Plasma membrane embedded system for spaciotemporal controlled payload delivery of biologics

Abstract

Disclosed is a peptide delivery system for delivering a peptide payload to a target cell or target locus and methods of delivering a peptide payload to a target locus. The peptide delivery system may include a cell, extracellular vesicle, or lipid-containing particle into which an exogenous polypeptide has been anchored, the exogenous polypeptide having a first and a second transmembrane domain connected by a loop region, which loop region comprises a first cleavage site, a peptide payload, and a second cleavage site and optionally, a targeting moiety for targeting the peptide delivery system to a target cell or target locus.

Description

[0001] TITLE

[0002]

[0001] Plasma Membrane Embedded System for Spaciotemporal Controlled Payload Delivery of Biologies

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004]

[0002] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 467,518 filed May 18, 2023, which application is expressly incorporated by reference herein in its entirety.

[0005] INCORPORATION BY REFERENCE STATEMENT REGARDING SEQUENCE LISTING

[0006]

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 16, 2024, is named “P71267_SL.xml” and is 451,938 bytes in size.

[0007] BACKGROUND

[0008]

[0004] Living organisms are increasingly being treated with peptide-based therapeutics. These therapeutics comprise hormones, cytokines, chemokines, pro-drugs, and antibodies in various formats, such as chimeric antigen receptors (CARs), bi-specific killer cell engager (BIKE), tri- specific killer cell engager (TRIKE), etc. Any peptide or protein that serves a function within the body is a potential therapeutic. While the therapeutic potential of peptides and proteins is promising, several factors limit their bioavailability, and thus their function in vivo.

[0009]

[0005] One limiting factor is the in vitro production, stability, and multiple dose-delivery of these biological products. These biologies are often synthesized in mammalian cells, bacteria, or, more recently, in unicellular eukaryotes like Pichia pastoris. The biologies are purified from the producer cells, formulated in a delivery system, and administered to the patients. Therapeutic levels often require continuous delivery and / or repeated peptide dosing.

[0010]

[0006] Systemic delivery of the biologies further lowers the efficacy depending on the drug's pharmacokinetics and dynamics because only a small fraction of the administered peptide may reach the target sites. Most of the biologic may be degraded and metabolized before reaching the intended site of action.

[0011]

[0007] A third limiting factor is the biotoxicity related to the mechanism of action (MO A) of these biologies.

[0008] There is a need in the art for improved protein and peptide delivery methods to increase safety and efficacy of biologies.

[0012] SUMMARY OF THE INVENTION

[0013]

[0009] Disclosed herein is a peptide delivery system for delivering a peptide payload to a target cell or target locus, the peptide delivery system comprising: a cell, extracellular vesicle, or lipid- containing particle into which an exogenous polypeptide has been anchored, which exogenous polypeptide comprises: a first and a second transmembrane domain connected by a loop region, which loop region comprises a first cleavage site, a peptide payload, and a second cleavage site; and optionally, a targeting moiety for targeting the peptide delivery system to the target cell or the target locus.

[0014]

[0010] In such a peptide delivery system, the first and / or second cleavage sites may comprise proteolytic cleavage substrates, preferably sheddase cleavage sites, MMP cleavage sites, GrzB cleavage sites, and Casp3 cleavage sites.

[0015] [Oi l] Such a peptide delivery system may be configured to release the peptide payload upon cleavage by a protease, preferably aby a sheddase, which may be endogenous or exogenous (e.g., included as a component of the peptide delivery system).

[0016]

[0012] Such a peptide delivery system is also contemplated, wherein the sheddase is present at the target locus and / or released by the target cell.

[0017]

[0013] In embodiments of the above peptide delivery system, the sheddase may comprise a full- time sheddase selected from ADAM proteases (metalloproteases), BACE proteases, serine- protease Granzyme-B and Site-1 proteases. The ADAM proteases may include membrane- anchored type 1 proteases such as ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, ADAMthr20, ADAM21, ADAM28, ADAM30, and ADAM33.

[0018]

[0014] Also contemplated herein is such a peptide delivery system, wherein the sheddase is a part- time sheddase selected from Meprin B, MT-MMPs (Membrane Type Matrix Metalloproteases), Pro-protein convertases, transmembrane serine proteases, Matrix Metalloproteases, Legumain and Cathepsin S and L. The MT-MMPs may be membrane-anchored type 1 or GPI-anchored and may include MT 1 -MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, and MT6-MMP. The Proprotein convertases 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 Matriptase, Matriptase-2, Matriptase-3, Polymerase- 1, Corin, Hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, Spinesin, Enteropeptidase, HAT, DESCL1, TMPRSS11A, HAT-like 4 and HAT -like 5. The Matrix Metalloproteases 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, MMP21, MMP 23A-B, MMP24, MMP25, MMP26, MMP27 and MMP28. In an embodiment the Matrix Metalloproteases are selected from MMP2, MMP9 and / or MMP25. In an embodiment the MMP2 may be selected from SEQ ID NOS: 2-6. In an embodiment the MMP9 may be selected from SEQ ID NOS: 7-8. In an embodiment the MMP25 may be selected from SEQ ID NOS: 9-17, 18 or 155, and 19-29.

[0019]

[0015] Also disclosed herein is such a peptide delivery system, wherein the transmembrane domain comprises a transmembrane domain from one or more of the following proteins: CD63, CD9, CD81, CD28, CD4, CD8, CD34, CD69, CD19, CD20, Integrins, TNFSF14, TNR1A, Aquaporins, NOTCH, NgRl, NRG1, GPi anchor, EGFR, or Rhodopsin.

[0020]

[0016] In certain embodiments, a peptide delivery system as above may further comprise an intracellular or extracellular domain selected from or derived from one or more of the following proteins: OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1, Programmed Death-I (PD- 1), Inducible T cell Co-Stimulator (ICOS), lymphocyte Function- associated Antigen-I (LFA-1 (CDI la / CD 18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, 1g alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), The costimulatory ligand ( PD-L2, 4-1 BBL, OX40L, Inducible Co-Stimulatory Ligand (ICOS-L), Inter-Cellular Adhesion Molecule (ICAM, CD30L, CD70, CD83, HLA-G, MICA, Ml CB, HVEM, lymphotoxin b receptor, 3 / TR6, ILT3, ILT4), activating NK cell receptors, BTLA, a 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, CDI Id, ITGAE, CD 103, ITGAL, CDI la, LFA-1, IT GAM, CDI lb, ITGAX, CDI le, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, a ligand that specifically binds with CD83, or any combination thereof.

[0021]

[0017] In embodiments such a peptide delivery system may comprise a targeting moiety and the targeting moiety may bind to BCMA, MUC16 (also known as CA125), EGFR, EGFRvin, MUCI, Flt-3, WT-1, CD38, CD70, CD90, CD133, MHC-WTI , TSPANI0, MHC-PRAME, MHC- NY-ESOI, HER2 (ERBB2), CA-IX (Carbonic anhydrase IX), LIVI, ADAMIO, CHRNA2, LeY, NKG2D, CSI, CD44v6, CD24, LGR5, ALDH, ALDH1, CD54, Seal, CD271, CD123, CD36, CD109, CD110, CD71 -negative, CCA, ABCG2, Claudin-18.2 (Claudin-18A2, or Claudinl8 isoform 2), PSCA, DLL3 (Delta-like protein 3, Drosophila Delta homolog 3, Delta3 ), Mud 7 (Mucinl 7, Muc3, Muc3), FAP alpha (Fibroblast Activation 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 scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGI, LI-CAM, integrin a5pi, integrin av|33, legumain, MORAb-009, MS4A1, MUC1, mucin CanAg, C-MET, CCR4, CD 152, CD 10, CD 19, CD20, CD200, N-glycolylneuraminic acid, NPC-IC, PDGF-R a, PDL192, phosphatidylserine, tumor antigen CTAA16.88, VEGF-A, VEGFR- 1, VEGFR2, vimentin, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF-P, TRAIL-R1, TRAIL-R2, folic acid receptor, transferrin receptors, or any combination thereof.

[0022]

[0018] Also contemplated herein is a peptide delivery system as above, wherein the second transmembrane domain is connected to an intracellular region comprising an intracellular domain from one or more of 41BB, ICOS, and CD3(^. In certain embodiments, phosphorylation by a kinase within the intracellular domain of 41BB, ICOS, or CD3(^ may activate GrzB and allow GrzB to be transported out of the cell and cleave the first and / or second cleavage site.

[0023]

[0019] In certain embodiments, the peptide delivery system as above may comprise a third and fourth transmembrane domain connected by a second loop region, wherein the third transmembrane domain is connected to an intracellular region comprising an intracellular domain from 4 IBB, ICOS, or CD3z. In such embodiments phosphorylation by a kinases within the intracellular domain of 4 IBB, ICOS or CD3(^ may activate the cell to release endogenous GrzB allowing the GrzB to be transported out of the cell and cleave GrzB cleavage present in the first or second loop region thereby releasing the peptide payload.

[0024]

[0020] In an embodiment the second transmembrane domain may be from a NOTCH protein having a cleavage site configured to release a functional domain to which the NOTCH protein transmembrane domain is connected in the intracellular milieu.

[0025]

[0021] Such a peptide delivery system as above is contemplated, wherein the payload may comprise: an antibody, an fragment of an antibody, a VHH, a cytokine or chemokine consisting a fraction or derivative of a sulfated xylan, which is an antagonist of a ligand selected from the list comprising IL-la, IL-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-3, IL-14, IL-15 or IL-17 family of cytokines including IL-25, interferon, G-CSF, M-CSF, GM-CSF, BDNF, CNTF, EGF, EPO, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF 11 , FGF 12, FGF 12, FGF 13 , FGF 14, FGF 15, FGF 16, FGF 17, FGF 18, FGF 19, FGF20, FGF21 , FGF22, FGF23, LIF, PDGF, SCF, TGFa, TGFB, TNFa, TNFB, TPO, VEGF, GH, NGF, NT3, NT4, NT5, NT6, NT7, Oncostatin M (OSM), insulin, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, a member of the MIP-1 family including MIP-la, MIP-2, eotaxin (eotaxin-1, -2 or -3), PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase; an enzyme of the cathepsin family, a cell adhesion molecules such as PECAM-1, a soluble or cell- or virus-bound receptor, cytokine- induced neutrophil chemoattractant (KC), TNF-a and IFN-g), 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, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, thrombin, urokinase, a vaccine or any combination thereof.

[0026]

[0022] In embodiments, a peptide delivery system as above may further comprise a linker between the first transmembrane domain and the first cleavage site. The linker may be 2-128 amino acids in length or 4-20 amino acids in length. In certain embodiments the linker may be selected from the linkers set forth in Table 4.

[0027]

[0023] In an embodiment, the peptide delivery system may be configured to be sorted specifically into extracellular vesicles.

[0028]

[0024] Also contemplated herein is a peptide delivery system as above which is configured to release two or more peptide payloads intracellularly or extracellularly.

[0029]

[0025] Also contemplated herein is such a peptide delivery system, which comprises two or more sheddase cleavage sites, and each cleavage site can be the same or different from any of the other cleavage sites.

[0030]

[0026] Also contemplated herein is such a peptide delivery system, which comprises an intracellular domain and the intracellular domain comprises a stalk domain of CD45 phosphatase.

[0027] Also contemplated herein is a peptide delivery system as above which comprises a single domain antibody or single chain of an antibody which binds a self-associated antigen (SAA) to bring the SAA in proximity, e.g., in cis or in trans.

[0028] Also contemplated herein is such a peptide delivery system, which comprises a single domain antibody or single chain of an antibody which binds the self-associated antigen (SAA), and which further comprises a CD45 phosphatase-binding moiety or an NKG2A-binding moiety, which activates an inhibitory signal in the target cell.

