Plasma-embedded systems for spatiotemporally controlled payload delivery of biologics

Abstract

A peptide delivery system for delivering a peptide payload to a target cell or target gene locus, and a method for delivering a peptide payload to a target gene locus are disclosed. The peptide delivery system may include cells, extracellular vesicles, or lipid-containing particles on which an exogenous polypeptide is immobilized, the exogenous polypeptide comprising first and second transmembrane domains connected by a loop region, the loop region comprising a first cleavage site, a peptide payload, and a second cleavage site; and optionally, a targeting portion for targeting the peptide delivery system to a target cell or target gene locus.

Description

Technical Field

[0005] , ,

[0001] Cross - reference to Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 467,518, filed on May 18, 2023, which is hereby incorporated by reference in its entirety.

[0002] Statement of Incorporation by Reference of a Sequence Listing This application includes a sequence listing submitted electronically in XML file format, which is hereby incorporated by reference in its entirety. The XML copy created on May 16, 2024, is named "P71267_SL.xml" and has a size of 451,938 bytes.

Background Art

[0003] Living organisms are increasingly being treated with peptide - based therapeutics. These therapeutics include hormones, cytokines, chemokines, prodrugs, and antibodies in various forms such as chimeric antigen receptors (CARs), bispecific killer cell engagers (BIKEs), and trispecific killer cell engagers (TRIKEs). Any peptide or protein that functions in the body can potentially be a therapeutic. The potential of peptides and proteins is promising, but multiple factors limit their bioavailability, i.e., their in - vivo function.

[0004]

[0005] Because only a small amount of the administered peptide can reach the target site, systemic delivery of biologics further reduces their efficacy depending on the drug's pharmacokinetics and pharmacodynamics. Most biologics can be degraded and metabolized before reaching their intended site of action.

[0006] The third limiting factor is the biotoxicity related to the mechanism of action (MOA) of these bioagents.

[0007] Improved protein and peptide delivery methods are needed in the art to enhance the safety and efficacy of biologics. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] Disclosed herein is a peptide delivery system for delivering a peptide payload to a target cell or target gene locus, the peptide delivery system comprising a cell, extracellular vesicle, or lipid-containing particle on which an exogenous polypeptide is immobilized, the exogenous polypeptide comprising first and second transmembrane domains connected by a loop region, the loop region comprising a first cleavage site, a peptide payload, and a second cleavage site; and optionally, a targeting portion for targeting the peptide delivery system to a target cell or target gene locus. [Means for solving the problem]

[0009] In such a peptide delivery system, the first and / or second cleavage sites may include proteolytic cleavage substrates, preferably schedase cleavage sites, MMP cleavage sites, GrzB cleavage sites, and Casp3 cleavage sites.

[0010] Such a peptide delivery system may be configured to release a peptide payload upon cleavage by a protease, preferably aby-α-schedase, which may be endogenous or exogenous (for example, may be included as a component of the peptide delivery system).

[0011] Such peptide delivery systems are also intended to involve the presence of a schedase at the target gene locus and / or release by the target cell.

[0012] In the above embodiment of the peptide delivery system, the schedase may include a full-time schedase selected from ADAM protease (metalloprotease), 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] Also intended herein is a peptide delivery system in which the schedase is a part-time schedase selected from meprin B, MT-MMP (membrane-type matrix metalloproteinase), proprotein converterase, transmembrane serine protease, matrix metalloproteinase, legmine, and cathepsin S and L. The MT-MMP may be membrane-anchored type 1 or GPI-anchored, and examples include MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, and MT6-MMP. The proprotein converterase may 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 matryptase, matryptase-2, matryptase-3, polymerase-1, choline, hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, spinesin, enteropeptidase, HAT, DESCL1, TMPRSS11A, HAT-L4, and HAT-L5. 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 sequence numbers 9-17, 18 or 155, and 19-29.

[0014] Also disclosed herein are peptide delivery systems in which the transmembrane domain comprises one or more transmembrane domains from the following proteins: CD63, CD9, CD81, CD28, CD4, CD8, CD34, CD69, CD19, CD20, integrins, TNFSF14, TNR1A, aquaporins, NOTCH, NgR1, NRG1, GPi anchors, EGFR, or rhodopsins.

[0015] In a particular embodiment, the peptide delivery system described above is used for the following protein: 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 costimulator (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signal transduction lymphocyte activating molecule (SLAM protein), costimulatory ligand (PD-L2, 4-1BBL, OX40L), inducible costimulatory ligand (ICOS) -L), cell adhesion molecules (ICAM, CD30L, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4), activated 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, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, IT GAM, CD11b, ITGAX, CD11e, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI(CD226), SLAMF4(CD244, 2B4), CD84, CD96(TACTILE), CEACAM1, CRTThe product may further include intracellular or extracellular domains selected from or derived from ligands that specifically bind to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83, or any combination thereof.

[0016] In embodiments, such a peptide delivery system may include a targeting moiety, which may contain 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, ADAM10, C HRNA2, 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 Claudin-18 isoform 2), PSCA, DLL3 (Delta-like protein 3, Drosophila delta homolog 3, Delta 3), Mud 7 (Mucin 17, Muc3, Muc3), FAP alpha (Fibroblast-activating protein alpha), Ly6G6D (Lymphocyte antigen 6 complex locus protein G6d, c6orf23, G6D, MEGTI, NG25), PSMA, MSLN, or RNF43 (E3 ubiquitin-protein ligase RNF43, RING finger protein 43), BAFF, C242 antigen, disialoganglioside (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 Dispersion Factor Receptor Kinase, IGF-1 Receptor, IGF-I, IgGI, LI-CAM, Integrin α5β1, Integrin ανβ3, Regmine, MORAb-009, MS4A1, MUC1, Mucin CanAg, C-MET, CCR4, CD152, CD10, CD19, CD20, CD200, N-Glycolylneuraminic Acid, NPC-IC, PDGF-Rα, PDL192, Phosphatidylserine, Tumor Antigen CTAA16.It can bind to 88, VEGF-A, VEGFR-1, VEGFR2, vimentin, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF-β, TRAIL-R1, TRAIL-R2, folate receptor, transferrin receptor, or any combination thereof.

[0017] Also contemplated herein is the peptide delivery system described above, wherein the second transmembrane domain is connected to an intracellular region comprising one or more intracellular domains of 41BB, ICOS, and CD3ζ. In certain embodiments, phosphorylation by kinases within the intracellular domains of 41BB, ICOS, or CD3ζ may activate GrzB, which is then transported outside the cell, causing GrzB to cleave the first and / or second cleavage sites.

[0018] In certain embodiments, the peptide delivery system may include third and fourth transmembrane domains connected by a second loop region, the third transmembrane domain being connected to an intracellular region containing an intracellular domain of 41BB, ICOS, or CD3Z. In such embodiments, phosphorylation by a kinase within the intracellular domain of 41BB, ICOS, or CD3Z may activate the cell to release endogenous GrzB, which is then transported outside the cell to cleave a GrzB cleavage site located within the first or second loop region, thereby releasing the peptide payload.

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

[0020] The above-described peptide delivery system is intended to have a payload comprising an antibody, an antibody fragment, VHH, a cytokine or chemokine comprising a fraction or derivative of sulfated xylan, wherein the cytokine or chemokine is the IL-17 family of cytokines including 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-25, interferon, G-CSF, M-CSF, GM-CSF, BDNF, CN TF, 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, TNFα, TNFB, TPO, VEGF, GH, NGF, NT3, NT4, NT5, NT6, NT7, Oncostatin M (OSM), Insulin, MCP-1, MCP-2, MCP -3, MCP-4, MCP-5, members of the MIP-1 family including MIP-1a, MIP-2, eotaxin (eotaxin-1, -2 or -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- or virus-binding receptors, cytokine-induced neutrophil chemoattractants (KC), TNF-α and IFN-γ), and other soluble mediators of inflammation 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, CCL19, CCL20, CCL21, CCL22, CCL23, C CL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15,It is an antagonist of a ligand selected from the list comprising CXCL16, CXCL17, XCL1, XCL2, CX3CL1, thrombin, urokinase, a vaccine, or any combination thereof.

[0021] In 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 to 128 amino acids long or 4 to 20 amino acids long. In certain embodiments, the linker may be selected from the linkers shown in Table 4.

[0022] In one embodiment, the peptide delivery system may be specifically localized (sor t) in extracellular vesicles.

[0023] Also contemplated herein is the above-described peptide delivery system configured to release two or more peptide payloads intracellularly or extracellularly.

[0024] Also contemplated herein is such a peptide delivery system comprising two or more sheddase cleavage sites, where each cleavage site can be identical to or different from any of the other cleavage sites.

[0025] Also contemplated herein is such a peptide delivery system comprising an intracellular domain, where the intracellular domain comprises the stalk domain of CD45 phosphatase.

[0026] Also contemplated herein is the above-described peptide delivery system comprising a single-domain antibody or single-chain of an antibody that binds to a self-associated antigen (SAA) and brings SAA in the vicinity, for example, in cis or in trans.

[0027] Also contemplated herein is such a peptide delivery system that includes a single domain antibody or single chain of an antibody that binds to a self-organizing antigen (SAA), and further includes a CD45 phosphatase binding portion or an NKG2A binding portion that activates an inhibitory signal within a target cell.

[0028] In an embodiment of the peptide delivery system, the peptide payload may be any peptide or protein that functions in the human body, including hormones, cytokines, chemokines, prodrugs, and antibodies in various forms such as chimeric antigen receptors (CARs), bispecific killer cell engagers (BIKEs), and trispecific killer cell engagers (TRIKEs).

[0029] In embodiments, the peptide delivery system described above includes 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), and TNFSF14-a-CD16-a-CD33-Myc-TNR1A-ADAM. 17-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-TNFα-Myc-MMP9-CD63-Cyt (SEQ ID NO: 88 or 295), CD63-MMP9-2L-TNFα-Myc-MMP9-CD63-D-Cyt (lacking EV localization (sort) motif) (SEQ ID NO: 103 or 311), CD63 -DL1-MMP2-2L-TNFα-Myc-MMP2-CD63-Cyt (SEQ ID NO: 104 or 368), CD63-MMP9-2L-TNFα-Myc-MMP9-aCD19CAR (SEQ ID NO: 105 or 369), CD63-MMP2-2L-TNFα-Myc-MMP2-CD63-D-Cyt (SEQ ID NO: 106 or 370 or 371), CD63-MMP2-2L-TNFα-Myc-MMP2-aCD19CAR (SEQ ID NO: 107 or 372 or 373), CD69cyt-T MII-TNFα-Myctag-CD28TM-cyt (SEQ ID NO: 119 or 370), CD69cyt-TMII-MMP9-TNFα-Myc-MMP9-CD28TM-cyt (SEQ ID NO: 120 or 377), CD69Cyt-TM-GrzB-TNFα-Myc-GrzB-a-CD19sc-Stalk-CD28-CD3z (SEQ ID NO: 107, 372, or 373), CD69Cyt-TM-GrzB-nLuc-Myc-GrzB-CD28-CD3z (Delta sc-GAGE B) (SEQ ID NO: 108 or 374), CD69Cyt-TM-nLuc-Myc-a-CD19sc-Stalk-CD28-CD3z (Delta GrzB, GAGE ​​B)A) (Sequence ID: 109 or 375), CD69Cyt-TM-GrzB(original)-nLuc-Myc-GrzB(original)-a-CD19sc-Stalk-CD28-CD3z (Sequence ID: 358 or 382), CD69Cyt-TM-GrzB(IEPD)-nLuc-Myc-GrzB(IEPD)-a-CD19sc-Stalk-CD28-CD3z (Sequence ID: 110 or 3 83) Alternatively, one of the following may be selected: CD69Cyt-TM-GrzB(SASA)-nLuc-Myc-,GrzB(SASA)-a-CD19sc-Stalk-CD28-CD3z (Sequence ID: 111 or 376), or CD69Cyt-TM-GrzB(ADKG)-nLuc-Myc-GrzB(ADKG)-a-CD19sc-Stalk-CD28-CD3z (Sequence ID: 112 or 384).

[0030] In certain embodiments, the peptide delivery system described above may include cells to which exogenous polypeptides are immobilized. In certain embodiments, the cells may be selected from hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs) and cell products derived therefrom, adoptive T cells, dendritic cells (DCs), natural killer (NK) cells, or any therapeutic immune or non-immune cells.

[0031] Also contemplated herein are adoptive cells or genetically engineered immune cells containing a peptide payload of such a peptide delivery system, wherein the immune cells are autologous or allogeneic.

[0032] In some embodiments, the exogenous polypeptide may be introduced into cells individually via a CRISPR system, or in the form of a viral or nonviral vector, or in the form of one or more mRNAs encoding the exogenous polypeptide.

[0033] In embodiments, the exogenous polypeptide of the peptide delivery system may be delivered using CPP, micelles, liposomes, nanoparticles, dendrimers, nanotubes, electroporation, viral transduction, nucleofection, transfection, cell fusion, or microinjection.

[0034] Also intended herein are gene constructs encoding exogenous polypeptides for peptide delivery systems.

[0035] Also contemplated herein is a method of treating a patient requiring the peptide delivery system described above, comprising administering the peptide delivery system to the patient.

[0036] Also disclosed herein is the delivery of peptides to target gene loci in patients that require them. A method comprising administering to a patient a peptide delivery system comprising a lipid-containing vesicle (e.g., a cell, an extracellular vesicle, or a lipid-containing particle) on which an exogenous polypeptide is immobilized, the system comprising: a peptide payload; first and second transmembrane regions, wherein the first transmembrane region comprises a transmembrane domain or other proteolytic cleavage site connected to at least one schidase, and the second transmembrane region comprises a transmembrane domain or other proteolytic cleavage site connected to at least one schidase; and optionally, a targeting region for targeting the exogenous polypeptide to a target gene locus, wherein once the lipid-containing vesicle reaches the target gene locus, the schidase and / or other proteases cleave the cleavage sites of the first and second transmembrane regions to release the peptide payload.

[0037] Also intended herein are such methods derived from naturally occurring proteolytic cleavage substrates in glycosylphosphatidylinositol (GPi)-anchored proteins, which are membrane proteins having one, two, three, four or more transmembrane domains, intracellular domains, or extracellular domains in the extracellular matrix or adjacent cells.

[0038] Also contemplated herein is a method in which the schedase is selected from full-time or part-time schedases. In embodiments, the full-time schedase may be selected from ADAM proteases, BACE proteases, serine proteases granzyme-B, and site 1 proteases. The ADAM proteases may be selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, and ADAM33. The part-time schidase may be selected from mepurin B, MT-MMP (membrane-type matrix metalloproteinase), proprotein converterase, transmembrane serine protease, matrix metalloproteinase, legmine, and cathepsin S and L, where mepurin B is a membrane-anchored type 1 metalloproteinase, and MT-MMP is either membrane-anchored type 1 or GPI-anchored, encompassing MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, and MT6-MMP. The proprotein converterase may be selected from PCSK1 / 3, KCSK2, furin, PCSK4, PSCK5 / 6, PACE4, PCSK7, and PCSK9. The transmembrane serine proteases may be selected from membrane-anchored type II proteases, including, for example, matryptase, matryptase-2, matryptase-3, polymerase-1, choline, hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, spinesin, enteropeptidase, HAT, DESCL1, TMPRSS11A, HAT-L4, and HAT-L5. Matrix metalloproteases may also be soluble proteases, and one or more of the following can be cited: 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 certain 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] Also intended herein is a transmembrane domain of the first and / or second transmembrane region being one of CD63, CD9, CD81, CD28, CD4, CD8, CD34, CD69, CD19, CD20, integrin, TNFSF14, TNR1A, aquaporin, NOTCH, NgR1, NRG1, GPi anchor, EGFR, and rhodopsin or The above method involves a transmembrane domain derived from multiple sources.

[0040] Also intended herein is the method described above, in which cutting at one or more of the cutting sites is performed by endogenous schedase.

[0041] Also intended herein is the above method, wherein the peptide delivery system further comprises a schedase, and the cleavage is performed at one or more cleavage sites of the peptide delivery system by the schedase.

[0042] Also intended herein are exogenous polypeptides whose intracellular or extracellular domains include: 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 costimulator (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signal transduction lymphocyte activating molecule (SLAM protein), costimulatory ligand (PD-L2, 4-1B BL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecules (ICAM, CD30L, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin b receptor, 3 / TR6, ILT3, ILT4), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(L IGHTR), KIRDS2, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha , ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, IT GAM, CD11b, ITGAX, CD11e, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI(CD226), SLAMF4(CD244, 2B4), CD84, CD96(TACTILE), CEACAM1, CRTThe above method involves selecting one or more intracellular or extracellular domains from among ligands that specifically bind to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0043] Also intended herein are targeted molecules such as 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, ADAM10, CHRNA2, LeY, and NK. G2D, CSI, CD44v6, CD24, LGR5, ALDH, ALDH1, CD54, Sca1, CD271, CD123, CD36, CD109, CD110, CD71 negative, CCA, ABCG2, Claudin-18.2 (Claudin-18A2, or Claudin-18 isoform 2), PSCA, DLL3 (Delta-like protein 3, Drosophila delta homolog 3, Delta 3), Mud 7 (Mucin 17, Muc3, Muc3), FAP alpha (Fibroblast-activating protein alpha), Ly6G6D (Lymphocyte antigen 6 complex locus protein G6d, c6orf23, G6D, MEGTI, NG25), PSMA, MSLN, or RNF43 (E3 ubiquitin-protein ligase RNF43, RING finger protein 43), BAFF, C242 antigen, disialoganglioside (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 Dispersion Factor Receptor Kinase, IGF-1 Receptor, IGF-I, IgGI, LI-CAM, Integrin α5β1, Integrin ανβ3, Regmine, MORAb-009, MS4A1, MUC1, Mucin CanAg, C-MET, CCR4, CD152, CD10, CD19, CD20, CD200, N-Glycolylneuraminic Acid, NPC-IC, PDGF-Rα, PDL192, Phosphatidylserine, Tumor Antigen CTAA16.The above method involves binding to 88, VEGF-A, VEGFR-1, VEGFR2, vimentin, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF-β, TRAIL-R1, TRAIL-R2, folate receptor, transferrin receptor, and any combination thereof.

[0044] Also intended herein is the method described above, wherein the first and / or second transmembrane domains comprise (i) an intracellular region comprising an intracellular region sequence from 41BB, ICOS, or CD3ζ, and (ii) a GrzB cleavage site, wherein the action of a kinase on the intracellular region sequence from 41BB, ICOS, or CD3ζ induces the release of GrzB, which is then transported outside the cell to cleave the GrzB cleavage site, thereby releasing a peptide payload, the peptide payload optionally comprising a bispecific antibody.

[0045] Also intended herein is the method described above, wherein the second transmembrane region comprises a transmembrane domain from a NOTCH protein and a cleavage site within the cell membrane, and cleavage at the cleavage site within the cell membrane releases a functional domain in the intracellular environment.

[0046] Also intended herein is a peptide payload comprising a cytokine or chemokine consisting of a fraction or derivative of sulfated xylan, wherein the cytokine or chemokine comprises the IL-17 family of cytokines including 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-25, interferon, G-CSF, M-CSF, GM-CSF, BDNF, CNTF, EGF, EPO, FGF1, FGF2, FG F3, 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, TNFα, 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-1a This includes members of the MIP-1 family, MIP-2, eotaxin (eotaxin-1, -2, or -3), PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase, cathepsin family enzymes, cell adhesion molecules such as PECAM-1, soluble receptors, or cell- or virus-binding receptors, cytokine-induced neutrophil chemoattractants (KC), TNF-α and IFN-γ), and other soluble mediators of inflammation 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, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, C CL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2,The above method is an antagonist of a ligand selected from a list including CX3CL1, thrombin, urokinase, vaccine, or any combination thereof.

[0047] Also intended herein is the above method, wherein the exogenous polypeptide further comprises at least one linker, the at least one linker optionally positioned between a cleavage site located in a first transmembrane region and a first binding site (e.g., an anti-CD3 VHH domain), between a cleavage site located in a second transmembrane region and a second binding site (e.g., an anti-TAA VHH domain), and / or between the first binding site and the second binding site.

[0048] Also intended herein is the above method, wherein the linker is 2 to 128 amino acid long, or preferably 4 to 20 amino acid long.

[0049] Also intended herein is the method described above, in which the linker is selected from Table 4.

[0050] Also intended herein is the above method, which can also be configured to specifically localize (sort) exogenous polypeptides to extracellular vesicles (EVs).

[0051] Also intended herein is the method described above, wherein the peptide payload comprises two or more bispecific engagementrs, and the bispecific engagementrs are released intracellularly or extracellularly.

[0052] Also intended herein is the above method, wherein the peptide delivery system includes schedase cleavage sites, and each cleavage site may be identical to or different from any of the other cleavage sites.

[0053] Also intended herein is the above method, wherein the peptide delivery system comprises an intracellular domain, and the intracellular domain comprises the stalk domain of CD45 phosphatase.

[0054] Also intended herein is the above method, comprising a peptide delivery system that binds to a self-associated antigen (SAA) and brings the SAA to the vicinity, for example, in a cis or trans configuration, a single-domain antibody or a single-chain antibody.

[0055] Also intended herein is the method described above, comprising the use of a peptide delivery system comprising a single-domain antibody or a single chain of antibody that binds to a self-associated antigen (SAA), and further comprising a CD45 phosphatase- or NKG2A-binding moiety that activates an inhibitory signal within a target cell.

[0056] Also intended herein is the method described above, in which the peptide payload is any peptide that performs a function in the body and is a potential therapeutic agent including hormones, cytokines, chemokines, prodrugs, and antibodies in various forms such as chimeric antigen receptors (CARs), bispecific killer cell engagers (BIKEs), and triplicate killer cell engagers (TRIKEs).

[0057] Also intended herein is a peptide delivery system 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 (Sequence ID: 71 or 252), CD63-MMP9-2L-TNFα-Myc-MMP9-CD63-Cyt (Sequence ID: 88 or 295), CD63-MMP9-2L-TNFα-Myc-MMP9-CD63-D-Cyt (lacking EV localization (sort) motif) (Sequence ID: 103 or 311), CD63-DL1-MMP2-2L-TNFα-Myc-MMP2-CD63-Cyt (Sequence ID: 71 or 252), CD63-MMP9-2L-TNFα-Myc-MMP2-CD63-Cyt9-CD63-Cyt (Sequence ID: 88 or 295), CD63-MMP9-2L-TNFα-Myc-MMP9-CD63-Cyt (lacking EV localization (sort) motif) (Sequence ID: 103 or 311), CD63-DL1-MMP2-2L-TNFα-Myc-MMP2-CD63-Cyt (Sequence ID: 7 (Sequence number: 104 or 368), CD63-MMP9-2L-TNFα-Myc-MMP9-aCD19CAR (Sequence number: 105 or 369), CD63-MMP2-2L-TNFα-Myc-MMP2-CD63-D-Cyt (Sequence number: 106 or 370 or 371), CD63-MMP2-2L-TNFα-Myc-MMP2-aCD19CAR (Sequence number: 107 or 372 or 373), CD69cyt-TMII-TNFα-Myctag-CD2 8TM-cyt (SEQ ID NO: 119 or 370), CD69cyt-TMII-MMP9-TNFα-Myc-MMP9-CD28TM-cyt (SEQ ID NO: 120 or 377), CD69Cyt-TM-GrzB-TNFα-Myc-GrzB-a-CD19sc-Stalk-CD28-CD3z (SEQ ID NO: 107, 372, or 373), CD69Cyt-TM-GrzB-nLuc-Myc-GrzB-CD28-CD3z (Delta sc-GAGE B) (SEQ ID NO: 108 or 374), CD69Cyt-TM-nLuc-Myc-a-CD19sc-Stalk-CD28-CD3z (Delta GrzB, GAGEA) (Sequence ID: 109 or 375), CD69Cyt-TM-GrzB(original)-nLuc-Myc-GrzB(original)-a-CD19sc-Stalk-CD28-CD3z (Sequence ID: 358 or 382), CD69Cyt-TM-GrzB(IEPD)-nLuc-Myc-GrzB(IEPD)-a-CD19sc-Stalk-CD28-CD3z (Sequence ID: 110 or 383) The above method is selected from CD69Cyt-TM-GrzB(SASA)-nLuc-Myc-,GrzB(SASA)-a-CD19sc-Stalk-CD28-CD3z (sequence number: 111 or 376), or CD69Cyt-TM-GrzB(ADKG)-nLuc-Myc-GrzB(ADKG)-a-CD19sc-Stalk-CD28-CD3z (sequence number: 112 or 384).

[0058] Also intended herein is the method described above, wherein the lipid-containing vesicles contain cells, the cells being 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. In certain embodiments, the immune cells may be autologous or allogeneic.

[0059] Also intended herein is the method by which the peptide delivery system is introduced into cells individually in the form of a virus or non-viral vector, mRNA, peptide, protein, antibody, nanobody, oligonucleotide, or extracellular vesicle (EV).

