudin18 isoform 2), PSCA, DLL3 (Delta-like protein 3, Drosophila Delta homolog 3, Delta3), Mud 7 (Mucinl 7, Muc3, Muc3), FAPα (fibroblast activator protein α), Ly6G6D (lymphocyte antigen 6 complex site protein G6d, c6orf23, G6D, MEGTI, NG25), PSMA, MSLN or RNF43 (E3 ubiquitin protein ligase RNF43, cyclic finger protein 43), BAFF, C242 antigen, disialotetine ganglioside (GD2), 4-IBB, 5T4, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44v6, CD44, CD51, CD52, CD56, CD74, CEA, CNT0888, CTLA-4, DR5, EpCAM, FAP, fibronectin extradomain-B, folate receptor 1, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scattering factor receptor kinase, IGF-1 receptor, IGF-I, IgGI, LI-CAM, integrin α5β1, integrin αvβ3, podocyte oleanolic acid, MORAb-009, MS4A1, MUC1, mucin CanAg, C-MET, CCR4, CD152, CD10, CD19, CD20, CD200, N-hydroxyacetylneuraminic acid, NPC-IC, PDGF-RA, PDL192, phosphatidylserine, tumor antigen CTAA16.

88. VEGF-A, VEGFR-1, VEGFR2, vimentin, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tendinin C, TGF-β, TRAIL-R1, TRAIL-R2, folate receptor, transferrin receptor, or any combination thereof.

61. The method of claim 43, wherein the first transmembrane region and / or the second transmembrane region comprises: (i) an intracellular region comprising an intracellular region sequence from 41BB, ICOS, or CD3ζ, and (ii) a GrzB cleavage site; and wherein the action of a kinase on the intracellular region sequence from 41BB, ICOS, or CD3ζ induces the release of GrzB, causing it to be transported out of the cell and cleaving the GrzB cleavage site, thereby releasing the peptide payload, the peptide payload optionally comprising a bispecific antibody.

62. The method of claim 61, wherein the second transmembrane region comprises a transmembrane region from the NOTCH protein and a cleavage site within the cell membrane, and cleavage of the cleavage site within the cell membrane releases the functional domain in the intracellular environment.

63. The method of claim 43, wherein the peptide loading comprises: a cytokine or chemokine composed of a portion or derivative of sulfated xylan, which is an antagonist selected from ligands comprising the following list: IL-1a, IL-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-3, IL-14, IL-15, or IL-17. The cytokine family includes IL-25, interferon, G-CSF, M-CSF, GM-CSF, BDNF, CNTF, and EGF. , EPO, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FG F18, FGF19, FGF20, FGF21, FGF22, FGF23, LIF, PDGF, SCF, TGFa, TGFB, TNFa, TNFB, TPO, VEGF, GH, NGF, NT3, NT4, NT5, NT6, NT7, Oncostatin M (OSM), insulin, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-1 family members including MIP-1a, MIP-2, eosinophil chemokines (eosinophil chemokine-1, eosinophil chemokine-2, or eosinophil chemokine-3), PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase; cathepsins, cell adhesion molecules such as PECAM-1, soluble receptors or cell-binding receptors or virus-binding receptors, cytokine-induced neutrophil chemotactic agents (KC), TNF-α and IFN-γ, and other soluble inflammatory mediators such as reactive oxygen species and nitric oxide, CCL1, CCL2, CCL3, CCL4, and CCL5. L5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, thrombin, urokinase, vaccine, or any combination thereof.

64. The method of claim 43, wherein the exogenous polypeptide further comprises at least one adapter, wherein the at least one adapter is optionally located between a cleavage site and a first binding moiety (e.g., an anti-CD3 VHH domain) present in the first transmembrane region, between a cleavage site and a second binding moiety (e.g., an anti-TAA VHH domain) present in the second transmembrane region, and / or between the first binding moiety and the second binding moiety.

65. The method of claim 64, wherein the length of the connector is 2-128 amino acids, or preferably 4-20 amino acids.

66. The method of claim 64, wherein the connector is selected from Table 4.

67. The method of claim 43, wherein the exogenous polypeptide is further configured for specific sorting into extracellular vesicles (EVs).

68. The method of claim 43, wherein the peptide load comprises two or more bispecific binders, and the bispecific binders are released intracellularly or extracellularly.

69. The method of claim 43, wherein the peptide delivery system comprises an abscission cleavage site, and each cleavage site may be the same as or different from any other cleavage site.

70. The method of claim 43, wherein the peptide delivery system comprises an intracellular domain, and the intracellular domain comprises a stem domain of CD45 phosphatase.

71. The method of claim 43, wherein the peptide delivery system comprises a single-domain antibody or antibody single chain that binds to an autoassociated antigen (SAA) to bring the SAA into contact, for example, in a cis or trans configuration.