[0031]

[0029] In embodiments of the above peptide delivery system, the peptide payload may be any peptide or protein that serves a function within the human body, including hormones, cytokines, chemokines, pro-drugs, and antibodies in various formats, such as chimeric antigen receptors (CARs), bi-specific killer cell engager (BIKE), tri-specific killer cell engager (TRIKE), etc.

[0032]

[0030] In embodiments, the peptide delivery system as above may be selected from: CD63- MMP2-a-CD16-a-CD33-Myc-MMP9-CD63-GPi (SEQ ID NO: 124), CD63-MMP2-a-CD16-a- CD33 -My c-MMP9-CytCD63 -EV Motif (SEQ ID NO : 155), CD63 -MMP2-a-CD 16-a-CD33 -My c- MMP9-CytCD63 (SEQ ID NO: 36 or 188), TNFSF14-a-CD16-a-CD33-Myc-TNRlA-ADAM17- a-CD38-41BB (SEQ ID NO: 54 or 366 or 367), CD63-GrzB-a-CD16-a-CD33-Myc-GrzB-a- CD38-CD28-CD3z (SEQ ID NO: 71 or 252), CD63-MMP9-2L-TNFa-Myc-MMP9-CD63-Cyt (SEQ ID NO: 88 or 295), CD63-MMP9-2L-TNFa-Myc-MMP9-CD63-D-Cyt (Lacks EVs sorting motif) (SEQ ID NO: 103 or 311), CD63-DLl-MMP2-2L-TNFa-Myc-MMP2-CD63-Cyt (SEQ ID NO: 104 or 368), CD63-MMP9-2L-TNFa-Myc-MMP9-aCD19CAR (SEQ ID NO: 105 or 369), CD63-MMP2-2L-TNFa-Myc-MMP2-CD63-D-Cyt (SEQ ID NO: 106)or 370 or 371, CD63- MMP2-2L-TNFa-Myc-MMP2-aCD19CAR (SEQ ID NO: 107 or 372 o4 373), CD69cyt-TMII- TNFa-Myctag-CD28TM-cyt (SEQ ID NO: 119 or 370), CD69cyt-TMII-MMP9-TNFa-Myc- MMP9-CD28TM-cyt (SEQ ID NO: 120 or 377), CD69Cyt-TM-GrzB-TNFa-Myc-GrzB-a- CD19sc-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, GAGE A) (SEQ ID NO: 109 or 375), CD69Cyt-TM- GrzB (Original)-nLuc-Myc-GrzB(Original)-a-CD19sc-Stalk-CD28-CD3z (SEQ ID NO: 358 or 382), CD69Cyt-TM-GrzB(IEPD)-nLuc-Myc-GrzB(IEPD)-a-CD19sc-Stalk-CD28-CD3z (SEQ ID NO: 110 or 383), CD69Cyt-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).

[0033]

[0031] In certain embodiments, the above peptide delivery system may comprises a cell into which the exogenous polypeptide has been anchored. In certain embodiments, the cell may be selected from a Hematopoietic Stem Cells (HSC), an induced Pluripotent Stem Cell (iPSC) and their derived cell products, an Adoptive T cell, a Dendritic cell (DC), Natural killer (NK) cells, or any therapeutic immune or non-immune cells.

[0032] Also contemplated herein is an adoptive cell or engineered immune cell comprising a peptide payload of such a peptide delivery system, wherein the immune cells are autologous or allogeneic.

[0034]

[0033] In embodiments, the exogenous polypeptide may be introduced into the cell individually via a CRISPR system, or in the form of viral or nonviral vectors, or one or more mRNAs that encode the exogenous polypeptide.

[0035]

[0034] In embodiments, the exogenous polypeptide of the above peptide delivery system may be delivered using CPPs, micelles, liposomes, nanoparticles, dendrimers, nanotubes, electroporation, viral transduction, nucleofection, transfection, cell fusion, or microinjection.

[0036]

[0035] Also contemplated herein is a genetic construct encoding the exogenous polypeptide of the peptide delivery system.

[0037]

[0036] Also contemplated herein is a method of treating a patient in need thereof comprising administering the peptide delivery system as above to the patient.

[0038]

[0037] Also disclosed herein is a method of delivering a peptide to a target locus in a patient in need thereof, the method comprising: administering to the patient a peptide delivery system comprising a lipid-comprising vescicle (e.g., cell, extracellular vesicle, lipid nanoparticle) into which an exogenous polypeptide has been anchored, the exogenous polypeptide comprising: a peptide payload; a first and a second transmembrane region; the first transmembrane region comprising a transmembrane domain connected to at least one sheddase or other proteolytic cleavage site; the second transmembrane region comprising a transmembrane domain connected to at least one sheddase or other proteolytic cleavage site; and optionally, a targeting moiety for targeting the exogenous polypeptide to a target locus, wherein when the lipid-comprising vescicle arrives at the target locus, a sheddase and / or other protease cleaves the cleavage sites of the first and second transmembrane regions releasing the peptide payload.

[0039]

[0038] Also contemplated herein is such a method, wherein the cleaveage sites are derived from proteolytic cleavage substrates naturally present in Glycosylphosphatidylinositol (GPi) anchored proteins, membrane proteins with a single, two, three, four, or more transmembrane domains, intracellular domains, or extracellular domains in the extracellular matrix or in neighboring cells.

[0040]

[0039] Also contemplated herein is such a method, wherein the sheddases are selected from fuller part-time sheddases. In embodiments, the full-time sheddases may be selected from ADAM proteases, BACE proteases, serine-protease 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 sheddases may be selected from: Meprin B, MT-MMPs (Membrane Type Matrix Metalloproteases), Pro- protein convertases, transmembrane serine proteases, Matrix Metalloproteases, Legumain and Cathepsin S and L, wherein the Meprin B is a membrane-anchored type 1 metalloprotease, and wherein the MT-MMPs are membrane-anchored type 1 or GPI-anchored, and include MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, and MT6-MMP. The Pro-protein convertases 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, Matriptase, Matriptase-2, Matriptase-3, Polymerase- 1, Corin, Hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, Spinesin, Enteropeptidase, HAT, DESCL1, TMPRSS 11 A, HAT -like 4 and HAT-like 5. The Matrix Metalloproteases may be soluble proteases and may include one or more of MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP21, MMP23A-B, MMP24, MMP25, MMP26, MMP27, and MMP28. In certain embodiments, the Matrix Metalloproteases may be selected from MMP2, MMP9, and / or MMP25. For example, the MMP2 may be selected from SEQ ID NOS: 2-6, the MMP9 may be selected from SEQ ID NOS: 7-8, and the MMP25 may be selected from SEQ ID NOS: 9-17, 18 or 155, 19-29.

[0041]

[0040] Also contemplated herein is a method as above, 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, Integrins, TNFSF14, TNR1A, Aquaporins, NOTCH, NgRl, NRG1, GPi anchor, EGFR, and Rhodopsin.

[0042]

[0041] Also contemplated herein is a method as above, wherein cleavage at one or more of the cleavage sites occurs by endogenous sheddases.

[0043]

[0042] Also contemplated herein is a method as above, wherein the peptide delivery system further comprises a sheddase, and cleavage occurs at one or more of the cleavage sites by the peptide delivery system sheddase.

[0044]

[0043] Also contemplated herein is a method as above, wherein an intracellular or extracellular domain of the exogenous polypeptide is selected from an intracellular or extracllular domain from one or more of the following: OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7- 2 (CD86), PDL-1 , Programmed Death-I (PD- 1), Inducible T cell Co-Stimulator (ICOS), lymphocyte Function-associated Antigen-I (LFA-1 (CDI la / CD 18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, 1g alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), The costimulatory ligand ( PD-L2, 4-1 BBL, OX40L, Inducible Co-Stimulatory Ligand (ICOS-L), Inter-Cellular Adhesion Molecule (ICAM, CD30L, CD70, CD83, HLA-G, MICA, Ml CB, HVEM, lymphotoxin b receptor, 3 / TR6, ILT3, ILT4), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 19, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, IT GAM, CD1 lb, ITGAX, CD1 le, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, a ligand that specifically binds with CD83, or any combination thereof.

[0045]

[0044] Also contemplated herein is a method as above, wherein the targeting moiety binds BCMA, MUC16 (also known as CA125), EGFR, EGFRvin, MUCI, Flt-3, WT-1, CD38, CD70, CD90, CD 133, MHC-WTI , TSPANIO, MHC-PRAME, MHC-NY-ESOI, HER2 (ERBB2), CA-IX (Carbonic anhydrase IX), LIVI, ADAMIO, CHRNA2, LeY, NKG2D, CSI, CD44v6, CD24, LGR5, ALDH, ALDH1, CD54, Seal, CD271, CD123, CD36, CD109, CD110, CD71 -negative, CCA, ABCG2, Claudin-18.2 (Claudin-18A2, or Claudinl8 isoform 2), PSCA, DLL3 (Delta-like protein 3, Drosophila Delta homolog 3, Delta3 ), Mud 7 (Mucinl 7, Muc3, Muc3), FAP alpha (Fibroblast Activation 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 scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGI, LI-CAM, integrin a5[31, integrin av[33, legumain, MORAb-009, MS4A1, MUCI, mucin CanAg, C-MET, CCR4, CD152, CD10, CD19, CD20, CD200, N-glycolylneuraminic acid, NPC-IC, PDGF-R a, PDL192, phosphatidylserine, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF-P, TRAIL-R1, TRAIL-R2, folic acid receptor, transferrin receptors, and any combination thereof.

[0046]

[0045] Also contemplated herein is a method as above, wherein the first and / or second transmembrane region comprises (i) an intracellular region comprising an intracellular region sequence from 4 IBB, ICOS, or CD3(^, and (ii) a GrzB cleavage site; and wherein the action of kinases on the intracellular region sequence from 41BB, ICOS, or CD3(^ induces the release of the GrzB allowing it to be transported out of the cell and cleave GrzB cleavage sites, thereby releasing the peptide payload, the peptide payload optionally comprising a bispecific antibody.

[0047]

[0046] Also contemplated herein is a method as above, 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 milieu.

[0048]

[0047] Also contemplated herein is a method as above, wherein the peptide payload comprises: a cytokine or chemokine consisting a fraction or derivative of a sulfated xylan, which is an antagonist of a ligand selected from the list comprising IL-la, IL-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-3, IL-14, IL-15 or IL-17 family of cytokines including IL-25, interferon, G-CSF, M-CSF, GM-CSF, BDNF, CNTF, EGF, EPO, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, LIF, PDGF, SCF, TGFa, TGFB, TNFa, TNFB, TPO, VEGF, GH, NGF, NT3, NT4, NT5, NT6, NT7, Oncostatin M (OSM), insulin, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, a member of the MIP-1 family including MIP-la, MIP- 2, eotaxin ( eotaxin-1, -2 or -3), , PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase; an enzyme of the cathepsin family, a cell adhesion molecules such as PECAM-1, a soluble or cell- or virus-bound receptor, cytokine-induced neutrophil chemoattractant (KC), TNF-a and IFN-g), 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, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, thrombin, urokinase, a vaccine or any combination thereof.

[0049]

[0048] Also contemplated herein is a method as above, wherein the exogenous polypeptide further comprises at least one linker, which at least one linker is optionally positioned between the cleavage site present in the first transmembrane region and a first binding moiety (e.g., anti-CD3 VHH domain), between a cleavage site present in the second transmembrane region and a second binding moiety (e.g., anti-TAA VHH domain), and / or between the first and second binding moieties.

[0050]

[0049] Also contemplated herein is a method as above, wherein the linker is 2-128 amino acids in length, or preferably 4-20 amino acids in length.

[0050] Also contemplated herein is a method as above, wherein the linker is selected from Table 4.

[0051]

[0051] Also contemplated herein is a method as above, wherein the exogenous polypeptide can also be configure for sorting specifically into extracellular vesicles (EVs).

[0052]

[0052] Also contemplated herein is a method as above, wherein the peptide payload comprises two or more bispecific engagers, and the bispecific engagers are released intracellularly or extracellularly.