[0060] Also intended herein are the methods described above, in which the peptide delivery system is delivered using CPP, micelles, liposomes, nanoparticles, dendrimers, nanotubes, electroporation, viral transduction, nucleofection, transfection, cell fusion, or microinjection.

[0061] The objective of this invention is to deliver peptides.

[0062] The objective of this invention is to target the payload to a specific cell type.

[0063] The objective of this invention is to deliver multiple peptides.

[0064] The objective of the present invention is to introduce a peptide delivery system into any cell that can deliver at least one peptide to a target cell or target gene locus.

[0065] The objective of this invention is to create a single-delivery system that can be incorporated into any cell. [Brief explanation of the drawing]

[0066] [Figure 1] Figure 1 shows a general embodiment of the present invention that includes two transmembrane domains, namely "dispanin". [Figure 2] Figure 2 shows an embodiment of the "dispanin" delivery system, which, in order, includes a type II transmembrane domain, a first MMP cleavage sequence, a first linker ("linker-a"), an anti-CD3 VHH domain, a second linker ("linker-b"), an anti-TAA VHH domain, a third linker ("linker-c"), a second MMP cleavage sequence, and a GPI anchor. [Figure 3] Figure 3 shows the amino acid sequence and various domains / motifs of the embodiment shown in Figure 2. [Figure 4A] Figure 4A illustrates a general embodiment of the present invention using a modified tetraspanin protein (CD63) containing a special motif GYEVM, which is linked to a specific engager (GAGE) (TNF-α in this specification) for EV-specific delivery. [Figure 4B] Figure 4B shows the amino acid sequence and various domains / motifs of the embodiment shown in Figure 4A. [Figure 5A] Figure 5A shows a general embodiment of the present invention using a modified tetraspanin protein (CD63) without an EV localization (sort) motif. [Figure 5B]Figure 5B shows the amino acid sequence and various domains / motifs of the embodiment shown in Figure 5A. [Figure 6] Figure 6 shows an embodiment of the present invention that uses modified transmembrane C-type lectin protein (CD69). [Figure 7] Figure 7 shows a general embodiment of the present invention using modified tetraspanins containing four different types of transmembrane domains (TMI to TMIV). [Figure 8] Figure 8 shows a general embodiment of the present invention using modified tetraspanin. [Figure 9] Figure 9 shows an embodiment of the dispanin of the present invention. [Figure 10] Figure 10 shows the amino acid sequence for an embodiment of CD63-loop-EV localization (sorting), illustrating the corresponding domains and motifs in the embodiment shown in Figure 9. [Figure 11] Figure 11 shows an embodiment of the dispanin of the present invention. [Figure 12] Figure 12 shows the amino acid sequence of the embodiment shown in Figure 11, and the corresponding domains and motifs. [Figure 13] Figure 13 shows an embodiment of the dispanin of the present invention. [Figure 14] Figure 14 shows the amino acid sequence of the embodiment shown in Figure 13, and the corresponding domains and motifs. [Figure 15] Figure 15 is a diagram illustrating an embodiment of the dispanin of the present invention. [Figure 16] Figure 16 shows the amino acid sequence for an embodiment of CD63-Grn-Ba-CD16-La-CD33-Grn-Ba-CD38CAR, illustrating the corresponding domains and motifs of the embodiment shown in Figure 15. [Figure 17A] Figure 17A is a diagram of an embodiment of the dispanin of the present invention encoded by CD69Cyt-TM-II-TNFα-Myc-CD28TM-CYT for TNFα delivery. [Figure 17B]Figure 17B shows the amino acid sequence of CD69Cyt-TM-II-TNFα-Myc-CD28TM-CYT. [Figure 18A] Figure 18A shows an embodiment of the present invention encoded by CD69cyt-TMII-9-TNFα-Myctag-9-CD28TM-cyt, which contains two MMP9 cleavage sites. [Figure 18B] Figure 18B shows the amino acid sequence of CD69cyt-TMII-9-TNFα-Myctag-9-CD28TM-cyt. [Figure 19] Figure 19 shows an embodiment of the present invention having α-TAA-αSAA as a payload that can be delivered during cutting. [Figure 20] Figure 20 shows the plasmid transmembrane domain, protease motif, and payload of various embodiments disclosed in this application. [Figure 21] Figure 21 is a diagram showing the experimental setup / model of the embodiment of the present invention as presented in this application. [Figure 22A] Figure 22A shows three options for experimental setups / models including TNFα and for measuring NF-κB luciferase activity. [Figure 22B] Figure 22B shows three options for experimental setups / models including TNFα and for measuring NF-κB luciferase activity. [Figure 22C] Figure 22C shows three options for experimental setups / models including TNFα and for measuring NF-κB luciferase activity. [Figure 23A] Figure 23A shows an experimental setup / model of an embodiment disclosed in this application in which granzyme B leads to the release of a TNFα payload from the surface of NK cells. [Figure 23B] Figure 23B shows an experimental setup / model of an embodiment disclosed in this application in which granzyme B results in the release of a TNFα payload from the surface of K562 cells. [Figure 24]Figure 24 is a graph showing that the TNFα payload becomes functional through direct intercellular contact with HEK293-NF-κB-Luc reporter cells. [Figure 25] Figure 25 is a graph showing the delivery of the TNFα payload after cleavage by MMP9. Note: Cotransfection between GAGE ​​and MMP9. The inventors have shown cases with and without EVs to control the movement of MMP through EVs. [Figure 26] Figure 26 is a graph mainly showing the delivery of the TNFα payload into the EV. [Figure 27] Figure 27 is a graph showing the delivery of the TNFα payload after cleavage by MMP2. In this experiment, supernatant from HEK293 cells expressing the GAGE ​​construct was used. [Figure 28] Figure 28 is a graph primarily showing the delivery of the TNFα payload into the EV. The segmentation is also done using MMP2. Note: EV missing vs. EV present. [Figure 29] Figure 29 is a graph showing differential CD63 and CD69 transmembrane GAGE ​​expression in HEK293 cells. [Figure 30] Figure 30 is a graph showing the results of an in vitro responsive assay to TNFα payload delivery mediated by MMP9 cleavage and NF-κB activation. [Figure 31] Figure 31 shows the percentage of K562 cells that were positive for surface TNFα expression three days after transduction. [Figure 32] Figure 32 is a graph showing the delivery of the TNFα payload from K562 cells during cleavage by granzyme B (GrzB) secreted by NK92 cells. [Figure 33] Figure 33 shows the percentage of K562 cells that were positive for surface TNFα and αCD19 / CAR expression 6 days after cell sorting. (Surface expression of GAGE ​​nanoluciferase in K562 transduced cells (MOI6)). [Figure 34A]Figure 34A is a graph showing the release of nanoluciferase (nLuc) payload from K562 cells upon cleavage by granzyme B (GrzB) secreted by NK92 cells. E:T 3:1, 4-hour incubation. [Figure 34B] Figure 34B is a graph showing the release of nanoluciferase (nLuc) payload from K562 cells upon cleavage by granzyme B (GrzB) secreted by NK92 cells. E:T 3:1, 4-hour incubation. [Figure 35] Figure 35 shows the percentage of KHYG-1 cells that were positive for surface nanoluciferase (nLuc) expression three days after transduction. [Figure 36] Figure 36 is a data graph showing the release of nanoluciferase (nLuc) payload from KHYG-1 cells upon cleavage with granzyme B (GrzB) after co-culture with Nalm-6 cells for 1–3 hours. Different time points after normalization to MFI. [Figure 37A] Figure 37A is a graph showing the percentage of KHYG-1 cells that were positive for surface nanoluciferase and MFI 6 days after cell sorting. [Figure 37B] Figure 37B is a graph showing the percentage of KHYG-1 cells that were positive for surface nanoluciferase and MFI 6 days after cell sorting. [Figure 38] Figure 38 is a graph showing the release of nanoluciferase (nLuc) payload from KHYG-1 cells upon cleavage with granzyme B (GrzB) after 4 and 6 hours of co-culture with K562 cells. [Figure 39A] Figure 39A is a graph showing the percentage of NK92 cells that were positive for surface nanoluciferase and MFI 10 days after transduction of NK92 cells. [Figure 39B] Figure 39B is a graph showing the percentage of NK92 cells that were positive for surface nanoluciferase and MFI 10 days after transduction of NK92 cells. [Figure 40]Figure 40 is a graph showing the release of nanoluciferase (nLuc) payload from NK92 cells upon cleavage with granzyme B (GrzB) after 4 hours of co-culture with Raji cells. [Figure 41] Figure 41 is a graph showing the percentage of NK92 cells (unsorted cells) and sorted cells that were positive for surface nanoluciferase three days after transduction. [Figure 42] Figure 42 is a graph showing the release of nanoluciferase (nLuc) payload from NK92 cells upon caspase-3 cleavage after 6 hours and 24 hours of co-culture with K562 cells. [Modes for carrying out the invention]

[0067] The details provided herein are illustrative and intended only to illustrate various embodiments, and are presented in order to provide what is considered to be the most useful and readily understandable description of the principles and conceptual aspects of the methods and compositions described herein. In this regard, no attempt has been made to provide more detail than is necessary for a basic understanding, and this specification makes it clear to those skilled in the art how various forms are embodied in practice.

[0068] The present invention is described herein by reference to more detailed embodiments. However, the present invention may be embodied in different forms and should not be construed as being limited to the embodiments shown herein. Rather, these embodiments are provided to fully convey to those skilled in the art that this disclosure is sufficient and complete and of its scope.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the invention pertains. The terminology used herein is for the purpose of describing specific embodiments and is not intended to be limiting. The singular forms “a,” “an,” and “the” used herein and in the appended claims are intended to encompass the plural forms unless otherwise clearly indicated in the context. All publications, patent applications, patents, and other reference materials referred herein are expressly incorporated by reference as a whole.

[0070] Unless otherwise shown, the numerical parameters expressed in the following specification and attached claims are approximations, which may vary depending on the desired characteristics sought to be obtained and may therefore be modified by the term “about”. At a minimum, and not as an attempt to limit the application of the theory equivalent to the claims, each numerical parameter should be interpreted in light of the number of significant figures and the usual rounding approach.

[0071] Although the numerical ranges and parameters representing a wide range are approximations, the numerical values ​​presented in the specific examples are reported as precisely as possible. However, each numerical value inherently contains certain errors arising from the standard deviation found in each test measurement. Each numerical range presented throughout this specification includes such narrow numerical ranges that fall within such broader numerical ranges, as if all of the narrow numerical ranges were explicitly described herein. The applicant intends to represent the range derived from the data points and expresses the range disclosed herein.

[0072] All references cited herein are incorporated by reference in their entirety.

[0073] Considered herein is a peptide or protein cargo delivery system having directed delivery and / or site-specific release and site-specific activation of a cargo. The cargo is targeted to an intended site and / or activated upon reaching the target. This approach makes it possible to increase local or targeted site doses while limiting the number of off-target side effects. The peptide or protein delivery system of the present invention may include specific safety and specificity mechanisms such that the biologic is released in high doses only to the target site and not to other tissues (e.g., tissues that may also express antigens for specific antibody-derived biologics). In addition to making drugs based on these peptides safer and more broadly applicable, our systems can reduce the cost of treatment and can be customized for more personalized or patient population-specific use. Furthermore, the delivery systems and methods disclosed herein can be equivalent to more frequent dosing and do not require repeated administration of the peptide. For example, delivery via cargo-carrying cells that store the peptide or protein to be delivered may provide an in vivo storage for inactive peptides or proteins (e.g., prodrugs).

[0074] Mammalian cells possess the hardware necessary for shedding many cell membrane-bound proteins. Shedding is a controlled, irreversible post-translational process carried out by the family of proteolytic enzymes, particularly schedases, described below. Cell biological processes cause cells to control the expression levels and functions of substrate proteins, as well as the location of those proteins on the cell surface.

[0075] Examples of proteolytic cleavage substrates include glycosylphosphatidylinositol (GPi)-anchored proteins that are membrane proteins having one, two, three, four, or more transmembrane domains, intracellular domains, or extracellular domains in the extracellular matrix or adjacent cells. Membrane proteins in the delivery system envisioned herein that have two transmembrane domains are referred to herein as “dispanins.” Membrane proteins in the delivery system envisioned herein that have four transmembrane domains are referred to herein as “tetraspanins.” Cleavage can target the near-membrane or distal region of membrane-bound proteins, resulting in the release of protein domains inside and / or outside the cell (ectodomain and / or endodomain release).

[0076] The inventors have found a way to directly deliver at least one peptide or protein peptide at a desired site of action. We designed a cell-based delivery system that enables the delivery and release of loads. The payload is uniquely fixed to the cell through a transmembrane domain (TMD). A TMD is part of a protein molecule that spans the entire cell membrane, typically consisting of one or more alpha-helices or beta-sheets that cross the membrane's lipid bilayer, fixing the protein in place and allowing it to interact with molecules on both sides of the membrane. Many important proteins have transmembrane domains, including receptors, transporters, and enzymes. These domains are often involved in transduction of cross-membrane signals or the transport of molecules into or out of the cell.

[0077] The delivery systems envisioned herein consist of, or include, single or multi-pass transmembrane (TM) proteins embedded in the membrane of a vehicle (e.g., cells, extracellular vesicles, or other lipid-containing particles or compositions). Additional motifs that modulate the delivery / release of the payload in the required environment may be present. Accordingly, the inventors aimed to infuse genetically engineered cells or EVs configured to deliver the cargo directly to the required site and release it at the correct time, as an alternative to direct systemic injection of biologics.

[0078] The delivery system envisioned herein is applicable to any cell type and can be used to deliver any relevant peptide. Suitable cell types include, but are not limited to, hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs) and cell products derived therefrom, adoptive T cells, dendritic cells (DCs), natural killer cells (NKs), NKT cells, or any therapeutic immune or non-immune cells. Such cells may be used in autologous or allogeneic cell therapy.

[0079] In one embodiment, the inventors modified NK cells to generate and deliver bispecific engagers (GAGEs) within the tumor microenvironment (TME). The TME consists of cellular and cell-free partners capable of modulating various aspects of tumorigenesis, including tumor initiation, 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 can be further regulated and induced by either NK cell activation (by cleavage induced by activation) or the tumor environment (by cleavage upon contact with proteases specifically elevated in the TME). In addition, such activation-stimulated release can lead to NK cell proliferation and activation depending on the availability of tumor antigens. In embodiments, the delivery systems described herein encompass next-generation cancer immunotherapy systems with enhanced ability to generate and release payloads under desired conditions and at specific sites.

[0080] The shedases that can be used in this payload delivery system are not particularly limited and are further described below. For example, the shedase may be selected from matrix metalloproteinases (MMPs) and granzyme B (GrzB) that enable environment-specific cargo release. Models based on the ADAM family of MMPs and the serine protease granzyme-B have also been presented. In these models, additional functions such as tumor targeting, proliferation, and / or activation were modulated by the addition of extracellular and / or intracellular domains. These additional domains may include extracellular cytokines, drugs or prodrugs, chimeric antigen receptor (CAR)-like or biantigen receptor (DAR)-like domains, or intracellular modulators, inhibitors, or signaling domain-containing proteins.

[0081] Furthermore, the system is induced by passive release in the tumor environment in response to specific MMPs released by cancer cells, or by activation of producer cells through receptor engagement. It was designed for active release.

[0082] These systems can also be designed in a way that allows them to be sorted into specific intracellular compartments. The inventors have designed protein molecules that can be actively sorted into extracellular vesicles (EVs), such as exosomes. Cells can release these exosomes in large quantities, releasing a payload upon protease cleavage within the TME or other target sites. These EVs can also be harvested from in vitro cultures and administered to patients as off-the-shelf therapeutics.

[0083] In its simplest form, the present invention includes a prodrug form of a peptide comprising first and second transmembrane regions, the first transmembrane region comprising at least one transmembrane unit connected to at least one cleavage site, and the second transmembrane region comprising one transmembrane unit connected to at least one cleavage site, and optionally the delivery system may include a targeting portion for targeting target cells to which the prodrug, in which case, upon contact of the target cells, a schedase cleaves the cleavage site and releases the peptide.

[0084] The inventors primarily used a set of TMDs within this multipath payload delivery system. The N-terminus of the TMD consists of a linker / payload containing a type II TMD connected to a type I TMD domain at the C-terminus. The TMD is preferably at least one or more of CD63, CD9, CD81, CD28, CD4, CD8, CD34, CD69, CD19, CD20, integrins, TNFSF14, TNR1A, aquaporins, NOTCH, NgR1, NRG1, GPi anchors, EGFR, rhodopsin, or any related TMD that provides similar functionality to the system may be used to express the inventors' invention.

[0085] In some cases, the second transmembrane region has an intracellular region containing 41BB, ICOS, and CD3ζ, and an MMP / GrzB that cleaves an extracellular MMP / GrzB cleavage site. The action of phosphatases at 41BB, ICOS, and CD3ζ cleaves and activates MMP / GrzB, transporting MMP / GrzB outside the cell, cleaving the MMP / GrzB cleavage site, thereby releasing a bispecific antibody / payload. In some cases, the first and second transmembrane regions are embedded with a NOTCH protein-dependent pulling system having a secretase cleavage site inside the cell membrane, along with multiple cargo / payloads connected to multiple extracellular or intracellular cleavage sites. In some cases, the system may be embedded by recruiting a self-associated antigen via the addition of a single-strand (α-SAA).

[0086] The genetically engineered system may be introduced into cells individually in the form of a virus or non-viral vector, mRNA, peptide, protein, antibody, nanobody, oligonucleotide, or extracellular vesicle (EV), which can be delivered using CPP, micelles, liposomes, nanoparticles, dendrimers, nanotubes, electroporation, viral transduction, nucleofection, transfection, cell fusion, or microinjection. The viral vector may contain genes / (multiple) encoding GAGE, and the viral particles thus produced may carry the genes(multiple). When the virus infects a cell, the genes / (multiple) are stably taken up and express the encoded proteins. The non-viral vector may be in the form of a plasmid carrying the GAGE ​​genes / (multiple). Cells are transfected, thereby resulting in the expression of genes / (multiple) encoding GAGE ​​proteins / (multiple). Cells can be selected to ensure stable expression. One possible gene delivery strategy that could be utilized is a site-specific uptake strategy such as CRISPR (e.g., Cas9, Cpf1, or other Cas effector protein complexes) or a transposon-transposase strategy. The CRISPR-Cas system can be introduced via electroporation or via LNPs and can take up the gene encoding GAGE.

[0087] The present invention relates to a peptide delivery system having site-directed release and activation of peptides. Site-directed means that the peptide-producing cells or extracellular viable cells (EVs) containing the peptide delivery system are targeted to the intended site. The cells or EVs are then activated and release the peptide at the target. This approach makes it possible to increase the dose delivered while limiting the number of off-target side effects. The peptide delivery system of the present invention incorporates specific safety and specificity mechanisms so that the biologic / payload is released in high doses only at the target site and not to other tissues where the antigen for the specific biologic (e.g., antibody-derived biologic) may also be expressed. In addition, the inventors' system can reduce the cost of treatment, in addition to making drugs based on these peptides safer, more applicable, and more specific. Moreover, since delivery via cargo cells that produce and store the peptides provides an in vivo storage of inactive peptides, the present invention can be equivalent to more frequent dosing without the need for repeated administration of the peptides.

[0088] Mammalian cells possess hardware necessary for shedding many cell membrane-bound proteins. Shedding is a controlled, irreversible post-translational process carried out by a family of proteolytic enzymes called proteases or schedases. Cell biological action causes cells to control the expression level and function of substrate proteins, as well as the location of those proteins on the cell surface. Schedases, as a classification, are well known and include full-time and part-time schedases. 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.

[0089] Full-time schidases include ADAM proteases, BACE proteases, and serine proteases such as granzyme-B and site 1 proteases. ADAM proteases (metalloproteases) are membrane-anchored type 1 proteases and include ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, and ADAM33. BACE (aspartyl) proteases are also membrane-anchored type 1 proteases and include BACE1 and BACE2. Site 1 (serine) proteases are membrane-anchored type 1 proteases that include SKI-a or SIP.

[0090] Examples of part-time schidases include meprin B, MT-MMP (membrane-type matrix metalloproteinase), proprotein converterase, transmembrane serine protease, matrix metalloproteinase, legmine, and cathepsin S and L. Meprin B is a membrane-anchored type 1 metalloproteinase. MT-MMP is either membrane-anchored type 1 or GPI-anchored and includes MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, and MT6-MMP. Proprotein converterase is either membrane-anchored type 1 or soluble protease and includes PCSK1 / 3, KCSK2, furin, PCSK4, PSCK5 / 6, PACE4, PCSK7, and PCSK9. Transmembrane serine proteases are membrane-anchored type II proteases and include matryptase, matryptase-2, matryptase-3, polymerase-1, choline, hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, spinesin, enteropeptidase, HAT, DESCL1, TMPRSS11A, HAT-L4, and HAT-L5. Matrix metalloproteases are soluble proteases that include 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.

[0091] For evaluation, substrate targets exhibiting the highest cleavage efficiency in one MMP and less than 4000 k(obs) cleavage efficiency in other MMPs were considered "specific" substrate sequences for the particular MMP. The substrate hexapeptide sequence is sandwiched between constant regions from the M13 phage gene III protein containing the FLAG epitope: ADVGGTDYKDDDDKPGGRPTWPSSGGSGXXXXXXTASGAET (Sequence ID: 365).

[0092] Tables 1-3 below show the selected MMP substrates and their binding affinity sequences specific to MMP2, MMP9, and MMP25.

[0093] [Table 1]

[0094] [Table 2]

[0095] [Table 3]

[0096] Examples of protein lysis cleavage substrates include glycosylphosphatidylinositol (GPi)-anchored proteins, which are membrane proteins having one, two, three, four, or more transmembrane domains, intracellular domains, or extracellular domains in the extracellular matrix or adjacent cells. Cleavage can target the near-membrane or distal region of membrane-bound proteins, leading to the release of protein domains both inside and outside the cell (ectodomain and / or endodomain release) (Figures 1-20).

[0097] Membrane-bound proteins that cross the plasma membrane are called "single-pass" molecules and consist of type I, II, III, and IV members. Type I transmembrane proteins have an N-terminal signal peptide domain that targets the endoplasmic reticulum (ER) lumen during synthesis, leading to expression in the extracellular space. A stop-transfer anchor sequence maintains the type I transmembrane protein fixed to the lipid membrane. Both types II and III utilize signal-anchor sequences, but type II targets the ER lumen with its C-terminal domain, while the N-terminal domain causes type III to target the ER lumen. Type IV can be either IV-A, which has an N-terminal domain targeted to the cytosol, or IV-B, where those N-terminal domains are targeted to the lumen. The delivery system of the present invention may have one, two, three, four, or more transmembrane proteins (see, for example, Figures 4A, 4B, 5A, 5B, 6, 7, 8, 9, etc.).

[0098] Depending on the case, the transmembrane region following the first or second extracellular loop (ECL) has an intracellular region containing 41BB, ICOS, and CD3ζ, and 41BB, ICOS, CD28, and C The action of the kinase at D3ζ activates genetically engineered cells (NK and T cells) to release endogenous GrzB, which then cleaves GrzB cleavage sites, thereby releasing bispecific antibodies / payloads (Figures 6, 7, 8, and 15). In NK cells, for example, CD69TM is embedded in the membrane with at least two protease cleavage site motifs that regulate the release of payloads in the required environment. In this model, a single-stranded CAR protein is also expressed at the N-terminus for specific delivery. The first and second transmembrane regions are sometimes embedded with a NOTCH protein-dependent pulling system having secretase cleavage sites inside the cell membrane, along with multiple cargo / payloads connected to multiple cleavage sites outside or inside the cell (Figures 7 and 8). In this system, two or more protease cleavage site motifs are inserted along with multiple potential cargo delivery / releases. This system also has the ability to release intracellular payloads.

[0099] In addition to the model shown in Figure 7, the system may be embedded by recruiting a self-associated antigen via the addition of a single-stranded (α-SAA) (Figure 8). This system is the modification in Figure 7 in which the intracellular domain of CD45 has an extra single-stranded self-associated antigen (α-SAA) to which it is attached. "α-SAA" means a single-stranded antibody or antibody domain directed to or bound to the self-associated antigen, preferably scFv or a camelid antibody (VHH domain or VHH domain antibody) or a single-domain antibody. Examples of self-associated antigens include CD45, CD148, or CD43.

[0100] A linker is a peptide region that separates functional units within a fusion protein. X, Zaro JL, Shen WC. Fusion protein linkers: property, design, and functionality. Adv Drug Deliv Rev. 2013;65(10):1357-1369. doi:10.1016 / j.addr.2012.09.039 discloses various considerations for selecting linkers. Those skilled in the art will be able to select linkers based on the payload by following the teachings in this reference. In certain examples, when a polypeptide consisting of various functional domains needs to be maintained at a specific distance, a non-cleavable rigid linker is used. Human muscle aldolase (HMA) is an example of a rigid linker used to maintain protein domains separated at a set distance. Similarly, flexible non-cleavable linkers are often used in polypeptides that accommodate multiple functional domains that have freedom of movement. These include polyglycine / serine linkers, which are widely used in chimeric proteins. The linker may be of any size required to perform its function, but is preferably 2 to 128 amino acid lengths, most preferably 4 to 20 amino acid lengths. Other intended linker lengths include 3 to 100 amino acid lengths, 5 to 50 amino acid lengths, 6 to 45 amino acid lengths, 7 to 40 amino acid lengths, 8 to 30 amino acid lengths, 10 to 20 amino acid lengths, and 5 to 15 amino acid lengths. Table 4 below provides examples of linkers and specific constructs containing linkers. Linkers disclosed in iGEM (http: / / parts.igem.org / Protein_domains / Linker) are also intended.