72. The method of claim 43, comprising a single-domain antibody or antibody single chain that binds to an autoassociated antigen (SAA), and further comprising a CD45 phosphatase-binding moiety or an NKG2A-binding moiety that activates an inhibitory signal in the target cell.

73. The method of claim 43, wherein the peptide load is any potentially therapeutic peptide that functions in vivo, including hormones, cytokines, chemokines, prodrugs, and various forms of antibodies, such as chimeric antigen receptors (CARs), bispecific killer cell binders (BIKEs), trispecific killer cell binders (TRIKEs), etc.

74. The method of claim 43, wherein the peptide delivery system is selected from: CD63-MMP2-a-CD16-a-CD33-Myc-MMP9-CD63-GPi (SEQ ID NO: 124), CD63-MMP2-a-CD16-a-CD33-Myc-MMP9-CytCD63-EV motif (SEQ ID NO: 155), CD63-MMP2-a-CD16-a-CD33-Myc-MMP9-CytCD63 (SEQ ID NO: 36 or 188), TNFSF14-a-CD16-a-CD33-Myc-TNR1A-ADAM17-a-CD38-41BB (SEQ ID NO: 124). 54 or 366 or 367), CD63-GrzB-a-CD16-a-CD33-Myc-GrzB-a-CD38-CD28-CD3z (SEQ ID NO: 71 or 252), CD63-MMP9-2L-TNFa-Myc-MMP9-CD63-Cyt (SEQ ID NO: 88 or 295), CD63-MMP9-2L-TNFa-Myc-MMP9-CD63-D-Cyt (lacking EV sorting sequence) (SEQ ID NO: 103 or 311), CD63-DL1-MMP2-2L-TNFa-Myc-MMP2-CD63-Cyt (SEQ ID NO: 104 or 368), CD63-MMP9-2L-TNFa-Myc-MMP9-aCD19CAR (SEQ ID NO: 54 or 366 or 367), CD63-GrzB-a-CD16-a-CD33-Myc-GrzB-a-CD38-CD28-CD3z (SEQ ID NO: 71 or 252), CD63-MMP9-2L-TNFa-Myc-MMP9-aCD19CAR (SEQ ID NO: 88 or 295 ... 105 or 369), CD63-MMP2-2L-TNFa-Myc-MMP2-CD63-D-Cyt (SEQ ID NO: 106 or 370 or 371), CD63-MMP2-2L-TNFa-Myc-MMP2-aCD19CAR (SEQ ID NO: 107 or 372 or 373), CD69cyt-TMII-TNFa-Myctag-CD28TM-cyt (SEQ ID NO: 119 or 370), CD69cyt-TMII-MMP9-TNFa-Myc-MMP9-CD28TM-cyt (SEQ ID NO: 120 or 377), CD69Cyt-TM-GrzB-TNFa-Myc-GrzB-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 105 or 369 ...CD63-D-Cyt (SEQ ID NO: 106 or 370 or 371), CD63-MMP2-2L-TNFa-Myc-MMP2-aCD19CAR (SEQ ID 107 or 372 or 373), CD69Cyt-TM-GrzB-nLuc-Myc-GrzB-CD28-CD3z (δ sc-GAGE B) (SEQ ID NO:108 or 374), CD69Cyt-TM-nLuc-Myc-a-CD19sc-stem-CD28-CD3z (δ GrzB, GAGE ​​A) (SEQ ID NO: 109 or 375), CD69Cyt-TM-GrzB (primitive)-nLuc-Myc-GrzB (primitive)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 358 or 382), CD69Cyt-TM-GrzB(IEPD)-nLuc-Myc-GrzB(IEPD)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 110 or 383), CD69Cyt-TM-GrzB(SASA)-nLuc-Myc-,GrzB(SASA)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 109 or 374), CD69Cyt-TM-GrzB(primitive)-nLuc-Myc-,GrzB(SASA)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 109 or 375), CD69Cyt-TM-GrzB(primitive ... 111 or 376), or CD69Cyt-TM-GrzB(ADKG)-nLuc-Myc-GrzB(ADKG)-a-CD19sc-stem-CD28-CD3z (SEQ ID NO: 112 or 384).

75. The method of claim 43, wherein the lipid-containing vesicle comprises cells, and the cells are selected from hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs) and derived cell products, adoptive T cells, dendritic cells (DCs), natural killer (NK) cells, or any therapeutic immune or non-immune cells.

76. The method of claim 75, wherein the immune cells are autologous or allogeneic.

77. The method of claim 43, wherein the peptide delivery system is individually introduced into the cell in the form of a viral or non-viral vector, mRNA, peptide, protein, antibody, nanobody, oligonucleotide, or extracellular vesicle (EV).

78. The method of claim 43, wherein the peptide delivery system is delivered using CPP, micelles, liposomes, nanoparticles, dendritic polymers, nanotubes, electroporation, viral transduction, nuclear transfection, transfection, cell fusion, or microinjection.

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