[0053]

[0053] Also contemplated herein is a method as above, wherein the peptide delivery system comprises sheddase cleavage sites, and each cleavage site can be the same or different from any of the other cleavage sites.

[0054]

[0054] Also contemplated herein is a method as above, wherein the peptide delivery system comprises and intracellular domain, and the intracellular domain comprises a stalk domain of CD45 phosphatase.

[0055]

[0055] Also contemplated herein is a method as above, wherein the peptide delivery system comprises a single domain antibody or single chain of an antibody which binds a self-associated antigen (SAA) to bring the SAA in proximity, e.g., in cis or in trans.

[0056]

[0056] Also contemplated herein is a method as above, which comprises use of a peptide delivery system comprising a single domain antibody or single chain of an antibody which binds the selfassociated antigen (SAA), and which further comprises a CD45 phosphatase- or an NKG2A- binding moiety, which activates an inhibitory signal in the target cell.

[0057]

[0057] Also contemplated herein is a method as above, wherein the peptide payload is any peptide that serves a function within the body is a potential therapeutic, including hormones, cytokines, chemokines, pro-drugs, and antibodies in various formats, such as chimeric antigen receptors (CARs), bi-specific killer cell engager (BIKE), tri-specific killer cell engager (TRIKE), etc.

[0058]

[0058] Also contemplated herein is a method as above, wherein the peptide delivery system is selected from: CD63-MMP2-a-CD16-a-CD33-Myc-MMP9-CD63-GPi (SEQ ID NO: 124), CD63- MMP2-a-CD16-a-CD33-Myc-MMP9-CytCD63-EV Motif (SEQ ID NO: 155), CD63-MMP2-a- CD16-a-CD33-Myc-MMP9-CytCD63 (SEQ ID NO: 36 or 188), TNFSF14-a-CD16-a-CD33- Myc-TNRlA-ADAM17-a-CD38-41BB (SEQ ID NO: 54 or 366 or 367), CD63-GrzB-a-CD16-a- CD33-Myc-GrzB-a-CD38-CD28-CD3z (SEQ ID NO: 71 or 252), CD63-MMP9-2L-TNFa-Myc- MMP9-CD63-Cyt (SEQ ID NO: 88 or 295), CD63-MMP9-2L-TNFa-Myc-MMP9-CD63-D-Cyt (Lacks EVs sorting motif) (SEQ ID NO: 103 or 311), CD63-DLl-MMP2-2L-TNFa-Myc-MMP2- CD63-Cyt (SEQ ID NO: 104 or 368), CD63-MMP9-2L-TNFa-Myc-MMP9-aCD19CAR (SEQ ID NO: 105 or 369), CD63-MMP2-2L-TNFa-Myc-MMP2-CD63-D-Cyt (SEQ ID NO: 106 or 370 or 371), CD63-MMP2-2L-TNFa-Myc-MMP2-aCD19CAR (SEQ ID NO: 107 or 372 or 373), CD69cyt-TMII-TNFa-Myctag-CD28TM-cyt (SEQ ID NO: 119 or 370), CD69cyt-TMII-MMP9- TNFa-Myc-MMP9-CD28TM-cyt (SEQ ID NO: 120 or 377), CD69Cyt-TM-GrzB-TNFa-Myc- GrzB-a-CD19sc-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, GAGE A) (SEQ ID NO: 109 or 375), CD69Cyt-TM- GrzB, (Original)-nLuc-Myc-GrzB(Original)-a-CD19sc-Stalk-CD28-CD3z (SEQ ID NO: 358 or 382), CD69Cyt-TM-GrzB(IEPD)-nLuc-Myc-GrzB(IEPD)-a-CD19sc-Stalk-CD28-CD3z (SEQ ID NO: 110 or 383), CD69Cyt-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).

[0059]

[0059] Also contemplated herein is a method as above, wherein the lipid-comprising vesicle comprises a cell, and the cell is selected from a Hematopoietic Stem Cells (HSC), induced Pluripotent Stem Cell (iPSC) and a derived cell product, an Adoptive T cell, a Dendritic cell (DC), a Natural killer (NK) cell, or any therapeutic immune cell or non -immune cell. In certain embodiments, the immune cell may be autologous or allogeneic.

[0060]

[0060] Also contemplated herein is a method as above, wherein the peptide delivery system is introduced into a cell individually in the form of viral or nonviral vectors, mRNAs, peptides, proteins, antibodies, nanobodies, oligonucleotides, or extracellular vesicles (EVs).

[0061]

[0061] Also contemplated herein is a method as above, wherein the peptide delivery system is delivered using CPPs, micelle, liposomes, nanoparticles, dendrimer, nanotube, electroporation, viral transduction, nucleofection, transfection, cell fusion or microinjection.

[0062]

[0062] It is an object of the invention to deliver a peptide.

[0063]

[0063] It is an object of the invention to target the payload to a specific cell type.

[0064]

[0064] It is an object of the invention to deliver multiple peptides.

[0065]

[0065] It is an object of the invention to introduce the peptide delivery system into any cell that can deliver at least one peptide to a target cell or target locus.

[0066]

[0066] It is an object of the invention to create a single delivery system which can be incorporated into any cell.

[0067] BRIEF DESCRIPTION OF THE DRAWINGS

[0068]

[0067] Figure 1 is a drawing of a generic embodiment of the invention comprising two transmembrane domains, i.e., a “dispanin”.

[0068] Figure 2 is a drawing of an embodiment of a “dispanin” delivery system comprising, in order, 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.

[0069]

[0069] Figure 3 shows the amino acid sequence and various domains / motifs of the embodiment shown in Figure 2.

[0070]

[0070] Figure 4A is a drawing of a generic embodiment of the invention using a modified tetraspanin protein (CD63) linked to a specific engager (GAGE) (here, TNF-a) and comprising a particular motif, GYEVM, for the EVs specific delivery.

[0071]

[0071] Figure 4B shows the amino acid sequence and various domains / motifs of the embodiment shown in Figure 4A.

[0072]

[0072] Figure 5A shows a generic embodiment of the invention using a modified tetraspanin protein (CD63) without an EV sorting motif.

[0073]

[0073] Figure 5B shows the amino acid sequence and various domains / motifs of the embodiment shown in Figure 5A.

[0074]

[0074] Figure 6 shows an embodiment of the invention using, among other things, a modified transmembrane C-Type lectin protein (CD69).

[0075]

[0075] Figure 7 shows a generic embodiment of the invention using a modified tetraspanin comprising four different types of transmembrane domains (TMI - TMIV).

[0076]

[0076] Figure 8 shows a generic embodiment of the invention using a modified tetraspanin.

[0077]

[0077] Figure 9 is a drawing of a dispanin embodiment of the invention.

[0078]

[0078] Figure 10 is the amino acid sequence for a CD63-loop-EVs sorting embodiment and shows corresponding domains and motifs of the embodiment shown in Figure 9.

[0079]

[0079] Figure 11 is a drawing of a dispanin embodiment of the invention.

[0080]

[0080] Figure 12 is the amino acid sequence for the embodiment shown in Figure 11 and shows corresponding domains and motifs.

[0081]

[0081] Figure 13 is a drawing of a dispanin embodiment of the invention.

[0082]

[0082] Figure 14 is the amino acid sequence for the embodiment shown in Figure 13 and shows corresponding domains and motifs.

[0083]

[0083] Figure 15 is a drawing of an dispanin embodiment of the invention.

[0084]

[0084] Figure 16 is the amino acid sequence for a CD63-Gm-B-a-CD16-L-a-CD33-Gm-B-a- CD38CAR embodiment and shows corresponding domains and motifs of the embodiment shown in Figure 15.

[0085] Figure 17A is a drawing of a dispanin embodiment of the invention encoded by CD69Cyt- TM-n-TNFa-Myc-CD28TM- CYT for delivery of TNFa.

[0085]

[0086] Figure 17B is the Amino Acid Sequence of CD69Cyt-TM-II-TNFa-Myc-CD28TM- CYT.

[0086]

[0087] Figure 18A is a drawing showing an embodiment of the present invention encoded by CD69cyt-TMII-9-TNFa-Myctag-9-CD28TM-cyt containing two MMP9 cleavage sites.

[0087]

[0088] Figure 18B is the amino acid sequence of CD69cyt-TMII-9-TNFa-Myctag-9-CD28TM- cyt.

[0088]

[0089] Figure 19 is a drawing of an embodiment of the invention with aTAA- aSAA as a payload that can be delivered upon cleavage.

[0089]

[0090] Figure 20 is a drawing showing the plasmid transmembrane domains, protease motifs and payloads of various embodiments disclosed in this application.

[0090]

[0091] Figure 21 is a drawing showing the experimental setup / model of an embodiment of the invention shown in this application.

[0091]

[0092] Figures 22A, 22B, and 22C, show three options for experimental setups / models involving TNFa and measurement of NF-KB Luciferase activity.

[0092]

[0093] Figure 23A is a drawing showing the experimental setup / model of an embodiment disclosed in this application in which Granzyme B results in release of TNFa payload from the surface of an NK cell.

[0093]

[0094] Figure 23B is a drawing showing the experimental setup / model of an embodiment disclosed in this application in which Granzyme B results in release of TNFa payload from the surface of a K562 cell.

[0094]

[0095] Figure 24 is a graph showing that the TNFa payload is functional upon direct cell-to-cell contact with the HEK293 -NF-KB-LUC reporter cells.

[0095]

[0096] Figure 25 is a graph showing the delivery of TNFa payload after cleavage by MMP9 Note: co-transfection of GAGE and MMP9. We show EVs depleted vs. not depleted to control for transfer of the MMPs through EVs.

[0096]

[0097] Figure 26 is a graph showing the delivery of TNFa payload mainly into EVs.

[0097]

[0098] Figure 27 is a graph showing the delivery of TNFa payload after cleavage by MMP2. In this experiment, supernatant from HEK293 cells expressing GAGE constructs was utilized.

[0098]

[0099] Figure 28 is a graph showing the delivery of TNFa payload mainly into EVs. Cleavage is again by MMP2. Note: EVs depleted vs. not depleted.

[0099]

[0100] Figure 29 is a graph showing the differential CD63 and CD69 transmembrane GAGE expression on HEK293 cells.

[0101] Figure 30 is a graph showing the results of an in vitro responsive assay to TNFa payload delivery upon MMP9 cleavage and NF-KB activation.

[0100]

[0102] Figure 31 shows the percentage of K562 cells positive for surface TNFa expression 3 days after transduction.

[0101]

[0103] Figure 32 is a graph showing the delivery of TNFa payload from K562 cells upon cleavage by Granzyme B (GrzB) secreted by NK92 cells.

[0102]

[0104] Figure 33 shows the percentage of K562 cells positive for surface TNFa and aCD19 / CAR expression 6 days after sorting the cells. (Surface expression of GAGE Nanoluciferase in K562 transduced cells (MOI 6)).

[0103]

[0105] Figures 34A and 34B are graphs showing the release of NanoLuciferase (nLuc) payload from K562 cells upon cleavage by Granzyme B (GrzB) secreted by NK92 cells. E:T 3: 1, 4hr incubation.

[0104]

[0106] Figure 35 shows the percentage of KHYG-1 cells positive for surface NanoLuciferase (nLuc) expression 3 days after transduction.

[0105]

[0107] Figure 36 is a data graph showing the release of NanoLuciferase (nLuc) payload from KHYG-1 cells upon cleavage by Granzyme B (GrzB) after 1 -3 hrs co-culturing with Nalm-6 cells. Different time point after normalization to MFI.

[0106]

[0108] Figures 37A and 37B are graphs showing the percentage and MFI of KHYG-1 cells positive for surface NanoLuciferase 6 days after sorting the cells.

[0107]

[0109] Figure 38 is a graph showing the release of NanoLuciferase (nLuc) payload from KHYG- 1 cells upon cleavage by Granzyme B (GrzB) after 4 and 6 hrs co-culturing with K562 cells.