[0101] [Table 4-1]

[0102] [Table 4-2]

[0103] [Table 4-3]

[0104] Table 4-4

[0105] Table 4-5

[0106] Table 4-6

[0107] Table 4-7

[0108] Table 4-8

[0109] Table 4-9

[0110] Table 4-10

[0111] Table 4-11

[0112] Table 4-12

[0113] All of the relevant sequences disclosed in the following examples are listed in Table 5.

[0114] [Table 5-1]

[0115] [Table 5-2]

[0116] [Table 5-3]

[0117] [Table 5-4]

[0118] [Table 5-5]

[0119] [Table 5-6]

[0120] [Table 5-7]

[0121] [Table 5-8]

[0122] [Table 5-9]

[0123] [Table 5-10]

[0124] [Table 5-11]

[0125] [Table 5-12]

[0126] [Table 5-13]

[0127] [Table 5-14]

[0128] [Table 5-15]

[0129] In relation to the present invention, the payload is any amino acid sequence intended for delivery within a living organism. Suitable payloads include full-length proteins and peptides, enzymes, cytokines, chemokines, signaling proteins, and many other proteins with various functions. In the present invention, the payload is often a therapeutic antibody, a bispecific engager, and a cytokine. The payload is preferably less than 2000 amino acids in size.

[0130] The payload delivery system of the present invention is intended to be part of cell therapy. The payload delivery system can be delivered to cells via electroporation, extracellular vesicles, nanoparticles, and transient or stable gene modifications. The protein sequences disclosed herein may be extracellular and / or intracellular. The location on the cell membrane is determined by the type of transmembrane system used.

[0131] In some embodiments, schedase is a key component of a peptide delivery system. When cells meet the intended conditions for schedase release, the schedase is released, thereby delivering schedase to treat diseases unrelated to schedase. Inactivated or fragmented schedase can be expressed on the cell surface and activated under the correct conditions, triggering activation and / or reorganization of schedase, as well as assembly into activated schedase. Furthermore, schedase can also be expressed by systems based on artificial promoters that are activated under the correct conditions, including receptor ligation and activation or cleavage of membrane-bound transcription factors after receptor ligation. Specific schedases can be released from target cells, where they can lead to apoptosis involving caspase-3 via exogenous or endogenous pathways. Caspase-3 is a key protein involved in apoptosis or programmed cell death in mammalian cells. It belongs to the family of cysteine ​​proteases known as caspases and plays a vital role in the initiation and execution of apoptosis. Caspase 3 is considered an effector caspase because it acts downstream in the apoptosis signaling pathway and is responsible for carrying out the final stage of cell death. When caspase 3 is released from dead cells, it is released within the TME and releases peptides.

[0132] Several schedases have been found to be differentially expressed in the tumor microenvironment (TME) compared to normal tissues. For example, MMP, GrzB, and ADAM, ADAMTS are often overexpressed in many different types of cancer, including breast, lung, and pancreatic cancers. Differential expression of schedases within the TME may have important implications for tumor progression and metastasis. For instance, increased expression of MMP9, MMP2, ADAM10, and ADAM17 within the TME has been linked to increased release of tumorigenic growth factors and cytokines such as epidermal growth factor (EGF) and tumor necrosis factor alpha (TNF-α). This may lead to increased tumor cell proliferation, survival, and invasion. Overall, differential expression of schedases within the TME compared to normal tissues highlights the potential of targeting these enzymes as a therapeutic strategy for cancer treatment. Therefore, differential expression of schedases may be explored for site-specific delivery of payloads.

[0133] The inventors have designed a cell-based peptide delivery system that enables the direct delivery and release of payloads / cargoes at the desired site of action. The system consists of a single or multi-pass transmembrane (TM) protein, or a C-type lectin protein, embedded in the membrane of a vehicle cell with additional motifs that regulate payload delivery / release in the required environment. Therefore, instead of direct systemic injection of biologics, the inventors have developed a genetically engineered system that can deliver payloads to the required site and release them at the correct time. The goal was to infuse the cells or EVs derived therefrom into the fluid.

[0134] The delivery system, for example, is independent of cell type, but the inventors modified NK cells to generate and deliver bispecific engagers within the tumor microenvironment. This delivery can be further regulated and induced by either NK cell activation (activation-induced cleavage) or the tumor environment (cleavage upon contact with proteases specifically elevated in the TME) (Figure 19). NK cell activation may be due to the interaction of germline-encoded activating receptors or specifically genetically engineered receptors, such as chimeric antigen receptors / (multiple) against tumor antigens / (multiple). Cleavage induced by NK cell activation can be achieved either through granzyme B released after degranulation or by physical pulling, similar to NOTCH activation. In addition, such activation-stimulated release leads to NK cell proliferation and activation depending on the availability of tumor antigens. This represents a next-generation cancer immunotherapy system with enhanced ability to generate and release payloads under desired conditions and at specific sites.

[0135] While many shedases can be used in this payload delivery system, current research presents a general example of matrix metalloproteinases (MMPs) that can enable environment-specific cargo release. Models based on the ADAM family of MMPs and the serine protease granzyme-B have also been presented. In these models, auxiliary functions such as tumor targeting, proliferation, and / or activation were modulated by the addition of extracellular and / or intracellular domains. These additional domains may include any of the following: extracellular cytokines, drugs or prodrugs, chimeric antigen receptor (CAR)-like or biantigen receptor (DAR)-like domains or intracellular modulators, inhibitors, signaling domain-containing proteins, transcription factors, transcription activators or inhibitors, DNA modifying enzymes such as HDACs, proteins containing localization and dimerization domains, proteins containing disruption and dimerization domains, proteins containing hypoxia-responsive domains, proteins that activate immune cells, proteins that enhance the persistence and proliferation of immune cells, proteins that induce immune cell migration, and fragmented receptors such as fragmented CARs.

[0136] Furthermore, the system was designed for passive release in the tumor environment in response to specific MMPs released by cancer cells, or for active release induced by the activation of producer cells through receptor engagement.

[0137] The membrane-embedded system can also be designed to localize (sort) to specific intracellular compartments. In several embodiments, the inventors genetically engineered payloads that localize (sort) to extracellular vesicles (EVs), such as exosomes. Cells can release these exosomes in large quantities, releasing the payload upon cleavage by proteases in the TME or other target sites (Figures 4A-B, 5A-B, 9, 10, and 24-28). Theoretically, these EVs could also be harvested from in vitro cultures and administered to patients as off-the-shelf therapeutics.

[0138] In some embodiments, within NK or T cells, CD69TM is embedded in a membrane having at least two protease cleavage site motifs that regulate the release of the payload in the required environment (Figures 17A-B and 18A-B). In addition to the models described in Figures 17A-B and 18A-B, the system may also be equipped with a single-stranded CAR protein at the N-terminus for specific delivery (Figure 6).

[0139] In some embodiments, modified tetraspanins may be used in which two or more protease cleavage sites are inserted, along with multiple potential cargo delivery / release sites. This system also has the ability to release intracellular payloads (Figure 7).

[0140] In some embodiments, further modification of the self-associated antigen (a-SAA) linked to the intracellular domain of CD45 with a tetraspanin having an extra single strand may be used (Figure 8).

[0141] 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 cell death-I (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fe gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signal transduction lymphocyte activating molecule (SLAM protein), costimulatory ligand (PD-L2, 4-1BBL, OX40L, Inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin b receptor, 3 / TR6, I LT3, ILT4), activated 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, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, I TGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, IT GAM, CD11b, ITGAX, CD11e, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI(CD226), SLAMF4(CD244, 2B4), CD84, CD96(TACTILE), CEACAM1, CRTThis includes ligands that specifically bind to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0142] In some embodiments, the antigen-binding domains include 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, ADAM10, CHRNA2, LeY, and NK. G2D, CSI, CD44v6, CD24, LGR5, ALDH, ALDH1, CD54, Sca1, CD271, CD123, CD36, CD109, CD110, CD71 negative, CCA, ABCG2, Claudin-18.2 (Claudin-18A2, or Claudin-18 isoform 2), PSCA, DLL3 (Delta-like protein 3, Drosophila delta homolog 3, Delta 3), Mud 7 (Mucin 17, Muc3, Muc3), FAP alpha (Fibroblast-activating protein alpha), Ly6G6D (Lymphocyte antigen 6 complex locus protein G6d, c6orf23, G6D, MEGTI, NG25), PSMA, MSLN, or RNF43 (E3 ubiquitin-protein ligase RNF43, RING finger protein 43), BAFF, C242 antigen, disialoganglioside (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 Dispersion Factor Receptor Kinase, IGF-1 Receptor, IGF-I, IgGI, LI-CAM, Integrin α5β1, Integrin ανβ3, Regmine, MORAb-009, MS4A1, MUC1, Mucin CanAg, C-MET, CCR4, CD152, CD10, CD19, CD20, CD200, N-glycolylneuraminic acid, NPC-IC, PDGF-Rα, PDL192, phosphatidylserine, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin, RANKL, RON, ROR1,It specifically binds to SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF-β, TRAIL-R1, TRAIL-R2, folate receptor, transferrin receptor, and any combination thereof. These antigen-binding domains may be used within the CAR and DAR to target the payload. CARs and / or antibodies that target antigens are disclosed, for example, in: for BCMA, International Publication Nos. 201616630, 2020150339, 2019196713, 2016014565, and 2017025038; for MUC16, U.S. Patent No. 9,169,328, International Publication Nos. 2016149368, and 2020023888; for EGFRvIII, International Publication For FLT3, see International Publication Nos. 2017125830 and 2016016341; for CD20, see International Publication Nos. 2018222935, 2020010284 and 2017173410; for CD38, see International Publication Nos. 2018145649, 2020010235 and 2020123691; for CD38, see International Publication No. 2017025323; for CD70, see International Publication No. 201 For CD33, see International Publication No. 2018152181; for CD133, see International Publication No. 2016014576; for CD133, see International Publication No. 2018072025; for CSI, see International Publication No. 2019030240; for RORI, see International Publication No. 2016115559; for CD19, see International Publication No. 2002077029 and USI No. 1,077,144. Detailed information; for Claudine, see International Publication Nos. 2018006882 and 2021008463; for DLL3, see International Publication No. 2020180591; for WTI, see U.S. Patent Application Publication No. 20160152725 and U.S. Patent No. 7622119; for CD23, see U.S. Patent No. 6011138 and Chinese Patent Application Publication No. 1568198; for CD30, see U.S. Patent No. 10815301,For each specification of Patent Application No. 10808035; for PRAME, see U.S. Patent Application Publication No. 20180148503 and International Publication No. 2020186204; for LIVI, see U.S. Patent Application Publication No. 20200231699; for NKG2D, see International Publication No. 2021179353 and U.S. Patent Application Publication No. 20210269501; for FAP Alpha, see U.S. Patent Application Publication No. 20200246383 and No. 20210115102; for PSMA, see U.S. Patent Application Publication No. 20210277141 and International Publication No. 2020108646; for MSLN, see Chinese Patent Application Publication No. 109680002 and No. 109628492.

[0143] In some embodiments, the schedase cleavage site comprises one or more of a plurality of protease cleavage sites and is 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), matryptase (MT-SP1), regmine, disintegrin and metalloproteinase (ADAM), and transmembrane serine protease (TMPRSS).

[0144] In some embodiments, the shedase that recognizes a protease cleavage site, or recognizes one or more of a plurality of protease cleavage sites, is thrombin, trypsin, plasmin, prostate-specific antigen (PSA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase (MMP), matryptase (MT-SP1), regmine, disintegrin and metalloproteinase (ADAM), transmembrane serine protease (TMPRSS), granzyme B, activated tan. Protein C, caspase, cathepsin, chymase, elastase, guanidinobenzoate, HtrA1, human neutrophil elastase, lactoferrin, malapsin, NS3 / 4A, PACE4, tissue plasminogen activator (tPA), DESCl, DPP-4, hepsin, matryptase-2, secretase, kallikrein-related peptidase (KLK), and triplase, or one or more of serine proteases, cysteine-type lysosomal proteases, metalloproteases, coagulation factor proteases, or aspartyl-type lysosomal proteases.

[0145] In some embodiments, the payload comprises a cytokine or chemokine consisting of a fraction or derivative of sulfated xylan, wherein the cytokine or chemokine is the IL-17 family of cytokines including 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-25, interferon, G-CSF, M-CSF, GM-CSF, BDNF, CNTF, EGF, EPO, FGF1, FGF2, FGF3, FGF 4, 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, TNFα, 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 including MIP-1a -1 family members, MIP-2, eotaxin (eotaxin-1, -2 or -3), PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase, cathepsin family enzymes, cell adhesion molecules such as PECAM-1, soluble receptors, or cell- or virus-binding receptors, cytokine-induced neutrophil chemoattractants (KC), TNF-α and IFN-γ), and other soluble mediators of inflammation 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, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, C XCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1,Or an antagonist of a ligand selected from a list containing any combination thereof.

[0146] In some embodiments, the payload may include thrombin. In other embodiments, the payload may be a vaccine. In yet another embodiment, the payload may be a urokinase delivery substance. The constructs of the present invention may enable delivery of the payload across the blood-brain barrier.

[0147] As used herein, "α-TAA" means a single-chain antibody or antibody domain directed to or bound to a tumor-associated antigen, preferably an scFv or camelid antibody (VHH domain antibody or VHH domain), or a single-domain antibody or antibody-binding domain. Examples of tumor-associated antigens conjugated by the initiation polypeptide used in the present invention include, for example, pan-B antigens (e.g., CD20 found on the surface of both malignant and non-malignant B cells, such as B cells in non-Hodgkin lymphoma) and pan-T cell antigens (e.g., CD2, CD3, CD5, CD6, CD7). Other exemplary tumor-associated antigens include, but are not limited to, MAGE-1, MAGE-3, MUC-1, HPV16, HPV E6 & E7, TAG-72, CEA, α-Lewis y, 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 α-fetoprotein (AFP) are two examples of such tumor-associated antigens. Other targets include the MICA / B ligand for NKG2D. These molecules are expressed in many types of tumors but are not typically expressed in healthy cells.Additional 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 (e.g., human papillomavirus antigen), prostate-specific antigens (PSA, PSMA), RAGE (renal antigen), CAMEL (CTL-recognizing antigen on melanoma), and CT antigens (MAGE-B5, -B6, -C). Examples include 2, -C3, and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigens (e.g., MUC1, mucin-CA125), cancer-associated ganglioside antigens, tyrosinase, gp75, C-myc, Mart1, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7, Ep-CAM, tumor-derived heat shock proteins, and similar (e.g., Acres See also: 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 CA195 tumor-associated antigen-like antigen (see, e.g., U.S. Patent No. 5,324,822) and female urea squamous cell carcinoma-like antigen (see, e.g., U.S. Patent No. 5,306,811), and mammary cell tumor-associated antigens described in U.S. Patent No. 4,960,716.

[0148] 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, in 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, IL13R alpha, IL13R alpha 2, AIM-2, AIM-3, BRAP, RTN4, GLEA2, T Examples include 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.

[0149] Spatiotemporal adjustment and delivery

[0150] Embodiments of the present invention are expected to be utilized for targeted payload release in the tumor microenvironment or tissue-specific environments, thereby enabling optimal time points and anatomical placement for cargo release and activation, such as the release and demasking of pro-inflammatory cytokines (e.g., TNF, IL2, IL15, IL18) in the tumor microenvironment, or the conditional release of specific growth factors in non-malignant tumor diseases (e.g., GMCSF in bone marrow transplantation, IL2 in certain immunodeficiencies, insulin in diabetes). Furthermore, transient and conditional cargo release according to the present invention is expected to limit currently observed systemic off-target effects, and / or limit continuous exposure and response. Conditional induction of response is advantageous in numerous diseases in which a distinct circadian-like rhythm is formed in the symptoms, such as inflammatory bowel disease and diabetes.

[0151] It is also crucial that bispecific engagers and other proteins with short half-lives and / or high risk of off-target activity in other tissues at the right time in the right microenvironment are available. Embodiments of the present invention are applicable in such conditions where spatiotemporal release is critical. One such example is the creation of a dispanin encoding a bispecific CD38-CD3 engager within an ectodomain loop that is cleavable only in the bone marrow microenvironment along with an anti-BCMA CAR, and thus reduces the off-target activity of the CAR in addition to the released engager. In this model system, a bispecific CD38-CD3 engager with a very short half-life reduces the likelihood of systemic distribution and impact. Furthermore, in embodiments of the present invention, it is hypothesized that one-time administration of cells will be more potent due to continuous protein turnover and release by the delivering cells, potentially achieving similar efficacy to administration regimens with repeated doses of other delivery systems while simultaneously suppressing side effects.

[0152] Example 1: General Structure

[0153] In one embodiment, the present invention includes a delivery system for a protein payload released by a schedase present in the body. See Figures 1-2. The payload delivery system for single payload delivery includes a first transmembrane anchor, a cytoplasmic region, a first site cleavable by schedase, a first linker, a payload sequence, a second linker, a second site cleavable by schedase, and optionally a single-strand specific CAR.

[0154] The transmembrane anchor can be selected from any relevant TMD that provides similar functions such as CD63, CD9, CD81, CD28, CD4, CD8, CD34, CD69, CD19, CD20, integrins, TNFSF14, TNR1A, aquaporins, NOTCH, NgR1, NRG1, GPi anchors, EGFR, rhodopsin, or membrane fixation, and signal transduction modulation may be used in the expression of the present invention.

[0155] The cytoplasmic domain can be selected from CD63, CD69, CD9, CD28, CD81, CD34, CD3, CD4, and CD8, or any relevant cytoplasmic domain that provides similar functions to the system (e.g., in addition to the ability to fix, move within the cell membrane, and / or sort to EVs, is related to 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 cell death-I (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fe gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signal transduction lymphocyte activating molecule (SLAM protein), costimulatory ligand (PD-L2, 4-1BBL, OX40L, Inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin b receptor, 3 / TR6, I LT3, ILT4), activated 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, CD8 Alpha, CD8 Beta, IL-2R Beta, IL-2R Gamma, IL-7R Alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITThis includes ligands that specifically bind to GAM, CD11b, ITGAX, CD11e, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (TACTILE), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0156] The linker can be selected from any suitable linker, and the linkers disclosed herein, for example, those shown in Table 4, are preferred.

[0157] The schidases and their cleavable sites may be selected from part-time or full-time schidases. Full-time schidases may be selected from ADAM proteases, BACE proteases, serine proteases granzyme-B and site 1 proteases. ADAM proteases (metalloproteases) are membrane-anchored type 1 proteases and include ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, and ADAM33. BACE (aspartyl) proteases are also membrane-anchored type 1 proteases that include BACE1 and BACE2. Site 1 (serine) proteases are membrane-anchored type 1 proteases that include SKI-a or SIP.

[0158] Part-time schidases and their cleavable sites include meprin B, MT-MMP (membrane-type matrix metalloproteinase), proprotein converterase, transmembrane serine protease, matrix metalloproteinase, legmine, and cathepsin S and L. Meprin B is a membrane-anchored type 1 metalloproteinase. MT-MMP is either membrane-anchored type 1 or GPI-anchored and includes MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, and MT6-MMP. Proprotein converterase is either membrane-anchored type 1 or soluble protease and includes PCSK1 / 3, KCSK2, furin, PCSK4, PSCK5 / 6, PACE4, PCSK7, and PCSK9. Transmembrane serine proteases are membrane-anchored type II proteases and include matryptase, matryptase-2, matryptase-3, polymerase-1, choline, hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, spinesin, enteropeptidase, HAT, DESCL1, TMPRSS11A, HAT-L4, and HAT-L5. Matrix metalloproteases are soluble proteases that include 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.

[0159] In some embodiments, the payload comprises a cytokine or chemokine consisting of a fraction or derivative of sulfated xylan, wherein the cytokine or chemokine is the IL-17 family of cytokines including 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-25, interferon, G-CSF, M-CSF, GM-CSF, BDNF, CNTF, EGF, EPO, FGF1, FGF2, FGF3, FGF 4, 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, TNFα, 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 including MIP-1a -1 family members, MIP-2, eotaxin (eotaxin-1, -2 or -3), PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase, cathepsin family enzymes, cell adhesion molecules such as PECAM-1, soluble receptors, or cell- or virus-binding receptors, cytokine-induced neutrophil chemoattractants (KC), TNF-α and IFN-γ), and other soluble mediators of inflammation 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, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, C XCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1,Or an antagonist of a ligand selected from a list containing any combination thereof.

[0160] In other embodiments, further modifications may be applied to the system by adding more cleavable schedase and more payloads to the extracellular or extracellular environment. Here, schedase cleavage releases a peptide or protein. The peptide or protein may become active upon cleavage, or may still require further activation via intracellular mechanisms. The activation mechanism may be naturally occurring or can be genetically engineered. Figures 4A-B and 5A-B show embodiments of the present invention comprising four transmembrane domains. Multiple crossings of the plasma membrane are necessary to create an elaborate delivery system capable of accommodating different payloads in intracellular and extracellular environments. In this embodiment, the inventors used a tetraspanin TMD linked through a payload that spreads across the schedase sites constituting the delivery system. A first protease site cleavable MMP9, a TNF-α payload, and a second protease site cleavable by MMP9. Figures 4A-B and 5A-B differ in the presence of an EV localization (sort) sequence.

[0161] Example 2: Delivery of the bike

[0162] In one embodiment, the delivery system of the present invention is configured to deliver BIKE. See Figure 2. Embodiments of the present invention include BIKE, in which a protease cleavage sequence is laterally positioned and 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 represented as SEQ ID NO: 123 or SEQ ID NO: 1 below. This 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 this protein is bound to the plasma membrane through a GPi anchor. Schedase results in cleavage, thus releasing the payload into the extracellular environment. (Sequence ID: 1)

[0163] This sequence is also shown in Figure 3, along with a legend indicating the domains and motifs.

[0164] The corresponding cDNAs are shown below.

[0165] ATGTGCGGCGCATGCAAGGAAAACTACTGTCTGATGATCACATTCGCCATCTTCCTGAGCCTGATCATGCTGGTGGAAGTGGCCGCTGCCATCGCCGGATACGTGGGAGGCAGCGGGCCTGTGCGGCGGTACCAGGGAGGCCCACTGGGAGTTAGAGGCGGCATGGAAGTGCAGCTGGTCGAATCGGGAGGCGGCGTGGTGAGACCAGGAGGCAGCCTCCGGCTGTCTTGTGCCGCGAGTGGTTTCACCTTTGACGACTACGGCATGAGCTGGGTGAGACAGGCTCCTGGAAAGGGCCTGGAATGGGTTTCCGGCATCAACTGGAACGGCGGATCGACTGGCTACGCCGACAGCGTGAAAGGCAGATTCACCATCAGCAGAGATAACGCCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGGCAGAAGCCTGCTGTTCGACTACTGGGGACAGGGCACACTGGTGACCGTCAGCAGAGGCGGCGGCGGCTCTGGCGGCGGCGGAAGCGGAGGCGGCGGATCCTCCGAGCTGACACAGGACCCCGCTGTTAGCGTGGCTCTGGGCCAGACCGTGCGGATCACCTGTCAGGGCGACAGCCTCCGCTCCTACTACGCCAGCTGGTACCAACAAAAACCTGGCCAAGCTCCTGTGCTGGTCATCTACGGAAAGAACAACAGACCTAGTGGCATCCCTGATAGATTTAGCGGCAGCAGCAGCGGCAACACCGCCTCACTGACCATTACAGGCGCCCAGGCCGAGGATGAGGCTGACTATTACTGCAACTCCAGGGACAGCTCTGG

[0166] As shown in Figure 3, sequence number 123 or 1 is divided into the following functional motifs.

[0167] DNA CD63-cytoplasmic MCGACKENYC (SEQ ID NO: 125) encoded by ATGTGCGGCGCATGCAAGGAAAACTACTGT (SEQ ID NO: 126)

[0168] DNA CD63-TM-Helical (Inside-Out) LMITFAIFLSLIMLVEVAAAI (Sequence Number: 127) is coded by CTGATGATCACATTCGCCATCTTCCTGAGCCTGATCATGCTGGTGGAAGTGGCCGCTGCCATC (Sequence Number: 128).

[0169] DNA Linker-a:GGSGPVRRYQ (Sequence ID: 129) coded by GGAGGCAGCGGGCCTGTGCGGCGGTACCAG (Sequence ID: 130).

[0170] DNA MMP2:GGPLGVRGG (Sequence ID: 131) coded by GGAGGCCCACTGGGAGTTAGAGGCGGC (Sequence ID: 132).