[0108]

[0110] Figures 39Aand 39B are graphs showing the percentage and MFI of NK92 cells positive for surface NanoLuciferase 10 days after transduction ofNK92 cells.

[0109]

[0111] Figure 40 is a graph showing the release of NanoLuciferase (nLuc) payload from NK92 cells upon cleavage by Granzyme B (GrzB) after 4 hrs co-culturing with Raji cells.

[0110]

[0112] Figure 41 is a graph showing the percentage of NK92 cells positive for surface NanoLuciferase 3 days after transduction ofNK92 cells (unsorted cells) and sorted cells.

[0111]

[0113] Figure 42 is a graph showing the release of NanoLuciferase (nLuc) payload from NK92 cells upon cleavage by Caspase 3 after 6 and 24 hrs co-culturing with K562 cells.

[0112] DETAILED DESCRIPTION OF THE INVENTION

[0113]

[0114] The particulars shown herein are by way of example and for purposes of illustrative discussion of the various embodiments only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the methods and compositions described herein. In this regard, no attempt is made to show more detail than is necessary for a fundamental understanding, the description making apparent to those skilled in the art how the several forms may be embodied in practice.

[0114]

[0115] The present invention will now be described by reference to more detailed embodiments. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope to those skilled in the art.

[0115]

[0116] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting. As used in the description and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety.

[0116]

[0117] Unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained and thus may be modified by the term “about”. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0117]

[0118] Notwithstanding that the numerical ranges and parameters setting forth the broad scope are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. Applicant also contemplates ranges derived from data points and express ranges disclosed herein.

[0118]

[0119] Each and every reference cited herein is incorporated by reference in its entirety.

[0119]

[0120] Contemplated herein is a peptide or protein cargo delivery system having directed delivery and / or site-directed release and site-directed activation of the cargo. The cargo is targeted to its intended site and / or activated when it reaches the target. This approach enables increasing the local or target site dose while limiting the number of off-target side effects. The present peptide or protein delivery system may include certain safety and specificity mechanisms, so the biological is only released in high doses at the target site, but not in other tissues (e.g., tissue where antigens for a specific antibody-derived biological may also be expressed). In addition to making these peptide-based drugs safer, and more broadly applicable, our system could reduce treatment costs and be customized for more personalized or patient population-specific uses. Furthermore, delivery systems and methods as disclosed herein enable the equivalent of more frequent dosing without needing to administer the peptide repetitively. For example, delivery via cargo-carrying cells that store the peptides or proteins to be delivered may provide an in vivo reservoir of inactive peptides or proteins (e.g., prodrugs).

[0120]

[0121] Mammalian cells have the hardware required to shed many cell membrane-bound proteins. Shedding is a controlled, irreversible posttranslational process performed by families of proteolytic enzymes, among them sheddases, which are further described below. Cell biology allows cells to control the expression levels and functions of the substrate proteins and their location on the cell surface.

[0121]

[0122] Proteolytic cleavage substrates include Glycosylphosphatidylinositol (GPi) anchored proteins, membrane proteins with a single, two, three, four, or more transmembrane domains, intracellular domains, extracellular domains in the extracellular matrix or even in neighboring cells. Membrane proteins of the delivery systems contemplated herein having 2 transmembrane domains are referred to herein as “dispanins”. Membrane proteins of the delivery systems contemplated herein having 4 transmembrane domains are referred to herein as “tetraspanins.” Cleavage can target the juxta-membrane or distal regions of the membrane-bound proteins and can result in the release of protein domains inside and / or outside the cells (ectodomain and / or endodomain release).

[0122]

[0123] We have designed a cell-based delivery system that allows the delivery and release of at least one peptide or protein payload directly at a desired site of action. The payloads are characteristically anchored to the cell through transmembrane domains (TMD). A TMD is a part of a protein molecule that spans across the cell membrane and typically consists of one or more alpha-helices or beta-sheets that cross the lipid bilayer of the membrane, anchoring the protein in place and allowing it to interact with molecules on both sides of the membrane. Many important proteins have transmembrane domains, including receptors, transporters, and enzymes. These domains are often involved in the transduction of signals across the membrane or in the transport of molecules into or out of the cell.

[0123]

[0124] Delivery systems as contemplated herein consist of or comprise single or multi-pass transmembrane (TM) proteins embedded into the membrane of a vehicle (e.g., cell, extracellular vesicle, or other lipid-containing particle or composition). Additional motifs may be present that regulate the delivery / release of the payload in the required milieu. Thus, instead of direct systemic biologies injections, we aimed to transfuse engineered cells or EVs configured to deliver the cargo straight to the site where it is needed and release it at the right time.

[0124]

[0125] The delivery systems contemplated herein are applicable to any cell type and can be used to deliver any peptide of interest. Suitable cell types include but are not restricted to Hematopoietic Stem Cells (HSCs), induced Pluripotent Stem Cells (iPSCs) and their derived cell products, Adoptive T cell, Dendritic cell (DC), Natural killer cells (NK), NKT cell, or any therapeutic immune or non-immune cells. Such cells may be utilized in autologous or allogenic therapy.

[0125]

[0126] In one embodiment we have modified NK cells to produce and deliver a bi-specific engager (GAGE) in the tumor microenvironment (TME). The TME is composed of cellular and acellular partners, which can regulate various aspects of oncogenesis, 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 is further regulated and can be induced upon either NK cell activation (by activation-induced cleavage) or the tumor milieu (by cleavage upon encountering proteases enriched specifically in the TME. In addition, such an activation-stimulated release may lead to NK cell expansion and activation depending on the tumor antigen availability. In embodiments delivery systems as described herein include next-generation cancer immunotherapy systems with an augmented capacity to produce and release the payload under the desired conditions and at specific sites.

[0126]

[0127] The sheddases which can be used for this payload delivery system are not particularly limited and further described below. For example, the sheddases may be selected from matrix metalloproteinases (MMPs) and Granzyme-B (GrzB), which allow the milieu-specific cargo release. Also, a model based on the ADAM family of MMPs, and the serine-protease Granzyme- B have been presented. In these models, additional functions like tumor targeting, expansion, and / or activation were modulated by adding extracellular and / or intracellular domains. These additional domains can include anything from extracellular cytokines, drugs or pro-drugs, chimeric antigen receptor (CAR)-like or dual antigen receptor (DAR)-like domains or intracellular modulators, inhibitors, signaling domain-containing proteins, etc.

[0127]

[0128] Moreover, the system has been devised for passive release in the tumor milieu depending on the specific MMPs released by the cancer cells or active release induced by the activation of producer cells due to receptor engagement.

[0129] These systems can also be designed in such a way that they are sorted into specific subcellular compartments. We have designed a protein molecule that can be actively sorted into extracellular vesicles (EVs), such as exosomes. The cell releases these exosomes in huge quantities and can release the payload upon cleavage by proteases within the TME or other target sites. These EVs can also be harvested from in-vitro culture and given to patients as an off-the-shelf therapeutic.

[0128]

[0130] In its simplest form, the present invention comprises a prodrug form of a peptide comprising: a first and a second transmembrane region; the first transmembrane region comprising at least one transmembrane unit connected to at least one cleavage site; the second transmembrane region consisting of one transmembrane unit connected to at least one cleavage site; optionally the delivery system may include a targeting moiety for targeting the prodrug to a target cell; wherein when the prodrug contacts the target cell, sheddases cleave the cleavage sites releasing the peptide.

[0129]

[0131] We mainly used a set of TMDs in this multi-pass payload delivery system The N-terminus of the TMDs consist of a type II TMD harboring linker / payload that is connected to a type I TMD domain at the C-terminus. TMDs are preferably at least one or more of CD63, CD9, CD81, CD28, CD4, CD8, CD34, CD69, CD19, CD20, integrins, TNFSF14, TNR1A, aquaporins, NOTCH, NgRl, NRG1, GPi anchor, EGFR, Rhodopsin or any relevant TMDs that provide similar function to the system can be used for the expression of our invention.

[0130]

[0132] Optionally, the second transmembrane region has an intracellular region comprising 41BB, ICOS, and CD3(^ and an MMP / GrzB that cleaves the MMP / GrzB cleavage sites on the exterior of the cell, wherein the action of phosphatases on 41BB, ICOS, and CD3(^ cleaves and activates the MMP / GrzB allowing it to be transported out of the cell and cleave the MMP / GrzB cleavage sites thereby releasing the bispecific antibody / payloads. Optionally, the first and second transmembrane regions are embedded with a NOTCH protein-dependent pulling system with a secretase cleavage site inside the cell membrane with multiple cargos / payloads connected to several cleavage sites on the exterior or interior of the cell. Optionally, the system can be embedded by recruiting the selfassociated antigen via adding a single-chain (a-SAA).

[0131]

[0133] The engineered system may be introduced into the cell individually in the form of viral or nonviral vectors, mRNAs, peptides, proteins, antibodies, nanobodies, oligonucleotides, or extracellular vesicles. (EVs), which can be delivered using CPPs, micelle, liposomes, nanoparticles, dendrimer, nanotube, electroporation, viral transduction, nucleofection, transfection, cell fusion or microinjection. Viral vectors may contain the gene / s encoding the GAGE and viral particles thus produced may carry the gene(s). When viruses infect the cells, gene / s get stably integrated and express the protein encoded. Non -viral vectors may be in the form plasmids carrying the GAGE gene / s. Cells are transfected and this results in the expression of the gene / s encoding the GAGE protein / s. Cells can be selected for stable expression. It could be envisioned that one of the gene delivery strategies that could be utilized is site-specific integration utilizing strategies such as CRISPR (e.g., Cas9, Cpfl, or other Cas effector protein complex) or transposon-transposase strategy. The CRISPR Cas system can be introduced through electroporation or via LNPs and can integrate the gene coding for the GAGE.

[0132]

[0134] The present invention is a peptide delivery system having a site-directed release and activation of the peptide. Site-directed means that the peptide-producing cell or EV comprising the peptide delivery system is targeted to its intended site. The cell or EV is then activated to release the peptide at the target. This approach enables increasing the delivered dose while limiting the number of off-target side effects. The present peptide delivery system includes certain safety and specificity mechanisms so that the biological / payload is only released in high doses at the target site, but not in other tissues where antigens for the specific biological (e.g., antibody-derived biological) may also be expressed. In addition to making these peptide-based drugs safer, more applicable, and more specific, our system could reduce treatment costs. Furthermore, this invention enables the equivalent of more frequent dosing without needing to administer the peptide repetitively because delivery via cargo cells which will produce and store the peptides, provides an in vivo reservoir of inactive peptides.

[0133]

[0135] Mammalian cells have the hardware required to shed many cells’ membrane-bound proteins. Shedding is a controlled, irreversible posttranslational modification performed by families of proteolytic enzymes called proteases or sheddases. Cell biology allows cells to control the expression levels and functions of the substrate proteins and their location on the cell surface. Sheddases as a class are known and comprised of full-time sheddases and part-time sheddases. 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.

[0134]

[0136] Full-time sheddases include ADAM proteases, BACE proteases, serine-protease Granzyme-B and Site-1 proteases. ADAM proteases (metalloproteases) are membrane-anchored type 1 protease and include ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, and ADAM33. The BACE (aspartyl) proteases are also membrane-anchored typel proteases which include BACE1 and BACE2. The Site-1 (serine) proteases are membrane-anchored type 1 protease, including SKLa or SIP.

[0135]

[0137] Part-Time sheddases include Meprin B, MT-MMPs (Membrane Type Matrix Metalloproteases), Pro-protein convertases, Transmembrane serine proteases, Matrix Metalloproteases, Legumain and Cathepsin S and L. Meprin B is a membrane-anchored type 1 metalloprotease. MT-MMPs are membrane-anchored type 1 or GPI-anchored and include MT1- MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, and MT6-MMP. The Pro-protein convertases are membrane-anchored type 1 or soluble proteases and include PCSK1 / 3, KCSK2, furin, PCSK4, PSCK5 / 6, PACE4, PCSK7, and PCSK9. Transmembrane serine proteases are membrane-anchored type II proteases and include Matriptase, Matriptase-2, Matriptase-3, Polymerase- 1, Corin, Hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, Spinesin, Enteropeptidase, HAT, DESCL1, TMPRSS11A, HAT-like 4 and HAT-like 5. The Matrix Metalloproteases are soluble proteases including MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP21, MMP 23A-B, MMP24, MMP25, MMP26, MMP27 and MMP28.

[0136]

[0138] For evaluation, the substrate targets with the highest cleavage efficiency for one MMP and lower than 4000 k (obs) for the other MMPs were considered “specific” substrate sequences for that particular MMP. Substrate hexapeptide sequences are sandwiched between constant regions from Ml 3 phage gene III protein containing FLAG epitope: ADVGGTDYKDDDDKPGGRPTWPSSGGSGXXXXXXTASGAET (SEQ ID NO: 365).

[0137]

[0139] Tables 1-3 below show the selected MMP substrates and their binding affinity sequences specific for MMP2, MMP9, and MMP25.

[0138]

[0140] Table 1: MMP2 Specific Target Substrates* k(obs) (M-1sec1)

[0139]

[0141] T able 2: MMP 9 Specific Target Substrates* k(obs) (M-1sec1)

[0140]

[0142] Table 3: MMP 25 Specific Target Substrates* k(obs) -11)

[0141]

[0143] Proteolytic cleavage substrates include Glycosylphosphatidylinositol (GPi) anchored proteins, membrane proteins with a single, two, three, four, or more transmembrane domains, intracellular domains, extracellular domains in the extracellular matrix or even in neighboring cells. Cleavage can target the juxta -membrane or distal regions of the membrane-bound proteins and can result in the release of protein domains inside and outside the cells (ectodomain and / or endodomain release) (Figures 1-20).

[0142]

[0144] Membrane-bound proteins which cross the plasma membrane once 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 them to the endoplasmic reticulum (ER) lumen during synthesis leading to expression in the extracellular space. A stop-transfer anchor sequence keeps Type I transmembrane proteins anchored to the lipid membrane. Although both Type II and III use a signal-anchor sequence, type II targets the ER lumen with its C-terminal domain, while N-terminal domains target type III to the ER lumen. Type IV can be either IV-A, with their N- terminal domains targeted to the cytosol, and IV-B, if their N-terminal domain is targeted to the lumen. The delivery system of the present invention can have one, two, three, four or more transmembrane proteins (see, e.g., Figs. 4A, 4B, 5A, 5B, 6, 7, 8, 9, etc.).

[0143]

[0145] Optionally, the transmembrane regions following the first or second extra cellular loop (ECL) has an intracellular region comprising 41BB, ICOS, and CD3ζ , wherein the action of kinases on 41BB, ICOS, CD28 and CD3(ζ activates the engineered cells (NK and T cells) to release their endogenous GrzB allowing it to cleave the GrzB cleavage sites thereby releasing the bispecific antibody / payloads (Figures 6, 7, 8, and 15). In NK cells, for example, the CD69 TM is embedded into the membrane with at least two protease cleavage site motifs that regulate the release of the payload in the required milieu. In this model, a single-chain CAR protein is also expressed at N-terminus for a specific delivery. Optionally, the first and second transmembrane regions are embedded with a NOTCH protein-dependent pulling system with a secretase cleavage site inside the cell membrane with multiple cargos / payloads connected to several cleavage sites on the exterior or interior of the cell (Figure 7 and 8). In this system, more than two protease cleavage site motifs are inserted with several potential cargo delivery / releases. This system also has the possibility of releasing intracellular payloads.

[0144]

[0146] In addition to the model described in Figure 7, optionally, the system can be embedded by recruiting the self-associated antigen via adding a single-chain (a-SAA) (Figure 8). This system is a modification of Figure 7 with an extra single-chain against the self-associated antigen (a-SAA) connected with the intracellular domain of CD45. “a-SAA” means a single chain antibody or antibody domain directed against or that binds to a self-associated antigen, preferably an scFv or camelid antibody (VHH domain or VHH domain antibody) or single domain antibody. Self- associated antigens include CD45, CD148, or CD43.

[0145]

[0147] Linkers are peptide regions that separate functional units in a fusion protein. Chen 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 choosing linkers. One of the skill in the art will be able to follow the teachings of this reference and select linkers based on the payload. In certain cases, where polypeptides consisting of various functional domains are needed to be kept at a certain distance, non-cleavable rigid linkers are used. Human-muscle aldolase (HMA) is an example of a rigid linker used to keep the protein domains separated at a set distance. Similarly, flexible, non-cleavable linkers are frequently used in polypeptides harboring multiple functional domains with freedom of movement. These include polyglycine / serine linkers widely used in chimeric proteins. Linkers may be any size needed to perform the function but are preferably from 2 to 128 amino acids in length and most preferably 4-20 amino acids in length. Other contemplated linker lengths include 3-100 amino acids in length,

[0146] 5-50 amino acids in length, 6-45 amino acids in length, 7-40 amino acids in length, 8-30 amino acids in length, 10-20 amino acids in length, and 5-15 amino acids in length. Table 4 below provides examples of linkers and certain constructs comprising linkers. Linkers as disclosed in iGEM ( >http: / / parts. igem.org / Protein_domains / Linker) are also contemplated.

[0147]

[0148] Table 4: Linkers and Certain Constructs Comprising Linkers

[0148]

[0149]

[0149] All the relevant sequences disclosed in the examples below are listed in Table 5.

[0150]

[0150] Table 5: Exemplary Protein Sequences

[0151]

[0152]

[0153]

[0154]

[0151] In the context of this invention, a payload is any amino acid sequence intended for delivery inside a living organism. Suitable payloads include full-length proteins and peptides, enzymes, cytokines, chemokines, signaling proteins, and many more proteins with various functions. In the present invention, the payload is often a therapeutic antibody, bi-specific engagers, and cytokines. The payload is preferably less than 2000 amino acids in size.

[0155]

[0152] 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 genetic modification. The protein sequences disclosed here can be extracellular and / or intracellular. The location on the cell membrane is determined by the type of transmembrane system used.

[0156]

[0153] In some embodiments, the sheddase is an actual part of the peptide delivery system. The sheddase is released when the cell is under the intended condition for the release of the sheddase, thereby allowing the sheddase to be delivered to treat conditions that are not associated with sheddases. An inactivated or split sheddase can be expressed on the surface of the cell which can be activated under the right conditions where a trigger can result in the activation and / or reorganization and assembley of the shedasse into activated one. Moreover, sheddase can also be expressed through an artificial promoter-based system which is activated under the right conditions including the receptor ligation and activation or cleavage of a membrane bound transcription factor following the receptor ligation. Certain sheddases maybe released from the target cells where due to extrinsic or intrinsic pathways lead to the apoptosis including Caspase 3. Caspase-3 is a key protein involved in apoptosis, or programmed cell death, in mammalian cells. It belongs to a family of cysteine proteases known as caspases, which play essential roles in initiating and executing apoptosis. Caspase-3 is considered an effector caspase because it acts downstream in the apoptotic signaling pathways and is responsible for executing the final stages of cell death. Once the Capsase- 3 is released from the dying cells, it is released in the TME and releases the peptide.

[0157]

[0154] Several sheddases have been found to be differentially expressed in the tumor microenvironment (TME) compared to normal tissue. For example, MMPs, GrzB and ADAMs, AD AMTS s are often overexpressed in many different types of cancer, including breast, lung, and pancreatic cancer. The differential expression of sheddases in the TME can have important implications for tumor progression and metastasis. For example, increased expression of MMP9, MMP2, ADAM10 and ADAM17 in the TME has been associated with increased release of pro- tumorigenic growth factors and cytokines, such as epidermal growth factor (EGF) and tumor necrosis factor alpha (TNF-a). This can lead to increased tumor cell proliferation, survival, and invasion. Overall, the differential expression of sheddases in the TME compared to normal tissue highlights the potential for targeting these enzymes as a therapeutic strategy for cancer treatment. Therefore, the differential expression of the sheddases can be exploited for the site directed specific delivery of the payloads.

[0155] We have designed a cell-based peptide delivery system that allows the delivery and release of the payload / cargo directly at the desired site of action. The system consists of single or multi pass transmembrane (TM), or C-type lectin proteins embedded into the membrane of the vehicle cell with additional motifs that regulate the delivery / release of the payload in the required milieu. Thus, instead of direct systemic biologies injections, we aimed to transfuse engineered cells or EVs derived thereof that could deliver the payload to the site where it is needed and release it at the right time.

[0158]

[0156] While the delivery system is independent of cell type, as an example, we have modified NK cells to produce and deliver a bi-specific engager in the tumor environment. This delivery is further regulated and can be induced upon either NK cell activation (activation -induced cleavage) or the tumor milieu (by cleavage upon encountering proteases enriched specifically in the TME) (Figure 19). NK cell activation can be due to the interaction of its germline-encoded activation receptors or specifically engineered receptors, such as chimeric antigen receptor / s against tumor antigen / s. NK cell activation -induced cleavage can be achieved either through Granzyme-B, released following degranulation, or the physical pull, as in notch activation. In addition, such an activation-stimulated release will lead to NK cell expansion and activation depending on the tumor antigen availability. This is a next-generation cancer immunotherapy system with an augmented capacity to produce and release the payload under the desired conditions and at specific sites.

[0159]

[0157] Although many sheddases can be used for this payload delivery system, the current work presents general examples of matrix metalloproteinases (MMPs), which may allow the milieuspecific cargo release. Also, a model based on the ADAM family of MMPs, and the serine-protease Granzyme-B has been presented. In these models, supplementary functions like tumor targeting, expansion, and / or activation were modulated by adding extracellular and / or intracellular domains. These additional domains can include anything from extracellular cytokines, drugs or pro-drugs, chimeric antigen receptor (CAR)-like or dual antigen receptor (DAR)-like domains or intracellular modulators, inhibitors, signaling domain-containing proteins, transcription factors, transcriptional activators or inhibitors, DNA modifying enzymes such as HD AC, proteins containing localization and dimerization domains, proteins containing destruction and dimerization domains, proteins containing hypoxia-responsive domains, proteins that activate immune cells, proteins that increase persistence and proliferation of immune cells, proteins that induce migration of immune cells, split receptors such as split CARs, etc.

[0158] Moreover, the system has been devised for passive release in the tumor milieu depending on the specific MMPs released by the cancer cells or active release induced by the activation of producer cells due to receptor engagement.

[0160]

[0159] The membrane embedded systems can also be designed in such a way that they are sorted into specific subcellular compartments. In some embodiments, we engineered the payload that will be sorted into extracellular vesicles (EVs), such as exosomes. The cell releases these exosomes in huge quantities and can release the payload upon cleavage by proteases within the TME or other target sites (Figures 4A-B, 5A-B, 9, 10, and 24-28). In theory, these EVs can also be harvested from in-vitro culture and given to patients as an off-the-shelf therapeutic.

[0161]

[0160] In some embodiments, in NK or T cells, the CD69 TM is embedded into the membrane with at least two protease cleavage site motifs that regulate the release of the payload in the required milieu (Figure 17A-B and 18A-B). In addition to the model described in Figure 17A-B and 18A- B, optionally, the system can be equipped with a single-chain CAR protein at N-terminus for a specific delivery (Figure 6).

[0162]

[0161] In some embodiment, a modified tetraspanin can be used, where more than two protease cleavage site motifs are inserted with several potential cargo delivery / releases. This system also has the possibility of releasing intracellular payloads (Figure 7).

[0163]

[0162] In some embodiment, further modification of tetraspanin can be used with an extra singlechain against the self-associated antigen (a-SAA) connected with the intracellular domain of CD45 (Figure 8).