[0171] DNA ATGGAAGTGCAGCTGGTCGAATCGGAGGCGGCGTGGTGAGACCAGGAGGCAGCCTCCGGCTGTCTTGTGCCGCGAGTGGTTTCACCTTTGACGACTACGGCATGAGCTGGGTGAGACAGGCTCCTGGAAAGGGCCTGGAATGGGTTTCCGGCATCAACTGGAACGGCGGATCGACTGGCTACGCCGACAGCGT VL-a-CD16-118AA encoded by GAAAGGCAGATTCACCATCAGCAGAGATAACGCCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGGCAGAAGCCTGCTGTTCGACTACTGGGGACAGGGCACACTGGTGACCGTCAGCAGA (SEQ ID NO: 134) MEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (Sequence ID: 133).

[0172] Linker-b and linker-c encoded by DNA GGCGGCGGCGGCTCTGGCGGCGGCGGAAGCGGAGGCGGCGGATCC (Sequence ID: 136): GGGGSGGGGSGGGGS 15AA (Sequence ID: 135)

[0173] DNA TCCGAGCTGACACAGGACCCCGCTGTTAGCGTGGCTCTGGGCCAGACCGTGCGGATCACCTGTCAGGGCGACAGCCTCCGCTCCTACTACGCCAGCTGGTACCAACAAAAACCTGGCCAAGCTCCTGTGCTGGTCATCTACGGAAAGAACAACAGACCTAGTGGCATCCCTGATAGA VH-a-CD16-107AA encoded by TTTAGCGGCAGCAGCAGCGGCAACACCGCCTCACTGACCATTACAGGCGCCAGGCCGAGGATGAGGCTGACTATTACTGCAACTCCAGGGACAGCTCTGGAAATCATGTGGTCTTCGGCGGCGGCACAAAGCTGACCGTGCTG (SEQ ID NO: 138) SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVL(Sequence ID: 137).

[0174] DNA Flanking 20AA:PSGQAGAAASESLFVSNHAY (Sequence ID: 139) coded by CCTTCTGGCCAAGCCGGTGCCGCCGCGTCTGAGAGCCTGTTCGTGAGCAACCACGCCTAC (Sequence ID: 140).

[0175] DNA 7AA flanking peptide: EASGGPE (Sequence ID: 141), encoded by GAGGCCAGCGGCGGCCCCGAG (Sequence ID: 142).

[0176] DNA CAGGTGCAGCTGGTGCAGAGCGGCGCTGAGGTGAAAAAGCCCGGCTCTAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCACAGATTATAATATGCACTGGGTCAGACAGGCCCCTGGGCAGGGACTGGAATGGATCGGCTATATCTACCCCTACAACGGCGGAACAGGCTACAACCAGAAGTT VL-a-CD33-116AA encoded by CAAGAGCAAGGCCACCATCACAGCCGACGAGAGCACCAACACCGCTTATATGGAACTGTCTTCTCTGCGGAGCGAAGATACCGCCGTGTACTACTGTGCCAGAGGCCGGCCTGCCATGGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCA (SEQ ID NO: 144) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS(Sequence ID: 143).

[0177] DNA GATATCCAGATGACCCAGAGCCCCAGCAGCCTGTCCGCCAGTGTCGGAGATAGGGTGACCATCACCTGCAGAGCCTCTGAAAGCGTTGACAATTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAAACCTGGCAAAGCCCCTAAGCTTCTTATTTACGCCGCTTCCAACCAGGGCTCT VH-a-CD33 encoded by GGCGTGCCCAGCCGGTTCAGCGGCTCAGGATCTGGAACCGACTTTACGCTGACAATCAGCTCTCTGCAGCCTGATGACTTCGCCACATACTACTGCCAGCAAAGCAAAGAGGTGCCTTGGACCTTCGGCCAGGGCACCAAGGTGGAGATTAAG (SEQ ID NO: 146) DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (Sequence ID: 145).

[0178] DNA Myc-tag EQKLISEEDL (sequence number: 147) coded by GAGCAGAAGCTGATCTCTGAGGAAGACCTG (sequence number: 148).

[0179] DNA MMP9:GGPLGMTS (Sequence ID: 149) coded by GGAGGCCCTCTGGGCATGACCTCT (Sequence ID: 150).

[0180] DNA CD63-extracellular, encoded by CCTAAGAACAACCACACCGCCAGCATCCTGGACCGGATGCAGGCCGACTTTAAGTGCTGCGGCGCCGCTAATTACACCGACTGGGAGAAAATCCCCAGCATGAGCAAGAATAGAGTGCCCGATAGCTGTTGCATCAACGTGACAGTGGGCTGCGGCATCAATTTCAACGAGAAGGCTATCCACAAGGAGGGCTGCGTGGAAAAGATCGGCGGCTGGCTGAGAAAGAACGTG (Sequence ID: 152) PKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNV(Sequence ID: 151).

[0181] DNA GPi-anchor coded by GTGCTGAGAGACAAGCTCGTGAAATGCGAGGGCATCAGCCTGCTGGCCCAGAACACATCCTGGCTGCTGCTGCTGCTGCTCAGCCTGAGTCTGCTGCAAGCCACCGATTTCATGTCTCTG (Sequence ID: 154) is VLRDKLVKCEGISLLAQNTSWLLLLLLSLSLLQATDFMSL (Sequence ID: 153).

[0182] Example 3: Payload delivered to extracellular vesicles

[0183] Refer to Figures 4A-B. This embodiment is a payload having an MMP-cleavable sequence embedded in the extracellular loop between the third and fourth transmembrane domains of tetraspanin. This particular molecule is localized (sorted) into extracellular vesicles by the presence of the GYEVM motif (SEQ ID NO: 337).

[0184] The amine acid sequence in sequence number 155 below encodes the construct. (Sequence ID: 155)

[0185] The corresponding cDNA is shown below: ATGTGCGGCGCCTGTAAAGAAAACTACTGCCTGATGATCACATTCGCAATCTTCCTGTCCCTCATCATGCTGGTGGAGGTGGCCGCCGCCATCGCTGGCTACGTGGGCGGCAGCGGCCCTGTGAGGAGATACCAGGGCGGTCCACTGGGCGTGCGGGGCGGCATGGAAGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGAGACCTGGCGGGTCCCTGCGGCTGAGCTGTGCCGCCAGCGGATTCACATTCGACGATTACGGCATGAGCTGGGTCAGACAGGCCCCAGGAAAAGGCCTGGAATGGGTGTCTGGCATTAACTGGAACGGCGGCAGCACCGGCTACGCCGATAGCGTTAAGGGCAGATTCACCATCAGCAGAGATAACGCCAAGAACTCTCTGTACCTGCAGATGAATAGCCTGCGGGCCGAAGATACCGCCGTCTACTACTGTGCCCGGGGAAGAAGCTTGCTGTTTGACTACTGGGGACAGGGCACACTGGTCACCGTGTCTAGGGGCGGCGGCGGCAGCGGAGGAGGCGGGTCGGGCGGCGGCGGATC

[0186] Sequence ID: 155 is divided into the following functional motifs: DNA CD63-cytoplasm:MCGACKENYC (Sequence ID: 157) encoded by ATGTGCGGCGCCTGTAAAGAAAACTACTGC (Sequence ID: 158) DNA CD63-TM-Helical (Inside-Out) LMITFAIFLSLIMLVEVAAAI (Sequence ID: 159) coded by CTGATGATCACATTCGCAATCTTCCTGTCCCTCATCATGCTGGTGGAGGTGGCCGCCGCCATC (Sequence ID: 160) DNA Linker-a:GGSGPVRRYQ(sequence number:161) coded by GGCGGCAGCGGCCCTGTGAGGAGATACCAG(sequence number:162) DNA MMP2:GGPLGVRGG (sequence number: 163) coded by GGCGGTCCACTGGGCGTGCGGGGCGGC (sequence number: 164)

[0187] DNA ATGGAAGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGAGACCTGGCGGGTCCCTGCGGCTGAGCTGTGCCGCCAGCGGATTCACATTCGACGATTACGGCATGAGCTGGGTCAGACAGGCCCCAGGAAAAGGCCTGGAATGGGTGTCTGGCATTAACTGGAACGGCGGCAGCACCGGCTACGCCGATAGCGT VL-a-CD16-118AA encoded by TAAGGGCAGATTCACCATCAGCAGAGATAACGCCAAGAACTCTCTGTACCTGCAGATGAATAGCCTGCGGGCGAAGATACCGCCGTCTACTACTGTGCCCGGGGAAGAAGCTTGCTGTTTGACTACTGGGGACAGGGCACACTGGTCACCGTGTCTAGG (SEQ ID NO: 166) MEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (Sequence ID: 165) Linker-b and linker-c encoded by DNA GGCGGCGGCGGCAGCGGAGGAGGCGGGTCGGGCGGCGGCGGATCC (Sequence ID: 168): GGGGSGGGGSGGGGS 15AA (Sequence ID: 167)

[0188] DNA AGCGAGCTGACACAAGATCCTGCTGTGAGCGTCGCCCTGGGCCAGACCGTGCGGATCACTTGCCAGGGAGATAGCCTGAGGTCCTACTACGCCAGTTGGTATCAACAGAAGCCTGGACAGGCCCTGTACTGGTGATCTACGGCAAGAACAACAGACCTTCTGGCATCCCTGATAGA VH-a-CD16-107AA encoded by TTCAGCGGCTCCAGCAGCGGAAACACCGCTAGCCTGACAATCACCGGAGCTCAGGCTGAGGACGAGGCCGACTACTACTGCAACAGCCGGGACTCTTCAGGAAATCACGTGGTGTTCGGCGGCGGCACCAAGCTGACAGTGCTG (SEQ ID NO: 170) SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVL(Sequence ID: 169) DNA Flanking 20AA:PSGQAGAAASESLFVSNHAY (Sequence ID: 171) DNA encoded by CCTAGCGGCCAGGCCGGCGCCGCGGCCAGCGAAAGCCTGTTCGTGTCTAACCACGCCTAC (Sequence ID: 172) 7AA flanking peptide encoded by GAGGCTTCCGGCGGCCCCGAG (SEQ ID NO: 174): EASGGPE (SEQ ID NO: 173)

[0189] DNA CAGGTGCAGCTGGTGCAAAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAAGTGTCCTGCAAGGCCTCCGCTACACCTTTACCGACTACAATATGCACTGGGTTCGGCAGGCC VL-a-CD33-116AA coded by CCTGGCCAAGGTCTCGAGTGGATCGGCTACATCTATCCTTACAACGGCGGCACCGGCTACAACCAGAAGTTCAAAAGCAAGGCAACAATCAGCCGACGAGAGCACTAACACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCTGTTTACTACTGCGCCAGAGGCAGACCAGCTATGGACTACTGGGGCCAAGGAACCCTGGTGACCGTGTCATCT (Sequence ID: 176) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS(Sequence ID: 175)

[0190] DNA GATATCCAGATGACCCAGAGCCCTAGCAGCCTGTCTGCCTCTGTTGGCGACCGGGTGACCATCACCTGCAGAGCTTCTGAATCAGTTGACAACTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAAACCTGGCAAGGCCCCCAAGCTGCTGATCTATGCCGCTTCTAATCAGGGCAGC VH-a-CD33 encoded by GGCGTGCCTAGCAGATTTAGCGGCAGTGGATCGGGAACCGACTTCACCCTGACCATTTCTTCGCTTCAGCCCGACGATTTCGCCACATACTATTGTCAGCAGAGCAAGGAGGTGCCATGGACATTTGGCCAGGGCACTAAGGTGGAAATCAAG (SEQ ID NO: 178) DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (Sequence ID: 177) Linker and Myc-TAG GGSGEQKLISEEDLGG (Sequence ID: 179)GGCGGCAGCGGAGAGCAGAAGCTGATCTCCGAGGAAGACCTGGGTGGA (Sequence ID: 180) MMP9:PLGMTS(Sequence ID: 181) CCCCTGGGCATGACCTCT(Sequence ID: 182) CD63-extracellular PKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNV(Sequence ID: 183) CCTAAGAACAACCACACAGCCAGCATCCTGGACAGAATGCAGGCCGACTTCAAGTGCTGCGGCGCCGCTAATTACACCGACTGGGAGAAAATCCCCTCCATGAGCAAAAAACCGGGTGCCC GACAGCTGTGCATCAATGTGACCGTGGCTGTGGCATTAACTTCAACGAGAAGGCTATCCATAAGGAGGGCTGCGTCGAGAAGATCGGCGGCTGGCTGAGAAAGAACGTG (SEQ ID NO: 184)

[0191] CD63-TM-Helical (Outside-In) LVVAAAALGIAFVEVLGIVFA (Sequence ID: 185) CTGGTGGTGGCCGCCGCCGCCCTGGGTATCGCCTTCGTGGAAGTGCTGGGCATCGTGTTCGCC(Sequence code: 186)

[0192] CD63-Cytoplasmic CCLVKSIRSGYEVM (SEQ ID NO: 187) TGCTGCCTGGTGAAGTCTATCAGATCTGGCTATGAGGTGATG (Sequence number: 189)

[0193] Example 4: BIKE expressed on the cell surface

[0194] Refer to Figures 5A-B. One embodiment of the present invention is a BIKE having an MMP / GrzB cleavage sequence embedded in the extracellular loop between the third and fourth transmembrane domains of a tetraspanin. This molecule lacks the signaling motif GYEVM (SEQ ID NO: 337), which is necessary for localization (sorting) into extracellular vesicles, and is therefore expressed on the cell surface.

[0195] The construct has the amino acid sequence outlined in Sequence ID No. 188 below: MCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQGGPLGVRGGMEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRGGGGSGGGGSGGGGSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVLPSGQAGAAASESLFVSNHAYEASGGPEQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKGGSGEQKLISEEDLGGPLGMTSGGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKS(SEQ ID NO: 188).

[0196] The corresponding cDNA is represented as follows (SEQ ID NO: 189): ATGTGCGGCGCCTGTAAAGAAAACTACTGCCTGATGATCACATTCGCAATCTTCCTGTCCCTCATCATGCTGGTGGAGGTGGCCGCCGCCATCGCTGGCTACGTGGGCGGCAGCGGCCCTGTGAGGAGATACCAGGGCGGTCCACTGGGCGTGCGGGGCGGCATGGAAGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGAGACCTGGCGGGTCCCTGCGGCTGAGCTGTGCCGCCAGCGGATTCACATTCGACGATTACGGCATGAGCTGGGTCAGACAGGCCCCAGGAAAAGGCCTGGAATGGGTGTCTGGCATTAACTGGAACGGCGGCAGCACCGGCTACGCCGATAGCGTTAAGGGCAGATTCACCATCAGCAGAGATAACGCCAAGAACTCTCTGTACCTGCAGATGAATAGCCTGCGGGCCGAAGATACCGCCGTCTACTACTGTGCCCGGGGAAGAAGCTTGCTGTTTGACTACTGGGGACAGGGCACACTGGTCACCGTGTCTAGGGGCGGCGGCGGCAGCGGAGGAGGCGGGTCGGGCGGCGGCGGATCCAGCGAGCTGACACAAGATCCTGCTGTGAGCGTCGCCCTGGGCCAGACCGTGCGGATCACTTGCCAGGGAGATAGCCTGAGGTCCTACTACGCCAGTTGGTATCAACAGAAGCCTGGACAGGCCCCTGTACTGGTGATCTACGGCAAGAACAACAGACCT

[0197] The above SEQ ID: 188 is divided into the functional domains and motifs shown in FIG. 11 and below: cDNA: CD63 - Cytoplasm: MCGACKENYC (SEQ ID: 190) encoded by ATGTGCGGCGCCTGTAAAGAAAACTACTGC (SEQ ID: 191)

[0198] CD63 - TM - Helical (inside - out) LMITFAIFLSLIMLVEVAAAI (SEQ ID: 192)

[0199] cDNA: It is encoded by CTGATGATCACATTCGCAATCTTCCTGTCCCTCATCATGCTGGTGGAGGTGGCCGCCGCCATC (SEQ ID: 193).

[0200] cDNA: Linker - a: GGSGPVRRYQ (SEQ ID: 194) encoded by CGGCAGCGGCCCTGTGAGGAGATACCAG (SEQ ID: 195)

[0201] cDNA: MMP2: GGPLGVRGG (SEQ ID: 196) encoded by GGCGGTCCACTGGGCGTGCGGGGCGGC (SEQ ID: 197)

[0202] VL - a - CD16 - 118AA MEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (SEQ ID: 198)

[0203] It is, cDNA: ATGGAAGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGAGACCTGGCGGGTCCCTGCGGCTGAGCTGTGCCGCCAGCGGATTCACATTCGACGATTACGGCATGAGCTGGGTCAGACAGGCCCCAGGAAAAGGCCTGGAATGGGTGTCTGGCATTAACTGGAACGGCGGCAGCACCGGCTACGCCGA TAGCGTTAAGGGCAGATTCACCATCAGCAGAGATAACGCCAAGAACTCTCTGTACCTGCAGATGAATAGCCTGCGGGCCGAAGATACCGCCGTCTACTACTGTGCCCGGGGAAGAAGCTTGCTGTTTGACTACTGGGGACAGGGCACACTGGTCACCGTGTCTAGG (SEQ ID NO: 199).

[0204] cDNA: Linker-b and linker-c coded by GGCGGCGGCGGCAGCGGAGGAGGCGGGTCGGGCGGCGGCGGATCC (Sequence ID: 201): GGGGSGGGGSGGGGS 15AA (Sequence ID: 200)

[0205] VH-a-CD16-107AA SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVL(Sequence ID: 202)

[0206] That is cDNA: AGCGAGCTGACACAAGATCCTGCTGTGAGCGTCGCCCTGGGCCAGACCGTGCGGATCACTTGCCAGGGAGATAGCCTGAGGTCCTACTACGCCAGTTGGTATCAACAGAAGCCTGGACAGGCCCTGTACTGGTGATCTACGGCAAGAACAACAGACCTTCTGGCATCCCT GATAGATTCAGCGGCTCCAGCAGCGGAAACACCGCTAGCCTGACAATCACCGGAGCTCAGGCTGAGGACGAGGCCGACTACTACTGCAACAGCCGGGACTCTTCAGGAAATCACGTGGTGTTCGGCGGCGGCACCAAGCTGACAGTGCTG (SEQ ID NO: 203).

[0207] cDNA: The code is CCTAGCGGCCAGGCCGGCGCCGCGGCCAGCGAAAGCCTGTTCGTGTCTAACCACGCCTAC (Sequence ID: 205) Ranking 20AA:PSGQAGAAASESLFVSNHAY (Sequence ID: 204)

[0208] cDNA: 7AA flanking peptide encoded by GAGGCTTCCGGCGGCCCCGAG (SEQ ID NO: 207): EASGGPE (SEQ ID NO: 206)

[0209] VL-a-CD33-116AA QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS(Sequence ID:208)

[0210] That is cDNA: CAGGTGCAGCTGGTGCAAAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAAGTGTCCTGCAAGGCCTCCGGCTACACCTTTACCGACTACAATATGCACTGGGTTCGGCAGGCCCCTGGCCAAGGTCTCGAGTGGATCGGCTACATCTATCCTTACAACGGCGGCACC GGCTACAACCAGAAGTTCAAAAGCAAGGCAACAATCACAGCCGACGAGAGCACTAACACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCTGTTTACTACTGCGCCAGAGGCAGACCAGCTATGGACTACTGGGGCCAAGGAACCCTGGTGACCGTGTCATCT (SEQ ID NO: 209).

[0211] VH-a-CD33 DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK(Sequence ID: 210)

[0212] That is cDNA: GATATCCAGATGACCCAGAGCCCTAGCAGCCTGTCTGCCTCTGTTGGCGACCGGGTGACCATCACCTGCAGAGCTTCTGAATCAGTTGACAACTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAAACCTGGCAAGGCCCCCAAGCTGCTGATCTATGCCGCTTCTAATCAGGGC AGCGGCGTGCCTAGCAGATTTAGCGGCAGTGGATCGGGAACCGACTTCACCCTGACCATTTCTTCGCTTCAGCCGACGATTTCGCCACATACTATTGTCAGCAGAGCAAGGAGGTGCCATGGACATTTGGCCAGGGCACTAAGGTGGAAATCAAG (SEQ ID NO: 211).

[0213] cDNA: Myc-tag ggsgeqkliseedlgg (SEQ ID NO: 380) encoded by GGCGGCAGCGGAGAGCAGAAGCTGATCTCCGAGGAAGACCTGGGTGGA (SEQ ID NO: 180)

[0214] cDNA: MMP9: ggPLGMTS (SEQ ID NO: 149) encoded by CCCCTGGGCATGACCTCT (SEQ ID NO: 182)

[0215] CD63 - extracellular PKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNV (SEQ ID NO: 151)

[0216] It is encoded by cDNA: CCTAAGAACAACCACACAGCCAGCATCCTGGACAGAATGCAGGCCGACTTCAAGTGCTGCGGCGCCGCTAATTACACCGACTGGGAGAAAATCCCCTCCATGAGCAAAAACCGGGTGCCCGACAGCTGTTGCATCAATGTGACCGTGGGCTGTGGCATTAACTTCAACGAGAAGGCTATCCATAAGGAGGGCTGCGTCGAGAAGATCGGCGGCTGGCTGAGAAAGAACGTG (SEQ ID NO: 184).

[0217] cDNA: CD63 - TM - helical (outside - in) LVVAAAALGIAFVEVLGIVFA (SEQ ID NO: 185) encoded by CTGGTGGTGGCCGCCGCCGCCCTGGGTATCGCCTTCGTGGAAGTGCTGGGCATCGTGTTCGCC (SEQ ID NO: 186)

[0218] cDNA: CD63-cytoplasmic CCLVKS (SEQ ID NO: 325) encoded by TGCTGCCTGGTGAAGTCT (SEQ ID NO: 379)

[0219] Example 5: Delivery of TRIKE containing ADAM17

[0220] Refer to Figures 13-14. One embodiment of the present invention includes TRIKE having ADAM17 target sequences embedded in type II TM (TNFSF14) and type I TM (TNR1A) domains. This molecule is expressed on the cell surface and cleaved upon NK cell activation.

[0221] This construct is outlined in sequence number 212 below: MSCSVARVGLGLLLLLMGAGLAVQGWFLLQLHWRLGEMVTRLPDGPAGSWEQLIQERRSHEVNPAAGGSGPVRRYQMEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLL FPY VKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSGGGGSGGGGSGGGSDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRF SGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKGGSGEQKLISEEDLSGGSGSLECTKLCLPQIENVKGTEDSGGSGQVQLVQSGAEVKKPGSSVKVSCKAFGGTFSSYAISWVRQAPGQGLEWMGRIIRFLGIANYAQKFQGRVTLIADKSTNTAYMELSSLRSEDT AVYYCAGEPGREDPDAVDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKARKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSSGTTVLLPLVIFFGLCLLSLLFIGLMYRYGGSGKRGRKKLLYIFKQPFMRPVQTTQEEDG (Sequence ID: 212)

[0222] The corresponding cDNA is shown below:

[0223] Domains and motifs

[0224] Sequence ID: 212 is represented below and is divided into the functional domains and motifs shown in Figure 14.

[0225] cDNA: TNFSF14 cytoplasmic MSCSVAR (SEQ ID NO: 214) encoded by ATGAGCTGCAGCGTGGCGAGA (SEQ ID NO: 215)

[0226] cDNA: TNFSF14 transmembrane (inside-out) VGLGLLLLLMGAGLAVQGWFL (Sequence ID: 216) coded by GTGGCGAGAGTAGGGCTGGGCCTGCTCCTGCTGCTGATGGGCGCCGGCCTGGCCGTGCAAGGATGGTTCCTG (Sequence ID: 217)

[0227] cDNA: TNFS having the cleavage sequence GSWEQLLQLHWRLGEMVTRLPDGPAGSWEQLIQERRSHEVNPAA (Sequence ID: 218), encoded by CTGCAGCTTCATTGGAGACTGGGAGAGATGGTGACTAGACTGCCTGACGGCCCTGCCGGCAGCTGGGAGCAGCTGATCCAGGAGAGAAGGAGCCACGAGGTGAACCCTGCTGCA (Sequence ID: 219). F14 extracellular

[0228] cDNA: Linker-a:GGSGPVRRYQ(sequence number:220) coded by GGTGGGAGCGGCCCTGTGCGGCGGTACCAA(sequence number:221)

[0229] cDNA: ATGGAAGTGCAGCTGGTCGAGTCTGGGGGCGGCGTGGTCCGGCCAGGCGGCAGCCTGCGTCTGTCTTGCGCCGCTAGCGGCTTCACTTTCGATGACTACGGCATGAGCTGGGTGCGGCAGGCCCCCGGCAAGGCCTCGAGTGGGTTTCTGGCATCAACTGGAACGGCGGCTCTACCGGCTACGCCGACAGCGT VL-a-CD16-118AA encoded by GAAGGGCCGCTTTACCATCTCCAGAGATAATGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCGAGGATACGGCCGTGTACTATTGTGCCCGGGGCAGAAGCCTCCTCTCGACTACTGGGGCCAGGGCACCCTGGTTACCGTGAGCCGG (SEQ ID NO: 223) MEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (Sequence ID: 222) cDNA: TRIKE-linker:GGGGSGGGGSGGGGS(sequence number:224) coded by GGCGGCGGCGGTTCTGGAGGCGGAGGCAGGCGGCGGGGGCTCC(sequence number:225)

[0230] VH-a-CD16-107AA SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVL(Sequence ID: 226)

[0231] That is cDNA: TCTGAGCTGACCCAGGACCCCGCCGTGAGCGTGGCCCTGGGCCAGACAGTCAGAATTACATGCCAGGGAGATAGCCTGAGATCCTACTATGCCAGCTGGTACCAGCAGAAACCTGGCCAAGCTCCTGTGCTGGTCATCTACGGCAAAAACAACAGACCTAGTGGAATCCCC GACAGATTTAGCGGATCCTCCAGCGGCAATACCGCCTCCTTGACAATCACCGGCGCTCAGGCCGAGGATGAAGCCGACTACTACTGCAACAGCAGAGACAGCTCTGGGAACCACGTGGTGTTCGGGGGGGGAACCAAGCTTACAGTGCTG (SEQ ID NO: 227).

[0232] cDNA: CCTTCTGGGCAGGCGGGAGCCGCCGCTAGCGAATCTCTGTTTGTGTCTAATCACGCCTAC (Sequence ID: 229) is used to code the flanking 20AA:PSGQAGAAASESLFVSNHAY (Sequence ID: 228).

[0233] cDNA: 7AA flanking peptide encoded by GAGGCCAGCGGCGGCCCCGAGCAGGTG (SEQ ID NO: 231): EASGGPE (SEQ ID NO: 230)

[0234] VL-a-CD33-116AA QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS(Sequence ID:232)

[0235] That is cDNA: CAGCTGGTGCAGAGTGGGGCTGAAGTGAAGAAACCTGGCAGCTCGGTGAAGGTGAGCTGCAAGGCCTCTGGCTACACCTTCACCGACTACAACATGCACTGGGTCAGACAGGCCCCTGGCCAGGGACTGGAATGGATCGGCTACATCTACCCCTACAACGGCGGCACTGGCTATAACCAA AAATTCAAGTCTAAGGCAACCATCACAGCCGACGAGAGCACCAACACCGCCTATATGGAGCTGTCTAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCCGGGGAAGACCTGCCATGGACTACTGGGGCCAAGGAACACTGGTGACCGTCAGC (SEQ ID NO: 233).

[0236] TRIKE-linker:GGGGSGGGGSGGGGS (Sequence ID: 234) encoded by cDNA:TCTGGAGGCGGCGGATCTGGCGGAGGAGGCAGCGGCGGCGGCGGTTCA (Sequence ID: 235)

[0237] TRIKEVH-a-CD33 DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (Sequence ID: 236)

[0238] That is cDNA: GACATCCAGATGACCCAGTCACCTAGCTCTCTGAGCGCCAGCGTGGGCGACCGGGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACAACTACGGAATTCTCATTCATGAACTGGTTCCAGCAGAAGCCCGGAAAGGCCCCGAAGCTGCTGATCTATGCTGCCAGCAACCAGGGT TCAGGGGTGCCTAGCAGATTTTCTGGGAGCGGCTCTGGCACCGACTTTACCCTGACAATCTCTAGCCTGCAGCCAGACGATTTCGCCACTTACTACTGTCAGCAGAGCAAGGAAGTGCCATGGACCTTCGGACAAGGAACAAAGGTTGAAATTAAG (SEQ ID NO: 237).

[0239] cDNA: Myc-tag GGSGEQKLISEEDLSGGSG (sequence number: 238) is coded by GGAGGCAGCGGGGAACAAAAGCTGATCAGCGAAGAGGACCTG (sequence number: 239).

[0240] cDNA: TNR1A-extracellular (SEQ ID NO: 240) containing the ADAM17 cleavage sequence SLECTKLCLPQIENVKGTEDS encoded by AGCCTGGAGTGCACCAAGCTGTGTCTGCCTCAGATCGAGAATGTGAAGGGCACAGAGGAT (SEQ ID NO: 241)

[0241] Vh-a-CD38 QVQLVQSGAEVKKPGSSVKVSCKAFGGTFSSYAISWVRQAPGQGLEWMGRIIRFLGIANYAQKFQGRVTLIADKSTNTAYMELSSLRSEDTAVYYCAGEPGREDPDAVDIWGQGTMVTVSS(Sequence ID:242)

[0242] That is cDNA: CAAGTGCAGCTGGTGCAGAGCGGCGCTGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGTAAAGCCTTCGGCGGAACCTTCAGCTCCTACGCTATCAGCTGGGTGAGACAGGCCCCTGGCCAGGGGCTAGAATGGATGGGCAGAATCATCCGGTTCCTGGGCATCGCCAATTACGCCCAGAAGTTT CAGGGCCGGGTGACGCTGATCGCCGATAAGAGCACCAACACCGCCTACATGGAACTGTCTTCCCTGCGATCAGAAGACACCGCCGTGTACTACTGCGCCGGCGAACCTGGAAGAGAAGATCCTGACGCCGTGGATATCTGGGGACAGGGCACAATGGTGACAGTGTCGAGC (SEQ ID NO: 243).

[0243] cDNA: Linker GGGGSGGGGSGGGGS (sequence number: 244) coded by GGCGGAGGCGGCTCTGGTGGCGGTGGCTCTGGCGGCGGCGGCTCC (sequence number: 245)

[0244] cDNA: GACATTCAGATGACACAGTCCCCTTCCTCTCTGTCCGCCTCCGTGGGCGACCGGGTGACCATCACCTGCAGAGCCAGTCAGGGCATCAGAAGCTGGCTGGCTTGGTACCAGCAGAAGCCTGAGAAGGCCAGGAAAAGCCTGATCTACGCCGCGAGCAGCCTGCAGTCTGGGGTG Vl-a-CD38 encoded by CCCTCTAGATTCAGCGGTTCTGGCAGCGGCACAGATTTCACACTGACAATAAGTTCGCTGCAGCCTGAAGACTTCGCCACCTATTACTGCCAGCATACAACAGCTACCCTCTGACCTTCGGCGGCGGAACCAAGGTTGAGATCAAG (SEQ ID NO: 247) DIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKARKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK(Sequence ID: 246)

[0245] cDNA: TNR1A-transmembrane SGTTVLLPLVIFFGLCLLSLLFIGLMYRY (Sequence ID: 248) encoded by TCCGGCACCACCGTCCTGCTGCCTCTGGTGATCTTCTTCGGCCTGTGCCTGCTGTCACTGCTGTTCATCGGACTCATGTACAGATAC (Sequence ID: 249)

[0246] cDNA: 41BB-intracellular:KRGRKKLLYIFKQPFMRPVQTTQEEDG(Sequence ID: 250) encoded by AAAAGAGGCCGAAAGAAGCTGCTGTACATCTTTAAACAGCCCTTCATGAGACCCGTGCAAACAACCCAGGAGGAAGATGGC (Sequence ID: 251)

[0247] Example 6: BIKE having anti-CD38 with granzyme B cleavage sequence

[0248] Refer to Figures 15 and 16. In some embodiments, the present invention includes BIKE+ anti-CD38 having a granzyme B cleavage sequence having an activation domain having 71 of SEQ ID NO: 252. This molecule is membrane-bound and is released upon granzyme B release following NK cell degranulation. Anti-CD38 remains on NK cells after the release of the remaining cargo and functions similarly to a chimeric antigen receptor (CAR).

[0249] The corresponding cDNA is represented as follows: ATGTGCGGCGCCTGCAAGGAAAACTACTGTCTGATGATCACTTTCGCCATCTTTCTGTCTCTGATCATGCTGGTGGAAGTGGCCGCTGCCATTGCCGGATACGTGGGCGGCTCTGGCCCTGTGCGAAGATACCAAATCGAGCCTGACGGCGGCAGCGGCATGGAAGTGCAGCTGGTTGAGTCCGGCGGCGGAGTGGTGCGGCCTGGCGGCTCTCTGAGACTGTCTTGCGCTGCCAGCGGTTTCACCTTCGACGATTACGGAATGAGCTGGGTGCGGCAGGCCCCAGGCAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGAAGCACCGGCTACGCAGACAGCGTGAAGGGCAGATTCACCATCTCCAGAGATAACGCCAAGAATTCACTGTACCTGCAGATGAACAGCCTGAGAGCTGAGGACACTGCAGTTTACTACTGCGCCAGAGGCCGGAGCCTGCTGTTCGACTACTGGGGACAGGGAACACTGGTGACCGTGAGCAGAGGCGGCG (Sequence number: 253)

[0250] Sequence ID: 252 is shown in Figure 15 and is divided into the functional domains and motifs shown below.

[0251] CD63-cytoplasmic MCGACKENYC (SEQ ID NO: 254) encoded by cDNA:ATGTGCGGCGCCTGCAAGGAAAACTACTGT (SEQ ID NO: 255)

[0252] cDNA: CD63-TM-Helical (Inside-Out) coded by CTGATGATCACTTTCGCCATCTTTCTGTCTCTGATCATGCTGGTGGAAGTGGCCGCT (Sequence ID: 257) LMITFAIFLSLIMLVEVAAAI (Sequence ID: 256)

[0253] cDNA: The granzyme B cleavage site: IEPD (Sequence ID: 258) coded by ATCGAGCCTGAC (Sequence ID: 259). Alternatively, the granzyme B cleavage site: EEEEVEADSEEEEEEE (Sequence ID: 260), the granzyme B cleavage site: AQGVISADASNLDDFY (Sequence ID: 261), or the granzyme B cleavage site: LEADKGKLEYD (Sequence ID: 262) may be used.

[0254] Linker-a:GGSG(Sequence ID:263)GGCGGCAGCGGC(Sequence ID:264)

[0255] VL-a-CD16-118AA MEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (Sequence ID: 265).

[0256] That is cDNA: ATGGAAGTGCAGCTGGTTGAGTCCGGCGGCGGAGTGGTGCGGCCTGGCGGCTCTCTGAGACTGTCTTGCGCTGCCAGCGGTTTCACCTTCGACGATTACGGAATGAGCTGGGTGCGGCAGGCCCCAGGCAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGAAGCACCGGCTACGCAGA CAGCGTGAAGGGCAGATTCACCATCTCCAGAGATAACGCCAAGAATTCACTGTACCTGCAGATGAACAGCCTGAGAGCTGAGGACACTGCAGTTTACTACTGCGCCAGAGGCCGGAGCCTGCTGTTCGACTACTGGGGACAGGGAACACTGGTGACCGTGAGCAGA (SEQ ID NO: 266).

[0257] VH-a-CD16-107AA SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVL(Sequence ID: 267)

[0258] That is cDNA: TCCGAGCTGACCCAGGACCCCGCCGTGTCCGTGGCCCTGGGCCAAACAGTGAGAATCACCTGTCAGGGCGACTCTCTGCGCTCTTACTACGCCAGCTGGTACCAACAGAAGCCCGGCCAGGCCCCTGTGCTGGTGATCTACGGCAAGAACAACAGACCTTCTGGTATCCCC GACAGATTTAGCGGCTCTTCTAGCGGCAACACCGCCTCCCTGACCATCACAGGCGCCCAGGCCGAGGACGAGGCTGACTACTATTGCAATAGTAGAGACAGCAGCGGAAACCATGTGGTGTTCGGCGGTGGCACAAAGCTGACAGTGCTT (SEQ ID NO: 268).

[0259] cDNA: Flanking 20AA:PSGQAGAAASESLFVSNHAY (Sequence ID: 269) coded by CCCAGCGGACAGGCCGGAGCCGCCGCCTCTGAAAGCCTGTTCGTGAGTAATCACGCCTAC (Sequence ID: 270)

[0260] cDNA:GAGGCCTCCGGAGGCCCTGAG (SEQ ID NO: 272) encodes 7AA flanking peptide:EASGGPE (SEQ ID NO: 271)

[0261] VL-a-CD33-116AA QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS(Sequence ID:273)

[0262] That is cDNA: CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCAGGCAGCAGCGTTAAGGTGAGTTGTAAAGCCTCCGGATACACCTTCACCGACTACAACATGCACTGGGTTAGACAGGCGCCTGGCCAGGGCCTGGAATG GATCGGCTACATCTACCCTTACAACGGCGGGACCGGCTATAATCAGAAGTTCAAGAGCAAGGCCACGATCACAGCCGACGAGAGCACCAACACAGCATACATGGAACTGAGCTCCCTGAGATCTGAGGACACCGCCGTGT It is coded by ACTACTGCGCCAGGGGTCGGCCGGCCATGGACTATTGGGGCCAGGGCACCCTGGTCACAGTGTCTAGC (Sequence ID: 274).

[0263] TRIKE VH-a-CD33 DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK(Sequence ID: 275)

[0264] That is cDNA: GATATTCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCTCTGTTGGAGATCGGGTGACAATCACATGTCGGGCCTCCGAGTCTGTGGACAACTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAAACCAGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCTAGCAACCAGGGC AGCGGCGTGCCTTCCCGCTTCAGCGGCAGCGGCAGCGGAACAGATTTCACCCTGACCATCTCTTCTCTCCAACCTGATGATTTCGCTACCTACTACTGCCAGCAGAGCAAGGAGGTGCCTTGGACCTTCGGACAGGGCACCAAAGTGGAGATCAAG (SEQ ID NO: 276).

[0265] cDNA: Myc-tag:GGSGEQKLISEEDLGG (sequence number: 277) is coded by GGCGGCTCTGGGGAACAGAAGCTGATCAGCGAGGAAGACCTGGGAGGC (sequence number: 278).

[0266] Granzyme B cleavage site. IEPD (Sequence ID: 279)ATCGAGCCTGAT (Sequence ID: 280)

[0267] CD38Vh: QVQLVQSGAEVKKPGSSVKVSCKAFGGTFSSYAISWVRQAPGQGLEWMGRIIRFLGIANYAQKFQGRVTLIADKSTNTAYMELSSLRSEDTAVYYCAGEPGREDPDAVDIWGQGTMVTVSS(Sequence ID:281)

[0268] That is cDNA: CAAGTGCAGCTGGTTCAAAGCGGCGCTGAGGTGAAAAAACCTGGGAGCAGCGTGAAGGTGTCCTGCAAGGCTTTCGGGGGCACCTTCAGCTACGCAATCAGCTGGGTGCGGCAGGCTCCCGGCCAGGGCCTGGAATGGATGGGCAGAATCATCCGGTTCCTCGGAATCGCCAACTACGCCCAGAAGTTC CAGGGTAGAGTGACCCTGATTGCCGACAAGAGCACAAACACCGCTTATATGGAACTGAGCTCTCTGCGCAGCGAAGATACCGCCGTGTACTACTGCGCCGGCGAACCCGGCGGGAAGATCCTGATGCCGTGGATATCTGGGGCCAGGGCACCATGGTGACAGTGTCATCC (SEQ ID NO: 282).

[0269] Linker encoded by cDNA:GGTGGCGGCGGAAGCGGCGGCGGCGGTAGTGGGGGAGGCGGCAGC (Sequence ID: 284):GGGGSGGGGSGGGGS (Sequence ID: 283)

[0270] CD38Vl: DIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKARKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK(Sequence ID: 285)

[0271] That is cDNA: GACATCCAGATGACCCAGTCACCTAGCTCTTTGTCGGCCAGCGTCGGAGATAGAGTGACCATTACCTGTCGGGCCTCTCAGGGAATCAGAAGCTGGCTCGCCTGGTACCAGCAAAAGCCTGAGAAGGCCAGAAAATCCCTAATCTATGCCGCTAGCAGCCTGCAGTCTGGC GTGCCCTCCAGATTTAGCGGAAGCGGAAGCGGCACAGACTTCACACTGACAATCAGCTCTCTGCAACCTGAGGATTTCGCCACCTACTACTGCCAACAGTACAACAGCTACCCACTGACATTTGGAGGCGGTACAAAGGTGGAAATCAAG (SEQ ID NO: 286).

[0272] cDNA: The hinge stalk region coded by GCCAAGCCCACCACAACCCCTGCCCCTAGACCCCCCACCCCAGCTCCCACCATCGCCAGCCCCTCTGTCCCTGCGGCCTGAAGCCTGCAGACCCGCTGCCGGCGGCGCCGTCCACACCCGGGGACTGGACTTCGCCCCTAGAAAGATCGAGGTGATGTACCCCCCTCCCTACCTGGATAATGAGAAGCAATGGCACAATCATCCACGTGAAAGGCAAACACCTGTGCCCTTCTCCTCTGTTTCCTGGACCTAGCAAGCCC (Sequence ID: 288): AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP(Sequence ID: 287)

[0273] CD28 transmembrane: FWVLVVVGGVLACYSLLVTVAFIIFWV (Sequence ID: 289) encoded by cDNA:TTCTGGGTCCTGGTCGTGGTCGCGGTCGCGCGCTCGCTACAGCCTCCTGGTGACCGTGGCCTTCATCATTTTTTGGGTG (Sequence ID: 290)

[0274] cDNA: CD28 intracellular code encoded by CGCAGCAAGAGATCCAGGCTGCTGCACTCTGACTACATGAACATGACCCCTAGACGGCCTGGTCCTACAAGAAAGCACTACCAGCCTTACGCCCCTCCCAGAGATTTCGCTGCCTACCGTAGC (Sequence ID: 292): RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (Sequence ID: 291)

[0275] cDNA: CGGGTGAAGTTCAGCAGATCTGCCGATGCCCCTGCTTACCAACAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCCGGCGGGAAGAGTACGACGTGCTGGACAAAAGAAGAGGCAGAGACCCTGAGATGGGGGGAAGCCTCGGAGAAAGAACCCCAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGAATGAAGGGAGAAAGA CD3z cells encoded by CGGAGAGGCAAAGGCCACGACGGCCTGTACCAGGGTCTGTCGACCGCTACCAAGGACACCTACGACGCCCTGCACATGCAGGCTCTGCCTCCAAGA (Sequence ID: 294): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(Sequence ID: 293)

[0276] Example 7: HEK293 cells containing TNF-α and MMP2 or MMP9 cleavage sites

[0277] Refer to Figures 4A-B, 5A-B, and 9-12. In some embodiments, following the above teachings, the inventors prepared the construct CD63-9-2L-TNFα-Myc-9-CD63-cyt (containing an EV localization (sort) motif) shown in Figures 4A-B, 9, and 10, and the construct CD63-9-2L-TNFα-Myc-9-CD63-D-Cyt (lacking an EV localization (sort) motif) shown in Figures 5A-B, 11, and 12. These constructs differ only in the extracellular vesicle localization (sort) motif.

[0278] Construct CD63-9-2L-TNFα-Myc-9-CD63-cyt (EV localization ( (sort) Motif) LeGo-CD63-9-2L-TNFα-Myc-9-CD63 - Center

[0279] This is represented by the following sequence number: 295. MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQGGPLGMTSSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGPLGMTSGGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM (Sequence ID: 295)

[0280] Refer to Figures 4A-4B, 9, and 10. The domains and motifs are encoded by the following amino acid sequences.

[0281] CD63-Cytoplasm: MAVEGGMKCVK (Sequence ID: 297)

[0282] CD63-TM-Helical (Inside-Out) FLLYVLLLAFCACAVGLIAVG (Sequence ID: 298)

[0283] CD63-Extracellular VGAQLVLSQTIIQGATPGS (Sequence ID: 299)

[0284] CD63-TM-Helical (Outside-In) LLPVVIIAVGVFLFLVAFVGC (Sequence ID: 300)

[0285] CD63-Cytoplasmic CGACKENYC (SEQ ID NO: 301)

[0286] CD63-TM-Helical (Inside-Out) LMITFAIFLSLIMLVEVAAAI (Sequence Number: 302)

[0287] Linker: GGSGPVRRYQ (Sequence ID: 303)

[0288] MMP9:GGPLGMTS(Sequence ID: 304)

[0289] TNFα: SSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIA(Sequence ID: 305)

[0290] Linker + Myc-tag:GGSGEQKLISEEDL(Sequence ID:306)

[0291] MMP9:GGPLGMTS(Sequence ID: 307)

[0292] CD63-extracellular PKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM (Sequence ID: 308)

[0293] CD63-TM-Helical (Outside-In) LVVAAAALGIAFVEVLGIVFA (Sequence ID: 309)

[0294] CD63-Cytoplasmic LVKSIRSGYEVM (SEQ ID NO: 310)

[0295] The construct CD63-9-2L-TNFα-Myc-9-CD63-D-Cyt (lacking the EV localization (sort) motif) is outlined in sequence number 311 below. MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQGGPLGMTSSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGPLGMTSGGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKS(Sequence ID: 311)

[0296] Refer to Figures 5A-B, 11, and 12. The domains and motifs of this construct are encoded by the following amino acid sequences.

[0297] CD63-cytoplasm: MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYV (Sequence ID: 313)

[0298] CD63-TM-Helical (Inside-Out) FLLYVLLLAFCACAVGLIAVG (Sequence ID: 314)

[0299] CD63-Extracellular VGAQLVLSQTIIQGATPGS (Sequence ID: 315)

[0300] CD63-TM-Helical (Outside-In)LLPVVIIAVGVFLFLVAFVGC (Sequence ID: 316)

[0301] CD63-Cytoplasmic CGACKENYC (SEQ ID NO: 317)

[0302] CD63-TM-Helical (Inside-Out) LMITFAIFLSLIMLVEVAAAI (Sequence Number: 318)

[0303] Linker: GGSGPVRRYQ (Sequence ID: 319)

[0304] MMP9:GGPLGMTS(Sequence ID: 320)

[0305] TNFα- SSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIA(Sequence ID: 321)

[0306] Linker + Myc-tag:GGSGEQKLISEEDL(Sequence ID:322)

[0307] MMP9:GGPLGMTS(Sequence ID: 323)

[0308] CD63-extracellular PKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM (Sequence ID: 324)

[0309] CD63-TM-Helical (Outside-In) LVVAAAALGIAFVEVLGIVFA (Sequence ID: 185)

[0310] CD63-cytoplasmic CCLVKS (SEQ ID NO: 325).

[0311] Functional assay

[0312] Example 8: Assay on TNFα using HEK293 cells

[0313] Refer to Figures 21 and 22. The inventors developed a functional assay to measure the presence of TNFα using HEK293 NF-κB luciferase reporter cells.

[0314] Refer to Figures 22 and 24. To test whether the GAGE ​​payload of this system is functional, HEK293 cells were transfected with the GAGE ​​plasmid using polyethyleneimine (PEI) in a transient transfection as follows. - Day 0: 400,000 cells were seeded into a 6-well plate. - Day 1: 2.5 μg of GAGE ​​plasmid was transfected into HEK293 wild-type cells. - Day 2: HEK293+GAGE and HEK293 NF-κB luciferase reporter cells were co-cultured for 24 hours or kept separately. - Day 3: Reporter cells were harvested and lysed, and the inventors assayed the functional activity of TNFα on HEK293 cells using an NF-κB luciferase reporter assay. Referring to Figure 26, it was immediately observed that TNFα expressed on HEK293 cells was functional regardless of the presence or absence of the EV localization (sort) motif.

[0315] Example 9: Assay for payload release using MMP9 or MMP2

[0316] Next, refer to Figures 25 and 27. To test whether GAGE-expressing cells are released in the presence of MMP9 or MMP2, HEK293 cells were transfected with GAGE ​​and MMP9 or MMP2 plasmids using polyethyleneimine (PEI) in transient transfection as follows. - Day 0: 400,000 cells were seeded into a 6-well plate. - Day 1: GAGE ​​and 2.5 μg each of MMP9 or MMP2 plasmid were separately transfected into HEK293 wild-type cells. - Day 2: HEK293+GAGE and HEK293+MMP9 or HEK293+MMP2 cells were co-cultured for 24 hours. - Day 3: 200 μL of supernatant was collected and given to HEK293 NF-κB reporter cells.

[0317] After a 6-hour incubation, reporter cells were harvested and lysed with 1% Triton. The inventors then assayed the functional activity of TNFα released from HEK293 cells using an NF-κB luciferase reporter assay. The substrate (D-luciferin) was automatically added to the sample, and luminescence was measured.

[0318] Referring to Figures 25 and 27, it is immediately observed that TNFα expressed on HEK293 cells is higher in EV-localized (sorted) plasmids, indicating that most of the GAGE ​​payload is more concentrated in EV-localized (sorted) plasmids than in non-EV-localized (sorted) plasmids.

[0319] Example 10: Delivery of payload to extracellular vesicles

[0320] To primarily verify the delivery of the GAGE ​​payload to EVs, the experiment in Example 9 was repeated using supernatant deprived of EVs by performing the procedure with an Amicon® Ultra 0.5 mL centrifugal filter. A luciferer eleporter test was performed using HEK293 NF-κB luciferase reporter cells. Supernatant from the co-culture was added to HEK293 reporter cells expressing NF-κB in response to the TNFα payload, either deprived of EVs or in its original state. Figures 26 and 28 show a significant reduction in luciferase activity after EV deprivation, suggesting the presence of GAGE ​​delivery in the supernatant containing extracellular vesicles.