[0164]

[0163] In some embodiments, the intracellular or extracellular domain comprises of OX-40, 4- 1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86),PDL-1 , programmed death-I (PD- 1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CDI la / CD 18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, 1g alpha (CD79a), DAP-10, Fe gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), The costimulatory ligand (PD-L2, 4-1 BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD70, CD83, HLA-G, MICA, Ml CB, HVEM, lymphotoxin b receptor, 3 / TR6, ILT3, ILT4), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI Id, ITGAE, CD 103, ITGAL, CDI la, LFA-1, IT GAM, CDI lb, ITGAX, CDI le, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, a ligand that specifically binds with CD83, or any combination thereof.

[0165]

[0164] In some embodiments, the antigen binding domain specifically binds BCMA, MUC16 (also known as CA125), EGFR, EGFRvin, MUCI, Flt-3, WT-1, CD38, CD70, CD90, CD133, MHC- WTI , TSPANIO, MHC-PRAME, MHC-NY-ESOI, HER2 (ERBB2), CA-IX (Carbonic anhydrase IX), LIVI, ADAMIO, CHRNA2, LeY, NKG2D, CSI, CD44v6, CD24, LGR5, ALDH, ALDH1, CD54, Seal, CD271, CD123, CD36, CD109, CD110, CD71 -negative, CCA, ABCG2, Claudin- 18.2 (Claudin-18A2, or Claudinl8 isoform 2), PSCA, DLL3 (Delta-like protein 3, Drosophila Delta homolog 3, Delta3 ), Mud 7 (Mucinl 7, Muc3, Muc3), FAP alpha (Fibroblast Activation 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 scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGI, LLCAM, integrin a5pi, integrin av|33, legumain, MORAb-009, MS4A1, MUCI, mucin CanAg, C-MET, CCR4, CD 152, CD 10, CD 19, CD20, CD200, N- glycolylneuraminic acid, NPC-IC, PDGF-R a, PDL192, phosphatidylserine, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF-P, TRAIL-R1, TRAIL-R2, folic acid receptor, transferrin receptors, and any combination thereof. These antigen binding domains may be used in CARs and DARs for targeting the payload. CARs and / or antibodies that target the antigens are disclosed, for example, in the following: BCMA- W0201616630, W02020150339, WO2019196713, WO2016014565, W02017025038; MUC16: US9, 169,328, WO2016149368, W02020023888; EGFRvIII: WO2017125830, W02016016341; Flt3: WO2018222935, W02020010284, W02017173410; CD20: WO2018145649, W02020010235, WO2020123691; CD38: WO2017025323; CD70: WO2019152742, WO2018152181; CD33: WO2016014576; CD133: W02018072025; CSI: W02019030240; RORI: WO2016115559; CD19:

[0166] W02002077029, USI 1,077,144; Claudin: W02018006882, W02021008463; DLL3: W02020180591; WTI: US20160152725 Al, US7622119B2; CD23: US6011138A, CN1568198A; CD30: US10815301B2, US10808035B2; PRAME: US20180148503A1, W02020186204A1; LIVI: US20200231699A1; NKG2D: WO2021 1 79353 Al, US20210269501A1; FAP Alpha: US20200246383A1, US20210115102A1; PSMA: US20210277141 Al, W02020108646 Al; MSLN: CN109680002A, CN109628492A.

[0167]

[0165] In some embodiments, the sheddase cleavage site comprises one or more of the multiple protease cleavage sites and is recognized by one or more of thrombin, trypsin, plasmin, prostatespecific antigen (PSA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase (MMP), matriptase (MT-SP1), legumain, a disintegrin and metalloproteinase (ADAM), and transmembrane Serine Protease (TMPRSS).

[0168]

[0166] In some embodiments, the sheddase that recognizes the protease cleavage site or that recognizes one or more of the multiple protease cleavage sites, is one or more of thrombin, trypsin, plasmin, prostate-specific antigen (PSA), urokinase plasminogen activator (uP A), urokinase plasminogen activator receptor (uP AR), matrix metalloproteinase (MMP), matriptase (MT-SP1), legumain, a disintegrin and metalloproteinase (ADAM), transmembrane Serine Protease (TMPRSS), Granzyme B, activated protein C, Caspase, Cathepsin, Chymase, Elastase, Guanidinobenzoatase, HtrAl, Human Neutrophil Elastase, Lactoferrin, Marapsin, NS3 / 4A, PACE4, tissue plasminogen activator (tPA), DESCI, DPP-4, Hepsin, Matriptase-2, secretase, kallikrein-related peptidase (KLK), and tryptase, or a serine protease, a cysteine-type lysosomal protease, a metalloproteinase, a coagulation factor protease, or an aspartyl -type lysosomal protease.

[0169]

[0167] In some embodiments, the payload comprise a cytokine or chemokine consisting a fraction or derivative of a sulfated xylan, which is an antagonist of a ligand selected from the list comprising IL-la, IL-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-3, IL-14, IL- 15 or IL- 17 family of cytokines including IL-25, interferon, G-CSF, M-CSF, GM-CSF, BDNF, CNTF, EGF, EPO, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, LIF, PDGF, SCF, TGFa, TGFB, TNFa, TNFB, TPO, VEGF, GH, NGF, NT3, NT4, NT5, NT6, NT7, oncostatin M (OSM), insulin, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, a member of the MIP-1 family including MIP-la, MIP-2, eotaxin ( eotaxin-1, -2 or -3), , PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase; an enzyme of the cathepsin family, a cell adhesion molecules such as PECAM-1, a soluble or cell- or virus-bound receptor, cytokine- induced neutrophil chemoattractant (KC), TNF-a and IFN-g), 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, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, or any combination thereof.

[0170]

[0168] In some embodiments the payload may comprise thrombin. In other embodiments the payload may be a vaccine. In yet another embodiment the payload can be Urokinase delivery. The present constructs may enable delivery of payloads through the blood brain barrier.

[0171]

[0169] As used herein “a-TAA” means a single chain antibody or antibody domain directed against or that binds to a tumor-associated antigen, preferably an scFv or camelid antibody (VHH domain antibody or VHH domain) or single domain antibody or antibody binding domain. Exemplary tumor-associated antigens bound by the starting polypeptides used in the invention include for example, pan B antigens (e.g. CD20 found on the surface of both malignant and non- malignant B cells such as those in non-Hodgkin's lymphoma) and pan T cell antigens (e.g. CD2, CD3, CD5, CD6, CD7). Other exemplary tumor associated antigens comprise but are not limited to MAGE-1, MAGE-3, MUC-1, HPV 16, HPV E6 & E7, TAG-72, CEA, a-Lewisy, L6-Antigen, CD19, CD22, CD25, CD30, CD33, CD37, CD44, CD52, CD56, mesothelin, PSMA, HLA-DR, EGF Receptor, VEGF Receptor, and HER2 Receptor. Carcinoembryonic antigen (CEA), and a- fetoprotein (AFP) are two examples of such tumor associated antigens. Other targets include the MICA / B ligands of NKG2D. These molecules are expressed on many types of tumors, but not normally on healthy cells. Additional specific examples of tumor associated antigens include epithelial cell adhesion molecule (Ep-CAM / TACSTDl), mesothelin, tumor-associated glycoprotein 72 (TAG-72), gplOO, Melan-A, MART-1, KDR, RCAS1, MDA7, cancer-associated viral vaccines (e.g., human papillomavirus antigens), prostate specific antigen (PSA, PSMA), RAGE (renal antigen), CAMEL (CTL-recognized antigen on melanoma), CT antigens (such as MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigens (e.g., MUC1, mucin-CA125, etc.), cancer-associated ganglioside antigens, tyrosinase, gp75, C-myc, Marti, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7, Ep- CAM, tumor-derived heat shock proteins, and the like (see also, e.g., Acres 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): S 161 -8; and Ohshima et al., Int J Cancer. 2001 Jul. 1; 93(1):91 -6). Other exemplary tumor associated antigen targets include CA 195 tumor-associated antigen-like antigen (see, e.g., U.S. Pat. No. 5,324,822) and female urine squamous cell carcinoma-like antigens (see, e.g., U.S. Pat. No. 5,306,811), and the breast cell tumor associated antigens described in U.S. Pat. No. 4,960,716.

[0172]

[0170] Preferably, such a tumor-associated antigen is located on or in the cell membrane of a tumor cell. Examples of tumor-associated antigens are described, e.g., in DeVita et al. (Eds., “Biological Therapy of Cancer”, 2. Edition, Chapter 3: Biology of Tumor Antigens, Lippincott Company, ISBN 0-397-51416-6 (1995)). Not -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, IL13Ralpha, IL13Ralpha2, AIM-2, AIM-3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, RBPSUH, NKTR, EGFRvin, LIC AM, Livin, LivinP, Nestin, OLIG2, ART1, ART4, Glil, Cav-1, CD74, E-Cadherin, GAGE-1, Ganglioside / GD2, PROXI, PSCA, phCG, WT1, mesothelin, melan-A, SSX-2, PLK1, VEGF-A, VEGFR2, and Tie-2.

[0173]

[0171] Spaciotemporal Regulation and Delivery

[0174]

[0172] It is envisioned to utilize embodiments of the invention for targeted payload release in the tumor microenvironment or tissue-specific environment, and this can enable optimal time-point and anatomical location for release and activation of cargos such as release and unmasking of pro- inflammatory cytokines (e.g., TNF, IL2, IL15, IL18) in the tumor microenvironment, or conditional release of certain growth factors in non-malignant disorders (e.g., GMCSF in bone marrow transplantation, IL2 in certain immune deficiencies, Insulin in diabetes). Furthermore, temporal and conditional cargo release according to the invention is envisioned to limit the currently observed systemic off-target effects and / or limit continuous exposure and response. Conditional induction of responses is beneficial in numerous diseases where a clear circadian-like rhythm in symptoms formation, such as inflammatory bowel disease and Diabetes Mellitus.

[0175]

[0173] It is also of great importance to be able to utilize bispecific engagers and other proteins with short half-lives and / or with high risk of off -target activity in other tissues in the right microenvironment at the right time. The embodiments of the invention are applicable in such conditions where spatio-temporal release is critical. One such example is the generation of a dispanin coding for a bispecific CD38-CD3 engager in the ectodomain-loop, which is cleavable only in the bone marrow micro-environment together with an anti -BCMA CAR and thus decrease off-target activity of the engager that is released as well as the CAR. In this model system, a bispecific CD38-CD3 engager with a very short half-life beneficially renders the systemic distribution and impact more unlikely. Furthermore, for embodiments of the invention, due to continuous protein turnover and release by the ferrying cells, one-time administration of the cells is estimated to be more potent and in some cases equivalent in efficacy to multiple-administration dosing regimes of other delivery systems while at the same time posing limited side effects.

[0176]

[0174] Example 1: General Structure

[0175] In one embodiment the present invention comprises a delivery system for a protein payload in which sheddases present in the body release the payload. Referring to Figures 1-2, the payload delivery system for delivery of a single payload comprises a first transmembrane anchor, a cytoplasmic region, a first site cleavable by a sheddase, a first linker, a payload sequence, a second linker, a second site cleavable by a sheddase, and optionally a single-chain specific CAR.

[0177]

[0176] The transmembrane anchor can be selected from CD63, CD9, CD81, CD28, CD4, CD8, CD34, CD69, CD19, CD20, integrins, TNFSF14, TNR1A, aquaporins, NOTCH, NgRl, NRG1, GPi anchor, EGFR, Rhodopsin or any relevant TMDs that provide similar function such as membrane anchoring, and signal transduction modulation can be used for the expression of our invention.