[0321] Example 11: Selection of TMDs for better cell surface expression

[0322] To evaluate another transmembrane (TM) protein that can be expressed primarily on the cell surface, the inventors replaced CD63TM with C-type lectin transmembrane CD69. Figure 31 shows CD63TM. In comparison, when CD69 is used as the TM protein, a higher percentage of TNFα-positive cells are observed.

[0323] Next, to evaluate whether GAGE ​​TNFα expressed on HEK293 cells can be released in the presence of MMP9, MMP9 cleavage was performed by direct co-culture of GAGE ​​TNFα-expressing cells with MMP-expressing cells, supernatant from MMP9-transfected cells, or the provided MMP9 recombinant protein. The success of MMP9 cleavage of the MMP9 protease substrate linked to TNFα GAGE ​​was verified by a luciferase assay using HEK293 NF-κB luciferase reporter cells. As shown in Figures 17A-B, 18A-B, 29, and 30, luciferase activity was observed only in clones containing the MMP9 cleavage substrate, indicating that TNFα can be released by specific protease cleavage.

[0324] Example 12: Payload release using Granzyme B

[0325] To further evaluate their invention, the inventors used a different schedase cleavage substrate for a granzyme B (GrzB) protein linked to TNFα GAGE ​​as a payload. First, both clones of CD69-GrzB-TNFα-Myc-GrzB-aCD19-CAR and CD69-TNFα-Myc-aCD19-CAR were produced and transduced into K562 cells (Figure 31).

[0326] Next, we evaluated whether GAGE ​​TNFα, which contains a GrzB cleavage substrate expressed on K562 cells, could be released in co-culture with NK92 cells. Contact of NK92 cells with target cells led to the induction of degranulation of NK92 cells containing granzyme B granules. GrzB cleavage accompanied by TNFα release was performed via co-culture of GAGE ​​TNFα-expressing K562 cells with NK92 cells. After 4 hours of incubation, the cells were centrifuged and the supernatant was collected. The success of cleavage of the GrzB protease substrate linked to TNFα GAGE ​​was verified by a luciferase assay using HEK293 NF-κB luciferase reporter cells. As shown in Figure 32, luciferase activity was higher in clones containing the GrzB cleavage substrate, indicating that TNFα is specifically released by specific protease cleavage.

[0327] Example 13: Functional assay of nanoluciferase delivery as proof of concept

[0328] To increase the readout sensitivity, the inventors repeated the experiment of Example 12 using a different GAGE ​​payload, this time using nanoluciferase as the payload.

[0329] The inventors created three types of clones as follows. - CD69-GrzB-nLuc-Myc-GrzB-aCD19-CAR (GAGE1 or GAGE ​​original) - CD69-nLuc-Myc-aCD19-CAR(GAGE A) - CD69-GrzB-nLuc-Myc-GrzB-CAR(GAGE B)

[0330] These plasmids were transduced into K562 cells at MOI6 and subsequently assayed using a luciferase assay. As shown in Figure 33, surface expression of GAGE ​​nLuc was observed in K562 cells.

[0331] Next, we evaluated whether GAGE ​​nLuc, which has a GrzB cleavage substrate expressed on K562 cells, could be released during co-culture with NK92. GrzB cleavage substrate release associated with nLuc release. B cleavage was performed via co-culture of GAGE ​​nLuc-expressing K562 cells and NK92 cells. After 4 hours of incubation, the cells were centrifuged and the supernatant was collected. The success of cleavage of the GrzB protease substrate linked to nLuc GAGE ​​was verified by a direct luciferase assay. As shown in Figure 34A, luciferase activity was higher in clones containing the GrzB cleavage substrate, indicating that nLuc is particularly released by specific protease cleavage. Further activation of NK92 cells with PAM / ionomycin did not have a significant effect on further substrate cleavage and showed no difference in the luciferase activity assay. Figure 34B.

[0332] Example 14: Additional clones to evaluate payload cleavage and release

[0333] The inventors created the following clones in KHYG-1 cells: CD69-GrzB-nLuc-Myc-GrzB-aCD19-CAR (GAGE1 or GAGE ​​original), CD69-nLuc-Myc-aCD19-CAR (GAGE A), and CD69-GrzB-nLuc-Myc-GrzB-CAR (GAGE B). Specific cleavage and payload release were further evaluated. The inventors transduced the three clones into KHYG-1 cells at an MOI of 30. As shown in Figure 35, surface expression of GAGE ​​nLuc in KHYG-1 cells was observed.

[0334] After co-culturing KHYG-1 cells expressing GAGE ​​nLuc with Nalm-6 cells (highly expressing CD19) at three different time points shown in Figure 36, luciferase activity was higher in clones containing a GrzB cleavage substrate with nLuc linked to sc-aCD19CAR, indicating that nLuc was particularly released by specific protease cleavage during the co-culturing period, with maximum cleavage occurring at 3 hours of incubation.

[0335] Next, the inventors sorted the cells to reduce the background activity of nanoluciferase. As shown in Figure 37A, KHYG-1 cells were positive in over 99% of the three clones. Interestingly, the MFI of nLuc GAGE-positive cells was higher in the clone without cleavage substrate sites compared to the other two clones (Figure 37B).

[0336] The inventors repeated the experiment with sorted cells, and two different time points (4 hours and 6 hours) were selected for co-culturing sorted KHYG-1 cells expressing GAGE ​​nLuc with K562 cells. As shown in Figure 38, luciferase activity was higher in clones containing nLuc and a GrzB cleavage substrate, indicating that nLuc was particularly released by specific protease cleavage during the co-culturing period, with the greatest cleavage occurring at 6 hours of incubation.

[0337] To evaluate the specificity and sensitivity of cleavage by granzyme B, the inventors created three clones of CD69-GrzB-nLuc-Myc-GrzB-aCD19-CAR in NK92 cells, each possessing a different GrzB cleavage substrate: GrzB(IEPD (SEQ ID NO: 258)), GrzB(AQGVISADASNLDDFY (SEQ ID NO: 261)), and GrzB(LEADKGKLEYD (SEQ ID NO: 262)). The inventors transduced these clones into NK92 cells at an MOI of 30. As shown in Figures 39A and 39B, surface expression of GAGE ​​nLuc was observed in NK92 cells.

[0338] After co-culturing NK92 cells expressing GAGE ​​nLuc with Raji cells for 4 hours, luciferase activity was increased, and compared to the control clone (GAGE A), clones (LEADKGKLEYD) containing GrzB as a cleavage substrate to which nLuc was attached were found to have higher luciferase activity. In column 262), the affinity was 9 times higher, indicating that this particular substrate has a higher affinity for GrzB cleavage during the co-culture period (Figure 40).

[0339] Example 15: Payload release via caspase 3

[0340] The inventors generated the following clones in NK92 cells: CD69-Casp3-nLuc-Myc-Casp3-aCD19-CAR (GAGE Casp3) and CD69-nLuc-Myc-aCD19-CAR (GAGE A). Specific cleavage and payload release were further evaluated. The inventors transduced the two clones described above into NK92 cells at an MOI of 30. As shown in Figure 41, surface expression of GAGE ​​nLuc was observed within NK92 cells.

[0341] After co-culturing K562 cells with NK92 cells expressing GAGE ​​Casp3-nLuc at two different time points shown in Figure 42, luciferase activity was higher in clones containing a Casp3 cleavage substrate with nLuc, indicating that nLuc is particularly released by specific protease cleavage during the co-culturing period.

[0342] CD69cyt-TMII-TNFα-Myctag-CD28TM-cyt

[0343] The cDNA of CD69Cyt-TM-II-TNFα-Myc-CD28TM-Cyt is represented by the following sequence number: 326. (query number:326)

[0344] Corresponding to amino acid sequence, sequence number: 327 is displayed. MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALSVGQYNGGSGPVRRYQSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRD NQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGSGCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLL (Sequence ID: 327)

[0345] Sequence ID: 327 is also shown in Figures 17A-B, along with the domain and motif legend.

[0346] CD69-Cytoplasm MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVP (Sequence ID: 328)

[0347] CD69-TM-II type (inside-out) VLCAVMNVVFITILIIALIAL (Sequence ID: 329)

[0348] CD69-Extracellular SVGQYNGGSGPVRRYQ (Sequence ID: 330)

[0349] TNFα RTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (Sequence ID: 331)

[0350] Linker GGSG (Sequence ID: 332)

[0351] Myc-tag EQKLISEEDL (SEQ ID NO: 333)

[0352] CD28TM FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 334)

[0353] CD69cyt-TMII-9-TNFα-Myctag-9-CD28TM-cyt

[0354] The cDNA of CD69cyt-TMII-9-TNFα-Myctag-9-CD28TM-cyt is represented as SEQ ID NO: 335 below. ATCCGCCGCCACCATGAGTTCCGAAAACTGCTTTGTCGCCGAGAATAGTAGTCTGCACCCCGAATCAGGACAGGAGAATGACGCAACCAGCCCCCACTTTTCAACAAGGCACGAAGGGAGCTTCCAGGTCCCTGTGCTGTGCGCAGTGATGAACGTGGTCTTTATCACTATTCTGATCATTGCTCTGATCGCACTGAGTGTCGGACAGTACAATGGCGGGTCAGGCCCCGTGCGGAGATATCAGGGAGGGTCTGGGGGAGGACCACTGGGAATGACCTCCGGAGGGTCTGGGAGCTCCTCTCGCACCCCTTCCGACAAGCCAGTGGCCCATGTGGTCGCTAACCCTCAGGCAGAGGGACAGCTGCAGTGGCTGAACAGGCGAGCCAATGCTCTGCTGGCTAACGGCGTGGAACTGCGAGATAATCAGCTGGTGGTCCCTAGCGAGGGGCTGTACCTGATCTATTCCCAGGTCCTGTTCAAAGGGCAGGGATGCCCATCTACACACGTGCTGCTGACCCATACAATCTCTAGAATTGCCGTCAGTTACCAGACTAAGGTGAACCTGCTGAGCGCCATCAAGTCACCATGTCAGAGGGAGACACCCGAAGGAGCAGAGGCCAAGCCCTGGTACGAGCCTATC TATCTGGGAGGCGTGTTTCAGCTGGAAAAAGGCGACCGACTGTCTGCCGAGATTAATCGGCCAGACTACCTGGATTTCGCTGAAAGTGGCCAGGTGTATTTTGGGATCATTGCACTGGGAGGCAGCGGAGAGCAGAAGCTGATCTCTGAGGAAGATCTGGGAGGACCACTGGGAATG ACCTCCGGAGGGAGCGGATGCCCATCCCCTCTGTTCCCAGGACCCAGCAAACCCTTTTGGGTCCTGGTGGTCGTGGGAGGCGTGCTGGCATGTTATAGCCTGCTGGTCACAGTGGCTTTCATTATCTTTTGGGTCCGCTCTAAACGGTCTCGGCTGCTGTAATGTACA (SEQ ID NO: 335)

[0355] The corresponding amino acid sequence is shown in sequence number 336 below, and is also shown in Figures 18A-B along with the domain and motif legend.

[0356] MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALSVGQYNGGSGPVRRYQGGSGGGPLGMTSGGSGSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGPLGMTSGGSGCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLL(Sequence ID: 336)

[0357] Domains and motifs

[0358] CD69-Cytoplasm MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVP (Sequence ID: 338)

[0359] CD69-TM-II type (inside-out) VLCAVMNVVFITILIIALIAL (Sequence ID: 339)

[0360] CD69-Extracellular SVGQYNGGSGPVRRYQ (Sequence ID: 340)

[0361] Linker GGSG (Sequence ID: 341)

[0362] MMP9 GGPLGMTSG (Sequence ID: 342)

[0363] Linker GSGSSS (Sequence ID: 343)

[0364] TNFα RTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (Sequence ID: 344)

[0365] Myc-tag EQKLISEEDL (Sequence ID: 345)

[0366] CD28TM FWVLVVVGGVLACYSLLVTVAFIIFW (Sequence ID: 346)

[0367] See Figure 20. Some of the DNA sequences of GAGE ​​are shown below:

[0368] LeGo-CD63-9-2L-TNFα-Myc-9-CD63-cyt MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQGGPLGMTSSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGPLGMTSGGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM (Sequence ID: 295)

[0369] That is cDNA:

[0370] LeGo-CD63-9-2L-TNFα-Myc-9-CD63-D-Cyt MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQGGPLGMTSSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGPLGMTSGGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKS (SEQ ID NO: 311).

[0371] It is cDNA:

[0372] LeGo-CD63-DL1-2-2L-TNFα-Myc-2-CD63-Cyt MAVEGGMKCVKCGACKENYCLMITFAIFLSLIMLVEVAAA IAGYVGGSGPVRRYQGGPLGVRGGSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGPLGVRGGGGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM (SEQ ID NO: 368)

[0373] It is cDNA:

[0374] LeGo-CD63-9-2L-TNFα-Myc-9-CD19CAR MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQGGPLGMTSSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGPLGMTSGGSGGGSGPDIQMTQTTSS LSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 369)

[0375] It is cDNA:

[0376] LeGo-CD63-2-2L-TNFα-Myc-2-CD63-D-Cyt MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQGGPLGVRGGSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGPLGVRGGGGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKS (SEQ ID NO: 371)

[0377] It is cDNA: GATCCGCCGCCACCATGGCCGTGGAGGGCGGGATGAAGTGCGTGAAATTCCTGCTGTACGTCCTGCTGCTGGCCTTTTGCGCATGTGCTGTGGGACTGATCGCAGTGGGAGTCGGAGCTCAGCTGGTGCTGAGCCAGACTATCATTCAGGGAGCAACCCC

[0378] LeGo_CD63-2-2L-TNFα-Myc-2-CD19CAR MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQGGPLGVRGGSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGPLGVRGGGGSGGGSGPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR (SEQ ID NO: 372)

[0379] It is cDNA: It is encoded by AGCAGTCCATACCCGAGGACTGGACTTCGCACCACGAAAGATTGAAGTGATGTACCCTCCACCCTATCTGGATAACGAAAAAAGCAATGGCACAATCATTCACGTGAAGGGGAAACATCTGTGCCCCAGTCCTCTGTTCCCAGGCCCCTCAAAGCCCTTTTGGGTCCTGGTGGTCGTGGGAGGGGTGCTGGCCTGTTACAGCCTGCTGGTCACAGTGGCTTTCATCATCTTCTGGGTGCGGAGCAAACGGTCCAGACTGCTGCACTCCGACTATATGAACATGACACCACGACGGCCTGGCCCAACTAGAAAGCATTACCAGCCCTATGCCCCTCCAAGGGACTTCGCCGCTTACAGGTCCCGCGTGAAATTTTCAAGAAGCGCAGATGCCCCAGCTTACCAGCAGGGACAGAATCAGCTGTATAACGAGCTGAATCTGGGCAGAAGGGAAGAGTATGACGTGCTGGATAAGCGACGAGGACGGGACCCCGAAATGGGAGGCAAGCCACGGAGAAAAAACCCTCAGGAGGGACTTTACAATGAACTGCAGAAGGACAAAATGGCAGAGGCCTATTCTGAAATCGGCATGAAGGGGGAGAGGCGCCGAGGAAAAGGCCACGATGGGCTGTACCAGGGACTGAGCACTGCCACCAAGGACACCTATGATGCCCTGCATATGCAGGCTCTGCCCCCTCGGTAATGTACA (SEQ ID NO: 351).

[0380] LeGo-CD63-2L-TNFα-Myc-CD63-Cyt MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVG GSGPVRRYQGGPLGVRGGSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKV NLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGPLGVRGGGGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM (Sequence ID: 364)

[0381] That is cDNA: GGATCCGCCGCCACCATGGCCGTGGAGGGCGGGATGAAGTGCGTGAAATTCCTGCTGTACGTCCTGCTGCTGGCCTTTTGCGCATGTGCTGTGGGACTGATCGCAGTGGGAGTCGGAGCTCAGCTGGTGCTGTCTCAGACTATCATTCAGGGGGCAACCCCTGGAAGTCTGCTGCCAGTGGTCATCATTGCCGTGGGCGTCTTCCTGTTTCTGGTGGCATTCGTCGGATGCTGTGGCGCCTGCAAGGAAAACTATTGTCTGATGATCACTTTCGCTATTTTTCTGTCCCTGATCATGCTGGTGGAGGTCGCCGCTGCAATTGCCGGATACGTGGGAGGCTCTGGCCCAGTCCGGAGATATCAGGGAGGACCACTGGGCGTGCGAGGAGGGAGCTCCTCTCGAACCCCAAGTGACAAGCCTGTGGCCCACGTGGTCGCTAATCCTCAGGCAGAAGGGCAGCTGCAGTGGCTGAACAGGCGCGCAAATGCCCTGCTGGCCAACGGAGTGGAACTGAGGGATAATCAGCTGGTGGTCCCAAGTGAGGGCCTGTACCTGATCTATTCACAGGTGCTGTTCAAAGGGCAGGGATGCCCCAGCACACACGTGCTGCTGACCCATACAATCTCAAGGATTGCTGTGA It is encoded by [[ID=]], SEQ ID NO: 352 GCTACCAGACAAAGGTCAACCTGCTGTCTGCCATCAAAAGTCCCTGTCAGCGCGAGACTCCTGAAGGGGCTGAGGCAAAGCCATGGTACGAGCCCATCTATCTGGGAGGCGTGTTTCAGCTGGAAAAAGGAGACAGACTGTCAGCAGAGATTAATAGGCCCGACTACCTGGATTTCGCCGAAAGCGGCCAGGTGTATTTTGGGATCATTGCTCTGGGAGGCAGCGGAGAGCAGAAGCTGATCTCTGAGGAAGATCTGGGAGGACCACTGGGGGTGCGCGGAGGAGGGGGAAGCGGACCTAAGAACAATCACACCGCTTCCATTCTGGACCGAATGCAGGCAGATTTCAAATGCTGTGGCGCCGCTAACTACACAGACTGGGAAAAGATCCCTAGTATGTCAAAAAACCGGGTGCCAGATTCCTGCTGTATTAATGTGACCGTCGGCTGCGGGATCAACTTTAATGAGAAGGCCATTCATAAAGAAGGCTGCGTGGAGAAGATCGGCGGGTGGCTGCGGAAAAATGTCCTGGTGGTCGCAGCAGCTGCACTGGGAATCGCCTTCGTGGAAGTCCTGGGGATCGTGTTCGCTTGCTGTCTGGTCAAAAGCATTAGATCCGGCTATGAAGTGATGTAATGTACAA.

[0382] LeGo-CD63-DL1-2L-TNFα-Myc-CD63-Cyt MAVEGGMKCVKCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQGGPLGVRGGSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGPLGVRGGGGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM (Sequence ID: 368).

[0383] That is cDNA: GGATCCGCCGCCACCATGGCAGTGGAAGGCGGGATGAAGTGCGTCAAATGTGGCGCCTGCAAGGAGAACTACTGTCTGATGATCACCTTCGCTATTTTTCTGAGCCTGATCATGCTGGTGGAAGTCGCCGCTGCAATTGCAGGATACGTGGGAGGCAGCGGACCCGTCCGGAGATATCAGAGCTCCTCTCGAACACCATCCGACAAGCCTGTGGCTCACGTGGTCGCAAATCCACAGGCAGAGGGACAGCTGCAGTGGCTGAACAGGCGAGCCAATGCTCTGCTGGCCAACGGAGTGGAACTGAGGGATAATCAGCTGGTGGTCCCCAGTGAGGGCCTGTACCTGATCTATTCACAGGTGCTGTTCAAAGGACAGGGCTGTCCTTCTACTCACGTGCTGCTGACCCATACAATCTCAAGGATTGCCGTGAGCTATCAGACTAAGGTCAACCTGCTGTCTGCTATCAAAAGTCCTTGCCAGCGAGAGACCCCAGAAGGAGCAGAGGCCAAGCCATGGTACGAGCCCATCTATCTGGGAGGCGTGTTTCAGCTGGAAAAAGGAGACAGACTGTCTGCAGAGATTAATAGGCCTGACTACCTGGATTTCGCCGAAAGTGGCCAGGTGTATTTTGGGATCATTGCTCTGGGAGGCAGCGGAGAGCAGAAGCTGATCTCTGAGGAAGATCTGGGGGGAAGCGGACCAAAGAACAATCACACAGCTTCCATTCTGGACCGAATGCAGGCAGATTTCAAATGCTGTG This is coded by GCGCCGCTAACTACACTGACTGGGAAAAGATCCCAAGTATGTCAAAAAACCGGGTGCCCGATAGCTGCTGTATTAATGTGACAGTCGGGTGTGGAATCAACTTTAATGAGAAGGCCATTCATAAAGAAGGCTGCGTGGAGAAGATCGGCGGGTGGCTGCGGAAAAATGTCCTGGTGGTCGCAGCAGCTGCACTGGGAATCGCCTTCGTGGAAGTCCTGGGGATCGTGTTCGCTTGCTGTCTGGTCAAAAGCATTAGATCCGGCTATGAAGTGATGTAATGTACAA (Sequence ID: 353).

[0384] LeGO-CD69Cyt-TM-GrnzB-TNFα-Myc-GrnzB-a-CD19sc-Stalk-CD28-CD3Z (Sequence number: 373).

[0385] That is cDNA: GGATCCATGTCATCCGAGAACTGCTTTGTCGCCGAAAACTCATCACTGCACCCCGAATCAGGCCAGGAAAACGACGCAACCTCCCCCCACTTCTCAACTAGACACGAGGGCAGCTTCCAGGTGCCAGTGCTGTGCGCCGTGATGAACGTGGTGTTTATCACCATTCTGATCATTGCTCTGATCGCCCTGAGCGTGGGGCAGTACAATGGCGGGTCCGGACCCGTGCGGAGATATCAGGGAGGCTCCGGAGGAGGAGAGGAAGAGGAAGAGGTGGAGGCCGACTCTGAAGAGGAAGAGGAAGAGGAATCCGGGGGATCTGGCAGCTCCTCTAGAACCCCTTCCGATAAGCCAGTGGCTCATGTGGTGGCCAACCCTCAGGCTGAGGGACAGCTGCAGTGGCTGAACAGGAGGGCTAATGCTCTGCTGGCTAACGGAGTGGAGCTGAGGGACAATCAGCTGGTGGTGCCTTCTGAAGGGCTGTACCTGATCTATAGTCAGGTGCTGTTCAAAGGGCAGGGATGCCCATCTACACACGTGCTGCTGACCCATACAATCAGTCGCATTGCCGTGAGCTACCAGACCAAGGTGAACCTGCTGAGCGCCATCAAGAGCCCTTGTCAGCGGGAAACACCAGAGGGGG

[0386] LeGO-CD69Cyt-TM-II-9-TNFα-Myc-9-CD28TM-Cyt MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALSVGQYNGGSGPVRRYQGGSGGGPLGMTSGGSGSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGPLGMTSGGSGCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLL (SEQ ID NO: 377).

[0387] It is cDNA:

[0388] LeGO-CD69Cyt-TM-II-TNFα-Myc-CD28TM-Cyt MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALSVGQYNGGSGPVRRYQSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGSGCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLL (SEQ ID NO: 370)

[0389] It is cDNA: It is encoded by GGATCCGCCGCCACCATGAGTTCCGAAAACTGCTTTGTCGCCGAGAATAGTAGTCTGCACCCCGAATCAGGACAGGAGAATGACGCAACCAGCCCCCACTTTTCAACAAGGCACGAAGGGAGCTTCCAGGTCCCTGTGCTGTGCGCAGTGATGAACGTGGTCTTTATCACTATTCTGATCATTGCTCTGATCGCACTGAGTGTCGGACAGTACAATGGCGGGTCAGGCCCCGTGCGGAGATATCAGAGCTCCTCTCGCACCCCTTCCGACAAGCCAGTGGCCCATGTGGTCGCTAACCCTCAGGCAGAGGGACAGCTGCAGTGGCTGAACAGGCGAGCCAATGCTCTGCTGGCTAACGGCGTGGAACTGCGAGATAATCAGCTGGTGGTCCCTAGCGAGGGGCTGTACCTGATCTATTCCCAGGTCCTGTTCAAAGGGCAGGGATGCCCATCTACACACGTGCTGCTGACCCATACAATCTCTAGAATTGCCGTCAGTTACCAGACTAAGGTGAACCTGCTGAGCGCCATCAAGTCACCATGTCAGAGGGAGACACCCGAAGGAGCAGAGGCCAAGCCCTGGTACGAGCCTATCTATCTGGGAGGCGTGTTTCAGCTGGAAAAAGGCGACCGACTGTCTGCCGAGATTAATCGGCCAGACTACCTGGATTTCGCTGAAAGTGGCCAGGTGTATTTTGGGATCATTGCACTGGGAGGCAGCGGAGAGCAGAAGCTGATCTCTGAGGAAGATCTGGGAGGGAGCGGATGCCCATCCCCTCTGTTCCCAGGACCCAGCAAACCCTTTTGGGTCCTGGTGGTCGTGGGAGGCGTGCTGGCATGTTATAGCCTGCTGGTCACAGTGGCTTTCATTATCTTTTGGGTCCGCTCTAAACGGTCTCGGCTGCTGTAATGTACAA (SEQ ID NO: 356).

[0390] LeGO-CD69Cyt-TM-GrzB-TNFα-Myc-GrzB-a-CD19sc-Stalk-CD28-CD3z MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALSVGQYNGGSGPVRRYQGGSGGGEEEEEVEADSEEEEEEESGGSGSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALEQKLISEEDLGGSGEEEEEVEADSEEEEEEEGGSGGGSGGGSGPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 373)

[0391] It is cDNA: It is encoded by AACATCTGTGCCCTAGTCCACTGTTCCCCGGCCCTTCAAAGCCCTTTTGGGTGCTGGTGGTGGTGGGAGGGGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCTTTCATCATCTTCTGGGTGCGGAGCAAACGCAGTCGGCTGCTGCACTCTGACTATATGAACATGACCCCAAGACGGCCCGGACCAACAAGGAAGCATTACCAGCCATATGCTCCTCCAAGGGACTTCGCTGCTTACAGAAGCAGGGTGAAATTTTCACGCAGCGCCGATGCTCCTGCCTACCAGCAGGGACAGAATCAGCTGTATAACGAGCTGAATCTGGGCAGGCGCGAAGAGTATGACGTGCTGGATAAGCGGAGAGGACGGGACCCCGAAATGGGAGGCAAGCCAAGGCGCAAAAACCCCCAGGAGGGCCTGTATAACGAACTGCAGAAGGACAAAATGGCTGAGGCCTATAGCGAAATCGGAATGAAGGGCGAGCGGAGAAGGGGCAAAGGGCACGATGGGCTGTACCAGGGACTGTCCACCGCCACTAAAGATACCTATGATGCTCTGCACATGCAGGCTCTGCCACCCCGATAATGTACA (SEQ ID NO: 357).

[0392] LeGO-CD69Cyt-TM-GrzB-nLuc-Myc-GrzB-a-CD19sc-Stalk CD28-CD3z (Sequence number: 358)

[0393] That is cDNA: GCCAGTCCTCCGATTGACTGAGTCGCCCGGATCCCGCCACCATGAGCTCTGAGAACTGCTTCGTGGCCGAGAACAGCAGCCTCCACCCCGAGAGTGGTCAGGAGAACGACGCCACCAGCCCCCACTTCAGCACAAGACACGAGGGCTCATTCCAGGTGCCTGTGCTGTGCGCCGTGATGAACGTGGTGTTCATCACCATCCTGATCATCGCCCTGATTGCCCTTTCTGTGGGCCAATACAATGGCGGCTCCGGTCCTGTGCGCAGATACTCTAGACAGGG It is encoded by TGAAGTTCTCCAGATCCGCCGACGCCCCTGCTTACCAACAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCAGAAGAGAGGAGTACGACGTGCTGGATAAGCGGAGAGGAAGAGACCCTGAGATGGGCGGCAAGCCCCGGCGGAAGAACCCTCAGGAGGGCCTGTATAACGAGCTGCAAAAAGATAAGATGGCCGAGGCTTATAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGGGGCAAAGGCCACGACGGCCTGTACCAGGGACTGAGCACAGCCACAAAGGACACCTACGACGCTCTGCACATGCAGGCCCTGCCTCCTAGATAATGTACAAGTAAAGCGGCCGGCC (SEQ ID NO: 382).

[0394] LeGO-CD69Cyt-TM-GrzB-nLuc-Myc-GrzB-CD28-CD3z (Delta-sc, GAGE B) (Sequence ID: 374)

[0395] That is cDNA: ATGAGCTCTGAGAACTGCTTCGTGGCCGAGAACAGCAGCCTCCACCCCGAGAGTGGTCAGGAGAACGACGCCACCAGCCCCCACTTCAGCACAAGACACGAGGGCTCATTCCAGGTGCCTGTGCTGTGCGCCGTGATGAACGTGGTGTTCATCACCATCCTGATCATCGCCCTGATTGCCCTTTCTGTGGGCCAATACAATGGCGGCTCCGGTCCTGTGCGCAGATACTCTAGACAGGGCGGCTCTGGCGGAGGCGAAGAGGAAGAAGAGGTGGAAGCCGATAGCGAGGAAGAAGAAGAAGAGGAGAGCGGCGGCTCCGGCAGCAGCAGCTCTAGAGTGTTCACCCTCGAAGATTTCGTGGGCGACTGGAGACAGACCGCCGGCTACAACCTGGACCAGGTGCTGGAACAGGGCGGCGTGAGCTCTCTGTTTCAGAATCTGGGGGTTAGCGTGACACCAATCCAGAGGATCGTGCTGTCCGGCGAGAACGGGCTGAAGATCGACATCCACGTGATCATTCCATACGAGGGCCTGTCTGGCGATCAGATGGGCCAGATCGAGAAGATCTTCAAGGTGGTGTACCCTGTAGACGACCACCATTTTAAGGTGATCCTGCACTACGGCACCCTGGTCATCGACGGCGTGACACCCAACATGATCGACTACTTCGGCAGACCATATGAGGGTATTGCCGTGTTTGATGGAAAGAAGATCACAGTCACCGGCACCCTGTGGAACGGCAACAAGATTATCGATGAAC

[0396] CD69 Cyt-TM-nLuc-Myc-a-CD19sc-Stalk-CD28-CD3z (Delta-GrzB, GAGE A) MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALSVGQYNGGSGPVRRYSRVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAEQKLISEEDLLEPRGGSGVNPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSVNSGGGGSGGGGSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 375)

[0397] It is cDNA: It is encoded by GGGTCCTGGTGGTGGTCGGCGGCGTGCTGGCGTGCTACAGCCTGCTCGTGACCGTGGCCTTCATCATTTTCTGGGTGCGGAGCAAACGGAGCAGACTGCTGCATAGCGATTACATGAACATGACCCCAAGAAGACCTGGGCCAACCAGAAAGCACTACCAGCCTTACGCCCCCCCCAGAGATTTCGCCGCTTATAGAAGCCGGGTGAAGTTCTCCAGATCCGCCGACGCCCCTGCTTACCAACAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCAGAAGAGAGGAGTACGACGTGCTGGATAAGCGGAGAGGAAGAGACCCTGAGATGGGCGGCAAGCCCCGGCGGAAGAACCCTCAGGAGGGCCTGTATAACGAGCTGCAAAAAGATAAGATGGCCGAGGCTTATAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGGGGCAAAGGCCACGACGGCCTGTACCAGGGACTGAGCACAGCCACAAAGGACACCTACGACGCTCTGCACATGCAGGCCCTGCCTCCTAGAtaaTGTACA (SEQ ID NO: 360).

[0398] CD69Cyt-TM-GrzB(IEPD)-nLuc-Myc-GrzB(IEPD)-a-CD19sc-Stalk-CD28-CD3z (Sequence number: 383)

[0399] That is cDNA: GCCAGTCCTCCGATTGACTGAGTCGCCCGGATCCCGCCACCATGAGCTCTGAGAACTGCTTCGTGGCCGAGAACAGCAGCCTCCACCCCGAGAGTGGTCAGGAGAACGACGCCACCAGCCCCCACTTCAGCACAAGACACGAGGGCTCATTCCAGGTGCCTGTGCTGTGCGCCGTGATGAACGTGGTGTTCATCACCATCCTGATCATCGCCCTGATTGCCCTTTCTGTGGGCCAATACAATGGCGGCTCCGGTCCTGTGCGCAGATACTCTAGACAGGGCGGCTCTGGCGGAGGCATCGAGCCTGACAGCGGCGGCTCCGGCTCTAGAGTGTTCACCCTCGAAGATTTCGTGGGCGACT It is coded by CTGTATAACGAGCTGCAAAAAGATAAGATGGCCGAGGCTTATAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGGGGCAAAGGCCACGACGGCCTGTACCAGGGACTGAGCACAGCCACAAAGGACACCTACGACGCTCTGCACATGCAGGCCCTGCCTCCTAGATAATGTACAAGTAAAGCGGCCGGCC (Sequence ID: 361).

[0400] CD69Cyt-TM-GrzB(SASA)-nLuc-Myc-GrzB(SASA)-a-CD19sc-Stalk-CD28-CD3z (Sequence number: 376)

[0401] That is cDNA: GCCAGTCCTCCGATTGACTGAGTCGCCCGGATCCCGCCACCATGAGCTCTGAGAACTGCTTCGTGGCCGAGAACAGCAGCCTCCACCCCGAGAGTGGTCAGGAGAACGACGCCACCAGCCCCCACTTCAGCACAAGACACGAGGGCTCATTCCAGGTGCCTGTGCTGTGCGCCGTGATGAACGTGGTGTTCATCACCATCCTGATCATCGCCCTGATTGCCCTTTCTGTGGGCCAATACAATGGCGGCTCCGGTCCTGTGCGCAGATACTCTAGACAGGGCGGCTCTGGCGGAGGCGCCCAGGGCGTGATTAGCGCCAGCGCCAGCAATCTGGACGACTTTTACAGCGGCGGCTCCGGCTCTAGAGTGTTCACCCTCGAAGATTTCGTGGGCGACTGGAGACAGACCGCCGGCTACAACCTGGACCAGGTGCTGGAACAGGGCGGCGTGAGCTCTCTGTTTCAGAATCTGGGGGTTAGCGTGACACCAATCCAGAGGATCGTGCTGTCCGGCGAGAACGGGCTGAAGATCGACATCCACGTGATCATTCCATACGAGGGCCTGTCTGGCGATCAGATGGGCCAGATCGAGAAGATCTTCAAGGTGGTGTACCCTGTAGACGACCACCATTTTAAGGTGATCCTGCACTACGGCACCCTGGTCATCGACGGCGTGACACCC

[0402] CD69 Cyt-TM-GrzB(ADKG)-TNFα-Myc-GrzB(ADKG)-α-CD19sc-Stalk-CD28-CD3z MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALSVGQYNGGSGPVRRYSRQGGSGGGLEADKGKLEYDSGGSGSRVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAEQKLISEEDLLEGGSGLEADKGKLEYDGGSGGGSGLEPRGGSGVNPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSVNSGGGGSGGGGSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 384)

[0403] それは、cDNA: GCCAGTCCTCCGATTGACTGAGTCGCCCGGATCCCGCCACCATGAGCTCTGAGAACTGCTTCGTGGCCGAGAACAGCAGCCTCCACCCCGAGAGTGGTCAGGAGAACGACGCCACCAGCCCCCACTTCAGCACAAGACACGAGGGCTCATTCCAGGTGCCTGTGCTGTGCGCCGTGATGAACGTGGTGTTCATCACCATCCTGATCATCGCCCTGATTGCCCTTTCTGTGGGCCAATACAATGGCGGCTCCGGTCCTGTGCGCAGATACTCTAGACAGGGCGGCTCTGGCGGAGGCCTGGAGGCCGACAAGGGCAAGCTGGAGTACGATAGCGGCGGCTCCGGCTCTAGAGTGTTCACCCTCGAAGATTTCGTGGGCGACTGGAGACAGACCGCCGGCTACAACCTGGACCAGGTGCTGGAACAGGGCGGCGTGAGCTCTCTGTTTCAGAATCTGGGGGTTAGCGTGACACCAATCCAGAGGATCGTGCTGTCCGGCGAGAACGGGCTGAAGATCGACATCCACGTGATCATTCCATACGAGGGCCTGTCTGGCGATCAGATGGGCCAGATCGAGAAGATCTTCAAGGTGGTGTACCCTGTAGACGACCACCATTTTAAGGTGATCCTGCACTACGGCACCCTGGTCATCGACGGCGTGACACCCAACATGATCGACTACTTCGGCAGACCATATGAGGGTATTGCCGTGTTTGATGGAAAGAAGATCACAGTCACCGGCACCCTGTGGAACGGCAACAAGATTATCGATGAACGGCTGATCAACCCCGACGGCTCTCTGCTGTTTAGAGTGACCATCAATGGCGTGACAGGCTGGAGACTGTGCGAGAGAATCCTGGCTGAGCAGAAACTGATCAGCGAGGAAGACCTGCTCGAGGGCGGTAGCGGCCTGGAGGCCGAC

[0404] Example 19: Preparation of a lentiviral vector

[0405] A transfer vector containing the relevant gene was transfected into 293T cells along with lentiviral packaging vectors (pMDLg / pRRE and pRSV-Rev), and the envelope vector (phCMV-VSV-G) and viral supernatant were recovered.

[0406] Small-scale generation of VSV-G pseudotyped lentiviral vectors

[0407] This method describes the generation of a VSV-G pseudotyped lentiviral vector in a 6-well plate using a calcium phosphate transfection kit. The amount of viral supernatant per well was 4 ml, and the viral concentration depended on the vector used.

[0408] material [Table 6]

[0409] Common materials:

[0410] T75 flask, cell culture pipette, micropipette and tips, 1.5 ml microcentrifuge tube, 5 ml syringe, trypan blue, Virkon and M-ytdes.

[0411] plasmid Vector: Dependent on selection Gag-Pol plasmid: pMDLg / pRRE Rev plasmid: pRSV-Rev Envelope plasmid: phCMV-VSV-G

[0412] procedure

[0413] Maintenance of HEK293FT cells

[0414] Cells were maintained by dividing them into a 1:5 to 1:4 ratio every other day and holding them in T75 or T150 flasks. After thawing, the cells were cultured for at least three passages before being used for virus generation, allowing them to recover and initiate exponential growth. The use of multiple passaged cells is not recommended as it negatively impacts virus generation.

[0415] Day 1: Sowing & Transfection - 500,000 cells / well were seeded in complete growth medium. - The culture medium was pipetteed from the flask. - The cells were washed with 10 ml of PBS at room temperature. - Add 1 ml (T75) or 2 ml (T150) of TrypLE Express and incubate at 37°C for 5 minutes. - After incubation, 10-20 ml of complete medium was added to the flask, and the mixture was completely resuspended. The clumps were then dissolved by pipetting up and down several times. - Cells were counted using trypan blue to prepare a cell suspension with a concentration of 250,000 cells / ml. - 2 ml of this suspension was placed in wells (a 6-well plate with poly-L-lysine coating). One well was prepared for each vector. - The plates were placed in the incubator for at least 7-8 hours.

[0416] After incubation, the cells were transfected. After confirming cell attachment, an optimal confluence of 80% was achieved. If the cells were viable and at an appropriate density, the plate was returned to the incubator to prepare the transfection mix. If the confluence was below 60%, transfection was stopped. - A 1 ml complete growth medium was prepared by adding chloroquine to a final concentration of 25 μM. This was placed in a preheated incubator. It is important that the components of the callisme phosphate precipitation kit are at room temperature before the start of transfection.

[0417] - The plasmid mixture was prepared in a microcentrifuge tube as follows (total 4 μg): - LeGO-iG2 vector (including transgene) 2μg - pMDLg / pRRE(Gag / Pol) 1μg - pRSV-REV(Rev) 0.75μg - phCMV-VSV-G (envelope) 0.25μg - The plasmids were mixed and the volume was adjusted to 54 μl with ddH2O. - 6 μl of 2.5 M CaCl2 solution was added to the DNA mixture. - 60 μl of 2×HeBS buffer was placed in a separate microcentrifuge tube. CaCl2 / DNA mixture was added and vortexed. - This mixture was allowed to stand at room temperature for 15 minutes. Standing for longer than 30 minutes is not acceptable as it may reduce transfection efficiency. During these 15 minutes, the dish was removed, the medium was discarded, and 1 ml of preheated complete growth medium containing 25 μM chloroquine was added. - After 15 minutes of incubation, 120 μl of the mixture was added dropwise to the wells while gently stirring the dish in a circular motion. - I closed the lid on the dish and placed it back in the incubator for another 10-12 hours.

[0418] Day 2: Culture medium change - 10-12 hours after transfection: - The culture medium containing the transfection mix and chloroquine was aspirated from the wells and discarded. - 2 ml of complete growth medium was added per well. The medium was preheated to at least room temperature, preferably 37°C. The cells were then placed in an incubator.

[0419] Day 3: Recovery of the supernatant 1 - 24 hours after the culture medium change: - Cells were checked for GFP expression under a UV microscope. Transfection efficiency must exceed 90%. - A 0.45 μm filter, a 5 ml syringe, a 5 ml microcentrifuge tube, and a 1.5 ml tube were prepared for each well. - The culture medium was collected from the dish using a 5 ml syringe. A filter was attached, and the supernatant was filtered into a 5 ml microcentrifuge tube. Filtration should be performed gently, without creating air bubbles or applying excessive force. After completion, the syringe and filter were placed in Virkon solution. - A 100 μl aliquot from the filtered supernatant was taken into a 1.5 ml microcentrifuge tube (for viral titer measurement). If desired, the remaining supernatant was collected and frozen at -80°C for long-term storage. - 2 ml of complete growth medium was added per dish. The medium was preheated to at least room temperature, preferably 37°C. The cells were then placed in an incubator.

[0420] Day 4: Recovery of the supernatant 2 - 48 hours after the culture medium change: - The viral supernatant was collected using the same method as the previous day. - The plate was discarded.

[0421] cell line creation

[0422] The lentiviral particles recovered using the procedure described above were transduced into K562 and KHYG-1 cells, sorted, and grown. The resulting cells were tested for target gene expression using either the corresponding antibody or a fluorescently labeled protein. 1. Prepare culture medium RPMI 10% FBS = 400 μl / well. 2. A lentiviral vector and transduction medium were added. The date, name, cell type, and the virus used for transduction were labeled on the 3.24-well plate. 4. The centrifuge temperature was set to 32°C. 5. Cells were counted using trypan blue, and a cell suspension containing 10⁶ cells / ml in culture medium + 10% FBS was prepared. 6.50,000 cells were dispensed in 250 μl portions into 24-well plates. 7. The required protamine sulfate stock solution was extracted to a final concentration of 8 μg / ml. 8. Refreezing / thawing of the undiluted solution was avoided. 9. The culture medium was added according to the calculations. 10. The virus was kept on dry ice until use. The required amount of virus was rapidly thawed. 11. The viruses were carefully mixed to minimize contact with the air. 12. The calculated amount of virus was added to each well. 13. In the case of KHYG-1 cells, protamine sulfate (8 μg / ml) and IL-2 (100 IU / ml) were pipetted into each well. 14. The cells were carefully mixed by pipetting up and down. 15. The plates were centrifuged at 1000 × g and 32°C for 1 hour without interruption. 16. Remove the plate and incubate it in an incubator for 4 hours to overnight (the structure to be tested). Incubation was performed for a period of time (depending on the structure and viral titer). 17. At the end of incubation, the plate was centrifuged again at 1000 × g and 32°C for 1 hour. 18. Carefully remove 80% of the culture medium from all wells and fill with 500 μl of fresh pre-warmed medium containing serum. 19. The plate was placed back into the incubator.

[0423] Day 1 & 2: Cells were checked under a microscope to search for colonies.

[0424] Day 3: Cells were analyzed using flow cytometry.

[0425] HEK293 NF-κB Luciferase Assay Procedure 1. The day before the assay, NF-κB reporter (Luc)-HEK293 cells were harvested and seeded at a density of 20,000 cells per well in 100 μl of assay medium (DMEM + 10% FBS + 1% P / S) in a 96-well microplate. The cells were incubated overnight at 37°C and 5% CO2 for approximately 24 hours. 2. The following day, the culture medium on the 96-well microplate was aspirated, and 100 μL of supernatant from GAGE-expressing cells cultured alone or co-cultured with target cells was added to the corresponding wells and incubated at 37°C and 5% CO2 for 6 hours. 3. After 6 hours, the culture medium was aspirated from the cells, and 100 μL of 0.1% Triton buffer was added to each sample well. 4. Gently shake the plate several times to ensure that the cells are completely covered with lysis buffer. 5. Incubate at room temperature for 10 minutes to dissolve. 6. 30 μL of the lysate was transferred to a new white opaque 96-well plate for the luciferase assay system. The GLOMAX instrument was automatically set to add 50 μL of D-luciferase or nanoGLOW assay system (Promega) per well and measure luminescence using a luminometer according to the manufacturer's instructions. The resulting luciferase activity data was normalized to the percentage of intracellular GAGE ​​expression or MFI.

[0426] Flow cytometry

[0427] Staining and washing were performed in flow cytometry acquisition buffer. Single cell suspensions were incubated on ice with blocking reagents for 10 minutes, then stained with antibody, and viability staining was performed on ice for 30 minutes. Samples were analyzed using a Cytoflex flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (TreeStar, Ashland, Oregon). An AriaFusion instrument (BD Biosciences) was used for sorting. Sorted cells were cultured in antibiotic-containing medium for 2 weeks. Subsequently, the cells were cultured without antibiotics.

[0428] Example 20

[0429] Experiments were conducted in relation to the following three caspase sequences and constructs:

[0430] CD69Cyt-TM-Cas3(DEVD)-TNFα-Myc-Cas3(DEVD)-a-CD19sc-Stalk-CD28-CD3z

[0431] The Cas3(DEVDG) sequence is given in bold, uppercase, and underlined.

[0432] DNA sequence TCTGGGTCCTGGTGGTGGTCGGCGGCGTGCTGGCGTGCTACAGCCTGCTCGTGACCGTGGCCTTCATCATTTTCTGGGTGCGGAGCAAACGGAGCAGACTGCTGCATAGCGATTACATGAACATGACCCCAAGAAGACCTGGGCCAACCAGAAAGCACTACCAGCCTTACGCCCCCCCCAGAGATTTCGCCGCTTATAGAAGCCGGGTGAAGTTCTCCAGATCCGCCGACGCCCCTGCTTACCAACAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCAGAAGAGAGGAGTACGACGTGCTGGATAAGCGGAGAGGAAGAGACCCTGAGATGGGCGGCAAGCCCCGGCGGAAGAACCCTCAGGAGGGCCTGTATAACGAGCTGCAAAAAGATAAGATGGCCGAGGCTTATAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGGGGCAAAGGCCACGACGGCCTGTACCAGGGACTGAGCACAGCCACAAAGGACACCTACGACGCTCTGCACATGCAGGCCCTGCCTCCTAGAtaa(SEQ ID NO: 386)

[0433] CD69 Cyt-TM-Cas3(DEVD)-TNFα-Myc-Cas3(DEVD)-a-CD19sc-Stalk-CD28-CD3z

[0434] Protein sequence MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALSVGQYNGGSGPVRRYSRQGGSGGGDEVDGSGGSGSSSSRVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAEQKLISEEDLLEGGSGDEVDGGGSGGGSGLEPRGGSGVNPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSVNSGGGGSGGGGSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 387)

[0435] CD69 Cyt-TM-Cas3(EAALVDMVNDG)-TNFα-Myc-Cas3(EAALVDMVNDG)-a-CD19sc-Stalk-CD28-CD3z

[0436] The Cas3(EAALVDMVNDG(Sequence ID: 388)) sequence is given in bold, uppercase, and underlined.

[0437] DNA sequence GGTGGTCGGCGGCGTGCTGGCGTGCTACAGCCTGCTCGTGACCGTGGCCTTCATCATTTTCTGGGTGCGGAGCAAACGGAGCAGACTGCTGCATAGCGATTACATGAACATGACCCCAAGAAGACCTGGGCCAACCAGAAAGCACTACCAGCCTTACGCCCCCCCCAGAGATTTCGCCGCTTATAGAAGCCGGGTGAAGTTCTCCAGATCCGCCGACGCCCCTGCTTACCAACAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCAGAAGAGAGGAGTACGACGTGCTGGATAAGCGGAGAGGAAGAGACCCTGAGATGGGCGGCAAGCCCCGGCGGAAGAACCCTCAGGAGGGCCTGTATAACGAGCTGCAAAAAGATAAGATGGCCGAGGCTTATAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGGGGCAAAGGCCACGACGGCCTGTACCAGGGACTGAGCACAGCCACAAAGGACACCTACGACGCTCTGCACATGCAGGCCCTGCCTCCTAGAtaa(SEQ ID NO: 389)

[0438] > CD69Cyt-TM-Cas3(EAALVDMVNDG)-TNFα-Myc-Cas3(EAALVDMVNDG)-a-CD19sc-Stalk-CD28-CD3z

[0439] Protein sequence MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALSVGQYNGGSGPVRRYSRGGSGGGEAALVDMVNDGSGGSGGSRVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAEQKLISEEDLLEGGSGGGEAALVDMVNDGSGGSGGLEPRGGSGVNPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSVNSGGGGSGGGGSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 390)

[0440] CD69 Cyt-TM-Cas3(EDYGRDSGPP)-TNFα-Myc-Cas3(EDYGRDSGPP)-a-CD19sc-Stalk-CD28-CD3z

[0441] The Cas3(EDYGRDSGPP(Sequence ID: 391)) sequence is given in bold, uppercase, and underlined.