[0178]

[0177] The cytoplasmic domain can be selected from CD63, CD69, CD9, CD28, CD81, CD34, CD3, CD4, CD8 or any relevant cytoplasmic domain that provides similar function (e.g., with regard to anchoring, mobility within the plasma membrane, and / or the ability to sort to EVs, as well as signal transduction) to the system can be used. In some embodiments, the intracellular or extracellular domain comprises of OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86),PDL-1 , programmed death-I (PD- 1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-I (LFA-1 (CDI la / CD 18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, 1g alpha (CD79a), DAP-10, Fe gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), The costimulatory ligand (PD-L2, 4-1 BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD70, CD83, HLA-G, MICA, Ml CB, HVEM, lymphotoxin b receptor, 3 / TR6, ILT3, ILT4), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 19, CD8alpha, CD8beta, IL-2Rbeta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI Id, ITGAE, CD 103, ITGAL, CDI la, LFA-1, IT GAM, CDI lb, ITGAX, CDI le, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, a ligand that specifically binds with CD83, or any combination thereof.

[0179]

[0178] Linkers can be selected from any suitable linker with linkers as disclosed herein, e.g., the linkers set forth in Table 4, being preferred.

[0179] The sheddases and their cleavable sites may be selected from part or full-time sheddases. The full-time sheddases may be selected include ADAM proteases, BACE proteases, serine- protease Granzyme-B and Site-1 proteases. Adam proteases (metalloproteases) are membrane- anchored type 1 protease and include ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, and ADAM33. The BACE (aspartyl) proteases are also membrane-anchored typel proteases which include BACE1 and BACE2. The Site-1 (serine) proteases are membrane-anchored type 1 protease, including SKI-a or SIP.

[0180]

[0180] Part-Time sheddases and their cleavable sites include Meprin B, MT-MMPs (membrane type matrix metalloproteases), Pro-protein convertases, Transmembrane serine proteases, Matrix metalloproteases, Legumain and Cathepsin S and L. Meprin B is a membrane-anchored type 1 metalloprotease. MT-MMPs are membrane-anchored type 1 or GPI-anchored and include MT1- MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, and MT6-MMP. The Pro-protein convertases are membrane-anchored type 1 or soluble proteases and include PCSK1 / 3, KCSK2, furin, PCSK4, PSCK5 / 6, PACE4, PCSK7, and PCSK9. Transmembrane serine proteases are membrane-anchored type II proteases and include matriptase, Matriptase-2, matriptase-3, polymerase-1, Corin, Hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, Spinesin, Enteropeptidase, HAT, DESCL1, TMPRSS11A, HAT-like 4 and HAT-like 5. The Matrix Metalloproteases are soluble proteases including MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP21, MMP 23A-B, MMP24, MMP25, MMP26, MMP27 and MMP28.

[0181]

[0181] In some embodiments, the payload comprise a cytokine or chemokine consisting a fraction or derivative of a sulfated xylan, which is an antagonist of a ligand selected from the list comprising IL-la, IL-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-3, IL-14, IL- 15 or IL- 17 family of cytokines including IL-25, interferon, G-CSF, M-CSF, GM-CSF, BDNF, CNTF, EGF, EPO, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF 12, FGF 12, FGF 13 , FGF 14, FGF 15, FGF 16, FGF 17, FGF 18, FGF 19, FGF20, FGF21 , FGF22, FGF23, LIF, PDGF, SCF, TGFa, TGFB, TNFa, TNFB, TPO, VEGF, GH, NGF, NT3, NT4, NT5, NT6, NT7, oncostatin M (OSM), insulin, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, a member of the MIP-1 family including MIP-la, MIP-2, eotaxin ( eotaxin-1, -2 or -3), , PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase; an enzyme of the cathepsin family, a cell adhesion molecules such as PECAM-1, a soluble or cell- or virus-bound receptor, cytokine- induced neutrophil chemoattractant (KC), TNF-a and IFN-g), 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, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, or any combination thereof.

[0182]

[0182] In other embodiments, further modifications can be applied to the system by adding more cleavable sheddases and more payloads on the exterior or interior of the cell. Wherein cleavage by the sheddase releases a peptide or protein. The peptide or protein may be active when cleaved or may still require additional activation via mechanisms present in the cell. The activation mechanisms can be naturally present or genetically engineered. Figures 4A-B and 5A-B show an embodiment of the present invention comprising 4 transmembrane domains. To generate an elaborate delivery system that can harbor different payloads in the intra- and extra-cellular environment needs to cross the plasma membrane multiple times. In this example we used tetraspanin TMDs linked through payloads spanning the sheddases sites to compose the delivery system. A first protease site cleavable MMP 9, a TNF-a payload and a second protease site cleavable by MMP9. Figures 4A-B and 5A-B differ by the presence of the EV sorting sequence.

[0183]

[0183] Example 2: Delivery of a BIKE

[0184]

[0184] In one embodiment the delivery system of the present invention is configured to deliver a BIKE. Referring to Figure 2 an embodiment of the invention comprises a BIKE flanked by protease cleavage sequences embedded in the extracellular loop between a Type-II transmembrane protein and a GPI anchor. This particular embodiment is encoded by the amino acid sequence set forth 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, cleavage sequence, and payload followed by another cleavage sequence. The C-terminal of this protein is bound to the plasma membrane through the GPi anchor. Sheddases result in cleavage, thus releasing the payload in the extracellular milieu.

[0185] MCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQGGPLGVRGGMEVQ LVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGY ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRG GGGSGGGGSGGGGSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVL VIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKL TVLPSGQAGAAASESLFVSNHAYEASGGPEQVQLVQSGAEVKKPGSSVKVSCKASGYTF TDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLR SEDTAVYYCARGRPAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSAS VGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGT DFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKGGSGEQKLISEEDLGGPLGMTS GGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINF NEKAIHKEGCVEKIGGWLRKNVVLRDKLVKCEGISLLAQNTSWLLLLLLSLSLLQATDF MSL (SEQ ID NO: 1)

[0186]

[0185] This sequence is also set out in Figure 3, with a legend showing the domains and motifs.

[0187]

[0186] The corresponding cDNA is set forth below:

[0188]

[0187] ATGTGCGGCGCATGCAAGGAAAACTACTGTCTGATGATCACATTCGCCATCTT CCTGAGCCTGATCATGCTGGTGGAAGTGGCCGCTGCCATCGCCGGATACGTGGGAG GCAGCGGGCCTGTGCGGCGGTACCAGGGAGGCCCACTGGGAGTTAGAGGCGGCATG GAAGTGCAGCTGGTCGAATCGGGAGGCGGCGTGGTGAGACCAGGAGGCAGCCTCCG GCTGTCTTGTGCCGCGAGTGGTTTCACCTTTGACGACTACGGCATGAGCTGGGTGAG ACAGGCTCCTGGAAAGGGCCTGGAATGGGTTTCCGGCATCAACTGGAACGGCGGAT CGACTGGCTACGCCGACAGCGTGAAAGGCAGATTCACCATCAGCAGAGATAACGCC AAGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTA CTACTGCGCCAGAGGCAGAAGCCTGCTGTTCGACTACTGGGGACAGGGCACACTGG TGACCGTCAGCAGAGGCGGCGGCGGCTCTGGCGGCGGCGGAAGCGGAGGCGGCGG ATCCTCCGAGCTGACACAGGACCCCGCTGTTAGCGTGGCTCTGGGCCAGACCGTGCG GATCACCTGTCAGGGCGACAGCCTCCGCTCCTACTACGCCAGCTGGTACCAACAAAA ACCTGGCCAAGCTCCTGTGCTGGTCATCTACGGAAAGAACAACAGACCTAGTGGCAT CCCTGATAGATTTAGCGGCAGCAGCAGCGGCAACACCGCCTCACTGACCATTACAG GCGCCCAGGCCGAGGATGAGGCTGACTATTACTGCAACTCCAGGGACAGCTCTGGA AATCATGTGGTCTTCGGCGGCGGCACAAAGCTGACCGTGCTGCCTTCTGGCCAAGCC GGTGCCGCCGCGTCTGAGAGCCTGTTCGTGAGCAACCACGCCTACGAGGCCAGCGG CGGCCCCGAGCAGGTGCAGCTGGTGCAGAGCGGCGCTGAGGTGAAAAAGCCCGGCT CTAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCACAGATTATAATATGC ACTGGGTCAGACAGGCCCCTGGGCAGGGACTGGAATGGATCGGCTATATCTACCCCT ACAACGGCGGAACAGGCTACAACCAGAAGTTCAAGAGCAAGGCCACCATCACAGCC

[0189] GACGAGAGCACCAACACCGCTTATATGGAACTGTCTTCTCTGCGGAGCGAAGATACC GCCGTGTACTACTGTGCCAGAGGCCGGCCTGCCATGGACTACTGGGGCCAGGGCAC CCTGGTGACAGTGTCCTCAGGCGGCGGCGGTAGCGGCGGCGGCGGCAGCGGCGGCG GTGGTTCTGATATCCAGATGACCCAGAGCCCCAGCAGCCTGTCCGCCAGTGTCGGAG ATAGGGTGACCATCACCTGCAGAGCCTCTGAAAGCGTTGACAATTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAACCTGGCAAAGCCCCTAAGCTTCTTATTTACGCCG CTTCCAACCAGGGCTCTGGCGTGCCCAGCCGGTTCAGCGGCTCAGGATCTGGAACCG ACTTTACGCTGACAATCAGCTCTCTGCAGCCTGATGACTTCGCCACATACTACTGCC AGCAAAGCAAAGAGGTGCCTTGGACCTTCGGCCAGGGCACCAAGGTGGAGATTAAG GGCGGGTCCGGAGAGCAGAAGCTGATCTCTGAGGAAGACCTGGGAGGCCCTCTGGG CATGACCTCTGGAGGCAGCGGCCCTAAGAACAACCACACCGCCAGCATCCTGGACC GGATGCAGGCCGACTTTAAGTGCTGCGGCGCCGCTAATTACACCGACTGGGAGAAA ATCCCCAGCATGAGCAAGAATAGAGTGCCCGATAGCTGTTGCATCAACGTGACAGT GGGCTGCGGCATCAATTTCAACGAGAAGGCTATCCACAAGGAGGGCTGCGTGGAAA AGATCGGCGGCTGGCTGAGAAAGAACGTGGTGCTGAGAGACAAGCTCGTGAAATGC GAGGGCATCAGCCTGCTGGCCCAGAACACATCCTGGCTGCTGCTGCTGCTGCTCAGC CTGAGTCTGCTGCAAGCCACCGATTTCATGTCTCTGTAA (SEQ ID NO: 124)

[0190]

[0188] As shown in Figure 3, SEQ ID NO: 123 or 1 is divided into functional motifs as follows:

[0189] CD63 -CYTOPLASMIC MCGACKENYC (SEQ ID NO: 125), which is encoded by DNA ATGTGCGGCGCATGCAAGGAAAACTACTGT (SEQ ID NO: 126)

[0191]

[0190] CD63-TM-HELICAL(inside-out) LMITFAIFLSLIMLVEVAAAI (SEQ ID NO: 127), which is encoded by DNA

[0192] CTGATGATCACATTCGCCATCTTCCTGAGCCTGATCATGCTGGTGGAAGTGGCCGCT GCCATC (SEQ ID NO: 128).