[0442] DNA sequence CCTGGTGGTGGTCGGCGGCGTGCTGGCGTGCTACAGCCTGCTCGTGACCGTGGCCTTCATCATTTTCTGGGTGCGGAGCAAACGGAGCAGACTGCTGCATAGCGATTACATGAACATGACCCCAAGAAGACCTGGGCCAACCAGAAAGCACTACCAGCCTTACGCCCCCCCCAGAGATTTCGCCGCTTATAGAAGCCGGGTGAAGTTCTCCAGATCCGCCGACGCCCCTGCTTACCAACAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCAGAAGAGAGGAGTACGACGTGCTGGATAAGCGGAGAGGAAGAGACCCTGAGATGGGCGGCAAGCCCCGGCGGAAGAACCCTCAGGAGGGCCTGTATAACGAGCTGCAAAAAGATAAGATGGCCGAGGCTTATAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGGGGCAAAGGCCACGACGGCCTGTACCAGGGACTGAGCACAGCCACAAAGGACACCTACGACGCTCTGCACATGCAGGCCCTGCCTCCTAGAtaa(SEQ ID NO: 392)

[0443] CD69 Cyt-TM-Cas3(EDYGRDSGPP)-TNFα-Myc-Cas3(EDYGRDSGPP)-a-CD19sc-Stalk-CD28-CD3z

[0444] Protein sequence MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALSVGQYNGGSGPVRRYSRGGSGGGEDYGRDSGPPSGGSGSSSSRVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAEQKLISEEDLLEGGSGGGEDYGRDSGPPSGGSGSSSLEPRGGSGVNPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSVNSGGGGSGGGGSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 393)

[0445] D69Cyt-TM-Cas3(TPSEPDSGQGPPQ)-TNFα-Myc-Cas3(TPSEPDSGQGPPQ)-a-CD19sc-Stalk-CD28-CD3z

[0446] The Cas3(TPSEPDSGQGPPQ(Sequence ID: 394)) sequence is given in bold, uppercase, and underlined.

[0447] DNA sequence (Sequence number: 395)

[0448] CD69Cyt-TM-Cas3(TPSEPDSGQGPPQ)-TNFα-Myc-Cas3(TPSEPDSGQGPPQ)-a-CD19sc-Stalk-CD28-CD3z

[0449] The Cas3(TPSEPDSGQGPPQ(Sequence ID: 394)) sequence is given in bold, uppercase, and underlined.

[0450] Protein sequence (Sequence number: 396)

Claims

1. A peptide delivery system for delivering a peptide payload to a target cell or target gene locus, wherein the peptide delivery system is The exogenous polypeptide comprises cells, extracellular vesicles, or lipid-containing particles to which the exogenous polypeptide is immobilized, A first and second transmembrane domain connected by a loop region, wherein the loop region includes a first cleavage site, a peptide payload, and a second cleavage site; Depending on the circumstances, the peptide delivery system may include a targeting portion for targeting the target cell or the target gene locus, A peptide delivery system including this.

2. The peptide delivery system according to claim 1, wherein the first and / or second cleavage sites include a proteolytic cleavage substrate, preferably a schedase cleavage site, an MMP cleavage site, a GrzB cleavage site, and a Casp3 cleavage site.

3. The peptide delivery system according to claim 2, configured to release the peptide payload upon cleavage by a protease, preferably a shedase, which may be endogenous or exogenous (for example, which may be included as a component of the peptide delivery system).

4. The peptide delivery system according to claim 3, wherein the schedase is present at the target gene locus and / or released by the target cell.

5. The peptide delivery system according to claim 3 or 4, wherein the schedase is a full-time schedase selected from ADAM protease (metalloprotease), BACE protease, serine protease granzyme B, and site 1 protease.

6. The peptide delivery system according to claim 5, wherein the ADAM protease includes 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 according to claim 3 or 4, wherein the schidase is a part-time schidase selected from meprin B, MT-MMP (membrane-type matrix metalloproteinase), proprotein converterase, transmembrane serine protease, matrix metalloproteinase, regmine, and cathepsin S and L.

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

9. The peptide delivery system according to claim 7, wherein the proprotein converterase is selected from PCSK1 / 3, KCSK2, furin, PCSK4, PSCK5 / 6, PACE4, PCSK7, and PCSK9.

10. The peptide delivery system according to claim 7, wherein the transmembrane serine protease is selected from membrane-anchored type II proteases including matryptase, matryptase-2, matryptase-3, polymerase-1, choline, hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, spinesin, enteropeptidase, HAT, DESCL1, TMPRSS11A, HAT-L4, and HAT-L5.

11. The peptide delivery system according to claim 7, wherein the matrix metalloproteinase is selected from soluble proteases comprising 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 according to claim 11, wherein the matrix metalloproteinase is selected from MMP2, MMP9 and / or MMP25.

13. The peptide delivery system according to claim 12, wherein the MMP2 is selected from sequence numbers 2 to 6.

14. The peptide delivery system according to claim 12, wherein the MMP9 is selected from sequence numbers 7 to 8.

15. The peptide delivery system according to claim 12, wherein the MMP25 is selected from SEQ ID NOs: 9-17, 18 or 155, and 19-29.

16. The peptide delivery system according to any one of claims 1 to 15, wherein the transmembrane domain comprises one or more transmembrane domains from 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.

17. 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 costimulator (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signal transduction lymphocyte activator molecule (SLAM protein), costimulatory ligand (PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand) (ICOS-L), intercellular adhesion molecules (ICAM, CD30L, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KI RDS2, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD8 Alpha, CD8 Beta, IL-2R Beta, IL-2R Gamma, IL-7R Alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, IT GAM, CD11b, ITGAX, CD11e, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRA NCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (TACTILE), CEACAM1, CRTThe peptide delivery system according to claim 1, further comprising intracellular or extracellular domains selected from or derived from ligands that specifically bind to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

18. a targeting moiety, the targeting moiety being BCMA, MUC16 (also known as CA125), EGFR, EGFRvIII, MUCI, Flt-3, WT-1, CD38, CD70, CD90, CD133, MH C-WTI, TSPANI0, MHC-PRAME, MHC-NY-ESOI, HER2 (ERBB2), CA-IX (carbonic anhydrase IX), LIVI, ADAM10, 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 Claudin-18 isoform 2), PSCA, DLL3 (Delta-like protein 3, Drosophila delta homolog 3, Delta 3), Mud 7 (Mucin 17, Muc3, Muc3), FAP alpha (Fibroblast-activating protein alpha), Ly6G6D (Lymphocyte antigen 6 complex locus protein G6d, c6orf23, G6D, MEGTI, NG25), PSMA, MSLN, or RNF43 (E3 ubiquitin-protein ligase RNF43, RING finger protein 43), BAFF, C242 antigen, disialoganglioside (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, Folate Receptor 1, GD3 Ganglioside, Glycoprotein 75, GPNMB, HER2 / neu, HGF, Human Dispersion Factor Receptor Kinase, IGF-1 Receptor, IGF-I, IgG, LI-CAM, Integrin α5β1, Integrin ανβ3, Regmine, MORAb-009, MS4A1, MUC1, MucinA peptide delivery system according to claim 1, which binds to CanAg, C-MET, CCR4, CD152, CD10, CD19, CD20, CD200, N-glycolylneuraminic acid, NPC-IC, PDGF-Rα, PDL192, phosphatidylserine, tumor antigen CTAA16.88, VEGF-A, VEGF-1, VEGF-2, vimentin, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF-β, TRAIL-R1, TRAIL-R2, folate receptor, transferrin receptor, or any combination thereof.

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

20. The peptide delivery system according to claim 19, wherein phosphorylation by a kinase within the intracellular domain of 41BB, ICOS, or CD3ζ activates GrzB, which transports GrzB to the outside of the cell, causing GrzB to cleave the first and / or second cleavage sites.

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

22. Phosphorylation by kinases within the intracellular domain of 41BB, ICOS, or CD3ζ activates the cell, releasing endogenous GrzB and exporting the GrzB to the outside of the cell. The peptide delivery system according to claim 21, comprising sending a peptide to cleave a GrzB cleavage portion located within the first or second loop region, thereby releasing the peptide payload.

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

24. The payload comprises an antibody, an antibody fragment, VHH, a cytokine or chemokine consisting of a fraction or derivative of sulfated xylan, wherein the cytokine or chemokine is the IL-17 family of cytokines including 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-25, interferon, G-CSF, M-CSF, GM-CSF, BDNF, CNTF, EGF, EPO, FGF1, FGF2, FGF3 Includes 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, TNFα, 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-1a This includes members of the MIP-1 family, MIP-2, eotaxin (eotaxin-1, -2, or -3), PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase, cathepsin family enzymes, cell adhesion molecules such as PECAM-1, soluble receptors, or cell- or virus-binding receptors, cytokine-induced neutrophil chemoattractants (KC), TNF-α and IFN-γ), as well as other soluble mediators of inflammation such as reactive oxygen species and nitric oxide, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6 , CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL1 7, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, C CL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXC L10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2,A peptide delivery system according to any one of claims 1 to 23, wherein the ligand is an antagonist selected from a list including CX3CL1, thrombin, urokinase, vaccine, or any combination thereof.

25. The peptide delivery system according to any one of claims 1 to 24, further comprising a linker between the first transmembrane domain and the first cleavage site.

26. The peptide delivery system according to claim 25, wherein the linker has a length of 2 to 128 amino acids or 4 to 20 amino acids.

27. The peptide delivery system according to claim 25, wherein the linker is selected from the linkers shown in Table 4.

28. A peptide delivery system according to claim 1, configured to be specifically localized (sorted) to extracellular vesicles.

29. The peptide delivery system according to claim 1, configured to release two or more peptide payloads into or outside a cell.

30. The peptide delivery system according to claim 1, comprising two or more schedase cleavage sites, each of which may be identical to or different from any of the other cleavage sites.

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

32. The peptide delivery system according to claim 1, comprising a single-domain antibody or a single-chain antibody that binds to a self-associated antigen (SAA) and brings the SAA to the vicinity, for example, in a cis or trans configuration.

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

34. The peptide delivery system according to claim 1, wherein the peptide payload is any peptide or protein that performs a function in the human body, encompassing hormones, cytokines, chemokines, prodrugs, and antibodies in various forms such as chimeric antigen receptors (CARs), bispecific killer cell engagers (BIKEs), and triplicate killer cell engagers (TRIKEs).

35. The peptide delivery system includes 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-CD 38-41BB (Sequence ID: 54 or 366 or 367), CD63-GrzB-a-CD16-a-CD33-Myc-GrzB-a-CD38-CD28-CD3z (Sequence ID: 71 or 252), CD63-MMP9-2L-TNFα-Myc-MMP9-CD63-Cyt (Sequence ID: 88 or 295), CD63-MMP9-2L-TNFα-Myc-MMP9-CD63-D-Cyt (lacking EV localization (sort) motif) (Sequence ID: 103 or 311), CD63-DL1- MMP2-2L-TNFα-Myc-MMP2-CD63-Cyt (Sequence ID: 104 or 368), CD63-MMP9-2L-TNFα-Myc-MMP9-aCD19CAR (Sequence ID: 105 or 369), CD63-MMP2-2L-TNFα-Myc-MMP2-CD63-D-Cyt (Sequence ID: 106 or 370 or 371), CD63-MMP2-2L-TNFα-Myc-MMP2-aCD19CAR (Sequence ID: 107 or 372 or 373), CD69cyt-TMI I-TNFα-Myctag-CD28TM-cyt (Sequence ID: 119 or 370), CD69cyt-TMII-MMP9-TNFα-Myc-MMP9-CD28TM-cyt (Sequence ID: 120 or 377), CD69Cyt-TM-GrzB-TNFα-Myc-GrzB-a-CD19sc-Stalk-CD28-CD3z (Sequence ID: 107, 372 or 373), CD69Cyt-TM-GrzB-nLuc-Myc-GrzB-CD28-CD3z (Delta sc-GAGE B) (Sequence ID: 108 or 374), CD69Cyt-TM-nLuc-Myc-a-CD19sc-Stalk-CD28-CD3z(DeltaGrzB, GAGE)A) (Sequence ID: 109 or 375), CD69Cyt-TM-GrzB(original)-nLuc-Myc-GrzB(original)-a-CD19sc-Stalk-CD28-CD3z (Sequence ID: 358 or 382), CD69Cyt-TM-GrzB(IEPD)-nLuc-Myc-GrzB(IEPD)-a-CD19sc-Stalk-CD28-CD3z (Sequence ID: 110 or 383), CD69 A peptide delivery system according to claim 1, selected from Cyt-TM-GrzB(SASA)-nLuc-Myc-, GrzB(SASA)-a-CD19sc-Stalk-CD28-CD3z (SEQ ID NO: 111 or 376), or CD69Cyt-TM-GrzB(ADKG)-nLuc-Myc-GrzB(ADKG)-a-CD19sc-Stalk-CD28-CD3z (SEQ ID NO: 112 or 384).

36. The peptide delivery system according to any one of claims 1 to 35, comprising cells on which the exogenous polypeptide is immobilized.

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

38. Adoptive cells or genetically modified immune cells comprising the peptide payload described in claim 1, wherein the immune cells are autologous or allogeneic.

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

40. The peptide delivery system according to claim 39, wherein the exogenous polypeptide is delivered using CPP, micelles, liposomes, nanoparticles, dendrimers, nanotubes, electroporation, viral transduction, nucleofection, transfection, cell fusion, or microinjection.

41. A gene construct encoding an exogenous polypeptide as described in claim 1.

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

43. A method for delivering peptides to a target gene locus in a patient requiring a peptide, the method comprising administering to the patient a peptide delivery system comprising lipid-containing vesicles (e.g., cells, extracellular vesicles, lipid-containing particles) on which an exogenous polypeptide is immobilized, the exogenous polypeptide is Peptide payload and; The first and second transmembrane regions, The first transmembrane region includes a transmembrane domain or other proteolytic cleavage site connected to at least one schidase, The second transmembrane region includes a transmembrane domain or other proteolytic cleavage site connected to at least one schidase, The first and second transmembrane regions; Depending on the circumstances, the exogenous polypeptide may be targeted to a target gene locus, Includes, Once the lipid-containing vesicle reaches the target gene locus, schedase and / or other proteases cleave the cleavage sites in the first and second transmembrane regions, releasing the peptide payload. method.

44. The method according to claim 43, wherein the cleavage site is derived from a naturally occurring proteolytic cleavage substrate in glycosylphosphatidylinositol (GPi)-anchored proteins that are membrane proteins having one, two, three, four or more transmembrane domains, intracellular domains, or extracellular domains in the extracellular matrix or adjacent cells.

45. The method according to claim 43, wherein the schødase is selected from full-time or part-time schødase.

46. The method according to claim 45, wherein the full-time schedase is selected from ADAM protease, BACE protease, serine protease granzyme-B, and site 1 protease.

47. The method according to 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 according to claim 45, wherein the part-time schidase is selected from mepurin B, MT-MMP (membrane-type matrix metalloproteinase), proprotein converterase, transmembrane serine protease, matrix metalloproteinase, legmine, and cathepsin S and L, wherein the mepurin B is a membrane-anchored type 1 metalloproteinase, and the MT-MMP is a membrane-anchored type 1 or GPI-anchored type, and comprises MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, and MT6-MMP.

49. The method according to claim 48, wherein the proprotein converterase is selected from PCSK1 / 3, KCSK2, furin, PCSK4, PSCK5 / 6, PACE4, PCSK7, and PCSK9.

50. The method according to claim 48, wherein the transmembrane serine protease is selected from, for example, membrane-anchored type II proteases including matryptase, matryptase-2, matryptase-3, polymerase-1, choline, hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, spinesin, enteropeptidase, HAT, DESCL1, TMPRSS11A, HAT-L4, and HAT-L5.

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

53. The method according to claim 52, wherein the MMP2 is selected from sequence numbers 2 to 6.

54. The method according to claim 52, wherein the MMP9 is selected from sequence numbers 7 to 8.

55. MMP25 is selected from sequence numbers 9-17, 18 or 155, 19-29. The method described in item 52.

56. The method according to claim 43, wherein the transmembrane domain of the first and / or second transmembrane region comprises a transmembrane domain derived from one or more of 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 according to claim 43, wherein the cutting at one or more of the aforementioned cutting sites is performed by endogenous schedase.

58. The method according to claim 43, wherein the peptide delivery system further comprises a schidase, and the cleavage is performed by the schidase of the peptide delivery system at one or more of the cleavage sites.

59. The intracellular or extracellular domain of the exogenous polypeptide is one of the following: 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 costimulator (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CD11a / CD18), CD3 gamma, CD3 delta, CD3 i Psilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signal transduction lymphocyte activating molecule (SLAM protein), costimulatory ligand (PD-L2, 4-1BBL, OX40) L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin b receptor, 3 / TR6, ILT3, ILT4), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHT R), KIRDS2, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD8 Alpha, CD8 Beta, IL-2R Beta, IL-2R Gamma, IL-7R Alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, IT GAM, CD11b, ITGAX, CD11e, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRA NCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (TACTILE), CEACAM1, CRTThe method according to claim 43, wherein one or more intracellular or extracellular domains are selected from ligands that specifically bind to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

60. The targeting moiety is BCMA, MUC16 (also known as CA125), EGFR, EGFRvIII, MUCI, Flt-3, WT-1, CD38, CD70, CD90, CD133, MHC-WTI, T SPANI0, MHC-PRAME, MHC-NY-ESOI, HER2 (ERBB2), CA-IX (carbonic anhydrase IX), LIVI, ADAM10, 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 Claudin-18 isoform 2), PSCA, DLL3 (Delta-like protein 3, Drosophila delta homolog 3, Delta 3), Mud 7 (Mucin 17, Muc3, Muc3), FAP alpha (Fibroblast-activating protein alpha), Ly6G6D (Lymphocyte antigen 6 complex locus protein G6d, c6orf23, G6D, MEGTI, NG25), PSMA, MSLN, or RNF43 (E3 ubiquitin-protein ligase RNF43, RING finger protein 43), BAFF, C242 antigen, disialoganglioside (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, Folate Receptor 1, GD3 Ganglioside, Glycoprotein 75, GPNMB, HER2 / neu, HGF, Human Dispersion Factor Receptor Kinase, IGF-1 Receptor, IGF-I, IgG, LI-CAM, Integrin α5β1, Integrin ανβ3, Regmine, MORAb-009, MS4A1, MUC1, MucinThe method according to claim 43, which binds to CanAg, C-MET, CCR4, CD152, CD10, CD19, CD20, CD200, N-glycolylneuraminic acid, NPC-IC, PDGF-Rα, PDL192, phosphatidylserine, tumor antigen CTAA16.88, VEGF-A, VEGF-1, VEGF-2, vimentin, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF-β, TRAIL-R1, TRAIL-R2, folate receptor, transferrin receptor, and any combination thereof.

61. The method according to claim 43, wherein the first and / or second transmembrane region comprises (i) an intracellular region including an intracellular region sequence from 41BB, ICOS, or CD3ζ, and (ii) a GrzB cleavage site, wherein the action of a kinase on the intracellular region sequence from 41BB, ICOS, or CD3ζ induces the release of GrzB, which is then transported outside the cell to cleave the GrzB cleavage site, thereby releasing the peptide payload, the peptide payload optionally comprising a bispecific antibody.

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

63. The peptide payload comprises a cytokine or chemokine consisting of a fraction or derivative of sulfated xylan, wherein the cytokine or chemokine is the IL-17 family of cytokines including 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-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, TNFα, 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-1a and the MIP-1 family -Members, MIP-2, eotaxin (eotaxin-1, -2 or -3), PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase, cathepsin family enzymes, cell adhesion molecules such as PECAM-1, soluble receptors, or cell- or virus-binding receptors, cytokine-induced neutrophil chemoattractants (KC), TNF-α and IFN-γ), and other soluble mediators of inflammation such as reactive oxygen species and nitric oxide, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, C CL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL1 9, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, C XCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CX CL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, thrombin,The method according to claim 43, wherein the antagonist is a ligand selected from a list including urokinase, vaccine, or any combination thereof.

64. The method according to claim 43, wherein the exogenous polypeptide further comprises at least one linker, the at least one linker optionally positioned between the cleavage site and a first binding portion (e.g., an anti-CD3 VHH domain) located within the first transmembrane region, between the cleavage site and a second binding portion (e.g., an anti-TAA VHH domain) located within the second transmembrane region, and / or between the first binding portion and the second binding portion.

65. The method according to claim 64, wherein the linker has a length of 2 to 128 amino acids, or preferably 4 to 20 amino acids.

66. The method according to claim 64, wherein the linker is selected from Table 4.

67. The method according to claim 43, wherein the exogenous polypeptide can also be configured to specifically localize (sort) to extracellular vesicles (EVs).

68. The method according to claim 43, wherein the peptide payload comprises two or more bispecific engagers, and the bispecific engagers are released intracellularly or extracellularly.

69. The method according to claim 43, wherein the peptide delivery system includes schedase cleavage sites, and each cleavage site may be identical to or different from any of the other cleavage sites.

70. The method according to claim 43, wherein the peptide delivery system includes an intracellular domain, and the intracellular domain includes the talk domain of CD45 phosphatase.

71. The method according to claim 43, wherein the peptide delivery system comprises a single-domain antibody or a single-strand antibody, and the single-domain antibody or single-strand antibody binds to the self-associated antigen (SAA) to bring the SAA to the vicinity, for example, in a cis or trans configuration.

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

73. The method according to claim 43, wherein the peptide payload is any peptide that performs a function in the body and is a potential therapeutic agent comprising hormones, cytokines, chemokines, prodrugs, and antibodies in various forms such as chimeric antigen receptors (CARs), bispecific killer cell engagers (BIKEs), and triplicate killer cell engagers (TRIKEs).

74. The peptide delivery system is CD63-MMP2-a-CD16-a-CD33-Myc-MMP9-CD63-GPi (SEQ ID NO: 124), CD63-MMP2-a-C D16-a-CD33-Myc-MMP9-CytCD63-EV motif (Sequence ID: 155), CD63-MMP2-a-CD16-a-CD33-Myc-MMP9-CytCD63 (Sequence ID: 36 or 188), TNFSF14-a-CD16-a-CD33-Myc-TNR1A-ADAM17-a-CD38-41BB (Sequence ID: 54 or 366 or 367), CD63-GrzB-a-CD16-a-CD33-Myc-GrzB -a-CD38-CD28-CD3z (Sequence ID: 71 or 252), CD63-MMP9-2L-TNFα-Myc-MMP9-CD63-Cyt (Sequence ID: 88 or 295), CD63-MMP9-2L-TNFα-Myc-MMP9-CD63-D-Cyt (lacking EV localization (sort) motif) (Sequence ID: 103 or 311), CD63-DL1-MMP2-2L-TNFα-Myc-MMP2-CD63-Cyt (Sequence ID: 104 (or 368), CD63-MMP9-2L-TNFα-Myc-MMP9-aCD19CAR (Sequence ID: 105 or 369), CD63-MMP2-2L-TNFα-Myc-MMP2-CD63-D-Cyt (Sequence ID: 106 or 370 or 371), CD63-MMP2-2L-TNFα-Myc-MMP2-aCD19CAR (Sequence ID: 107 or 372 or 373), CD69cyt-TMII-TNFα-Myctag-CD28TM -cyt (SEQ ID NO: 119 or 370), CD69cyt-TMII-MMP9-TNFα-Myc-MMP9-CD28TM-cyt (SEQ ID NO: 120 or 377), CD69Cyt-TM-GrzB-TNFα-Myc-GrzB-a-CD19sc-Stalk-CD28-CD3z (SEQ ID NO: 107 or 372 or 373), CD69Cyt-TM-GrzB-nLuc-Myc-GrzB-CD28-CD3z (Delta sc-GAGE B) (SEQ ID NO: 108 or 374), CD69Cyt-TM-nLuc-Myc-a-CD19sc-Stalk-CD28-CD3z (Delta GrzB, GAGEA) (Sequence ID: 109 or 375), CD69Cyt-TM-GrzB(original)-nLuc-Myc-GrzB(original)-a-CD19sc-Stalk-CD28-CD3z (Sequence ID: 358 or 382), CD69Cyt-TM-GrzB(IEPD)-nLuc-Myc-GrzB(IEPD)-a-CD19sc-Stalk-CD28-CD3z (Sequence ID: 110 or 383), C The method according to claim 43, selected from D69Cyt-TM-GrzB(SASA)-nLuc-Myc-, GrzB(SASA)-a-CD19sc-Stalk-CD28-CD3z (Sequence ID: 111 or 376), or CD69Cyt-TM-GrzB(ADKG)-nLuc-Myc-GrzB(ADKG)-a-CD19sc-Stalk-CD28-CD3z (Sequence ID: 112 or 384).

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

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

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

78. The method according to claim 43, wherein the peptide delivery system is delivered using CPP, micelles, liposomes, nanoparticles, dendrimers, nanotubes, electroporation, viral transduction, nucleofection, transfection, cell fusion, or microinjection.

Images