[0193]

[0191] Linker-a: GGSGPVRRYQ (SEQ ID NO: 129), which is encoded by DNA

[0194] GGAGGCAGCGGGCCTGTGCGGCGGTACCAG (SEQ ID NO: 130)

[0195]

[0192] MMP2: GGPLGVRGG (SEQ ID NO: 131), which is encoded by DNA

[0196] GGAGGCCCACTGGGAGTTAGAGGCGGC (SEQ ID NO: 132)

[0197]

[0193] VL-a-CD16-118AA

[0198] MEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGG

[0199] STGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTV SR (SEQ ID NO 133) which is encoded by DNA

[0200] ATGGAAGTGCAGCTGGTCGAATCGGGAGGCGGCGTGGTGAGACCAGGAGGCAGCCT

[0201] CCGGCTGTCTTGTGCCGCGAGTGGTTTCACCTTTGACGACTACGGCATGAGCTGGGT

[0202] GAGACAGGCTCCTGGAAAGGGCCTGGAATGGGTTTCCGGCATCAACTGGAACGGCG

[0203] GATCGACTGGCTACGCCGACAGCGTGAAAGGCAGATTCACCATCAGCAGAGATAAC

[0204] GCCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGT

[0205] GTACTACTGCGCCAGAGGCAGAAGCCTGCTGTTCGACTACTGGGGACAGGGCACAC

[0206] TGGTGACCGTCAGCAGA (SEQ ID NO: 134)

[0207]

[0194] Linker-b and Linker-c: GGGGSGGGGSGGGGS 15AA (SEQ ID NO: 135), which is encoded by DNA GGCGGCGGCGGCTCTGGCGGCGGCGGAAGCGGAGGCGGCGGATCC (SEQ ID NO: 136)

[0208]

[0195] VH-a-CD16-107AA

[0209] SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRF

[0210] SGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVL (SEQ ID NO: 137), which is encoded by DNA

[0211] TCCGAGCTGACACAGGACCCCGCTGTTAGCGTGGCTCTGGGCCAGACCGTGCGGATC

[0212] ACCTGTCAGGGCGACAGCCTCCGCTCCTACTACGCCAGCTGGTACCAACAAAAACCT GGCCAAGCTCCTGTGCTGGTCATCTACGGAAAGAACAACAGACCTAGTGGCATCCCT GATAGATTTAGCGGCAGCAGCAGCGGCAACACCGCCTCACTGACCATTACAGGCGC CCAGGCCGAGGATGAGGCTGACTATTACTGCAACTCCAGGGACAGCTCTGGAAATC

[0213] ATGTGGTCTTCGGCGGCGGCACAAAGCTGACCGTGCTG (SEQ ID NO: 138)

[0214]

[0196] Flanking 20AA: PSGQAGAAASESLFVSNHAY (SEQ ID NO: 139), which is encoded by DNA

[0215] CCTTCTGGCCAAGCCGGTGCCGCCGCGTCTGAGAGCCTGTTCGTGAGCAACCACGCC

[0216] TAC (SEQ ID NO: 140)

[0217]

[0197] 7AA flanking peptide: EASGGPE (SEQ ID NO: 141), which is encoded by DNA

[0218] GAGGCCAGCGGCGGCCCCGAG (SEQ ID NO: 142)

[0219]

[0198] VL-a-CD33-116AA

[0220] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGG

[0221] TGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS (SEQ ID NO: 143) which is encoded by DNA

[0222] CAGGTGCAGCTGGTGCAGAGCGGCGCTGAGGTGAAAAAGCCCGGCTCTAGCGTGAA

[0223] GGTGTCCTGCAAGGCCAGCGGCTACACCTTCACAGATTATAATATGCACTGGGTCAG

[0224] ACAGGCCCCTGGGCAGGGACTGGAATGGATCGGCTATATCTACCCCTACAACGGCG

[0225] GAACAGGCTACAACCAGAAGTTCAAGAGCAAGGCCACCATCACAGCCGACGAGAGC

[0226] ACCAACACCGCTTATATGGAACTGTCTTCTCTGCGGAGCGAAGATACCGCCGTGTAC

[0227] TACTGTGCCAGAGGCCGGCCTGCCATGGACTACTGGGGCCAGGGCACCCTGGTGAC

[0228] AGTGTCCTCA (SEQ ID NO: 144)

[0229]

[0199] VH-a-CD33

[0230] DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGS

[0231] GVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 145), which is encoded by DNA

[0232] GATATCCAGATGACCCAGAGCCCCAGCAGCCTGTCCGCCAGTGTCGGAGATAGGGT

[0233] GACCATCACCTGCAGAGCCTCTGAAAGCGTTGACAATTACGGCATCAGCTTCATGAA

[0234] CTGGTTCCAGCAGAAACCTGGCAAAGCCCCTAAGCTTCTTATTTACGCCGCTTCCAA

[0235] CCAGGGCTCTGGCGTGCCCAGCCGGTTCAGCGGCTCAGGATCTGGAACCGACTTTAC

[0236] GCTGACAATCAGCTCTCTGCAGCCTGATGACTTCGCCACATACTACTGCCAGCAAAG

[0237] CAAAGAGGTGCCTTGGACCTTCGGCCAGGGCACCAAGGTGGAGATTAAG (SEQ ID NO: 146)

[0238]

[0200] Myc-tag EQKLISEEDL (SEQ ID NO: 147), which is encoded by DNA

[0239] GAGCAGAAGCTGATCTCTGAGGAAGACCTG (SEQ ID NO: 148)

[0240]

[0201] MMP9: GGPLGMTS (SEQ ID NO: 149), which is encoded by DNA GGAGGCCCTCTGGGCATGACCTCT (SEQ ID NO: 150)

[0202] CD63 -EXTRACELLULAR

[0241] PKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKA IHKEGCVEKIGGWLRKNV (SEQ ID NO: 151), which is encoded by DNA

[0242] CCTAAGAACAACCACACCGCCAGCATCCTGGACCGGATGCAGGCCGACTTTAAGTG

[0243] CTGCGGCGCCGCTAATTACACCGACTGGGAGAAAATCCCCAGCATGAGCAAGAATA

[0244] GAGTGCCCGATAGCTGTTGCATCAACGTGACAGTGGGCTGCGGCATCAATTTCAACG

[0245] AGAAGGCTATCCACAAGGAGGGCTGCGTGGAAAAGATCGGCGGCTGGCTGAGAAA

[0246] GAACGTG (SEQ ID NO: 152)

[0247]

[0203] GPi-anchor: VLRDKLVKCEGISLLAQNTSWLLLLLLSLSLLQATDFMSL (SEQ ID NO: 153), which is encoded by DNA

[0248] GTGCTGAGAGACAAGCTCGTGAAATGCGAGGGCATCAGCCTGCTGGCCCAGAACAC

[0249] ATCCTGGCTGCTGCTGCTGCTGCTCAGCCTGAGTCTGCTGCAAGCCACCGATTTCATG TCTCTG (SEQ ID NO: 154)

[0250]

[0204] Example 3: Payload Delivered into Extracellular Vesicles

[0251]

[0205] Referring to Figure 4A-B, this embodiment is a payload with MMP cleavable sequences embedded in the extracellular loop between the third and fourth transmembrane domain of tetraspanin. This particular molecule will get sorted into extracellular vesicles due to the presence of the GYEVM motif (SEQ ID NO: 337).

[0252]

[0206] The amino acid sequence in SEQ ID NO 155 below encodes the construct.

[0253] MCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQGGPLGVRGGMEVQ LVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGY ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRG GGGSGGGGSGGGGSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVL VIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKL

[0254] TVLPSGQAGAAASESLFVSNHAYEASGGPEQVQLVQSGAEVKKPGSSVKVSCKASGYTF TDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLR SEDTAVYYCARGRPAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSAS VGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGT DFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKGGSGEQKLISEEDLGGPLGMTS

[0255] GGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINF NEKAMKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM (SEQ ID NO: 155)

[0256]

[0207] The corresponding cDNA is set forth below:

[0257] ATGTGCGGCGCCTGTAAAGAAAACTACTGCCTGATGATCACATTCGCAATCTTCCT GTCCCTCATCATGCTGGTGGAGGTGGCCGCCGCCATCGCTGGCTACGTGGGCGGC AGCGGCCCTGTGAGGAGATACCAGGGCGGTCCACTGGGCGTGCGGGGCGGCATG GAAGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGAGACCTGGCGGGTCCCTG CGGCTGAGCTGTGCCGCCAGCGGATTCACATTCGACGATTACGGCATGAGCTGGG TCAGACAGGCCCCAGGAAAAGGCCTGGAATGGGTGTCTGGCATTAACTGGAACG GCGGCAGCACCGGCTACGCCGATAGCGTTAAGGGCAGATTCACCATCAGCAGAG ATAACGCCAAGAACTCTCTGTACCTGCAGATGAATAGCCTGCGGGCCGAAGATAC CGCCGTCTACTACTGTGCCCGGGGAAGAAGCTTGCTGTTTGACTACTGGGGACAG GGCACACTGGTCACCGTGTCTAGGGGCGGCGGCGGCAGCGGAGGAGGCGGGTCG GGCGGCGGCGGATCCAGCGAGCTGACACAAGATCCTGCTGTGAGCGTCGCCCTGG GCCAGACCGTGCGGATCACTTGCCAGGGAGATAGCCTGAGGTCCTACTACGCCAG TTGGTATCAACAGAAGCCTGGACAGGCCCCTGTACTGGTGATCTACGGCAAGAAC AACAGACCTTCTGGCATCCCTGATAGATTCAGCGGCTCCAGCAGCGGAAACACCG CTAGCCTGACAATCACCGGAGCTCAGGCTGAGGACGAGGCCGACTACTACTGCAA CAGCCGGGACTCTTCAGGAAATCACGTGGTGTTCGGCGGCGGCACCAAGCTGACA GTGCTGCCTAGCGGCCAGGCCGGCGCCGCGGCCAGCGAAAGCCTGTTCGTGTCTA ACCACGCCTACGAGGCTTCCGGCGGCCCCGAGCAGGTGCAGCTGGTGCAAAGCG GCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAAGTGTCCTGCAAGGCCTCCG GCTACACCTTTACCGACTACAATATGCACTGGGTTCGGCAGGCCCCTGGCCAAGG TCTCGAGTGGATCGGCTACATCTATCCTTACAACGGCGGCACCGGCTACAACCAG AAGTTCAAAAGCAAGGCAACAATCACAGCCGACGAGAGCACTAACACCGCCTAC ATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCTGTTTACTACTGCGCCAGAG GCAGACCAGCTATGGACTACTGGGGCCAAGGAACCCTGGTGACCGTGTCATCTGG GGGCGGCGGATCTGGCGGCGGCGGGAGCGGCGGCGGAGGAAGTGATATCCAGAT GACCCAGAGCCCTAGCAGCCTGTCTGCCTCTGTTGGCGACCGGGTGACCATCACC TGCAGAGCTTCTGAATCAGTTGACAACTACGGCATCAGCTTCATGAACTGGTTCC AGCAGAAACCTGGCAAGGCCCCCAAGCTGCTGATCTATGCCGCTTCTAATCAGGG CAGCGGCGTGCCTAGCAGATTTAGCGGCAGTGGATCGGGAACCGACTTCACCCTG ACCATTTCTTCGCTTCAGCCCGACGATTTCGCCACATACTATTGTCAGCAGAGCAA GGAGGTGCCATGGACATTTGGCCAGGGCACTAAGGTGGAAATCAAGGGCGGCAG CGGAGAGCAGAAGCTGATCTCCGAGGAAGACCTGGGTGGACCCCTGGGCATGAC CTCTGGCGGCAGCGGCCCTAAGAACAACCACACAGCCAGCATCCTGGACAGAAT GCAGGCCGACTTCAAGTGCTGCGGCGCCGCTAATTACACCGACTGGGAGAAAATC CCCTCCATGAGCAAAAACCGGGTGCCCGACAGCTGTTGCATCAATGTGACCGTGG GCTGTGGCATTAACTTCAACGAGAAGGCTATCCATAAGGAGGGCTGCGTCGAGAA GATCGGCGGCTGGCTGAGAAAGAACGTGCTGGTGGTGGCCGCCGCCGCCCTGGGT ATCGCCTTCGTGGAAGTGCTGGGCATCGTGTTCGCCTGCTGCCTGGTGAAGTCTAT CAGATCTGGCTATGAGGTGATG (SEQ ID NO: 156)

[0258]

[0208] SEQ ID NO 155 is divided into functional motifs as follows:

[0259] CD63 -CYTOPLASMIC: MCGACKENYC (SEQ ID NO: 157), which is encoded by DNA ATGTGCGGCGCCTGTAAAGAAAACTACTGC (SEQ ID NO: 158)

[0260] CD63-TM-HELICAL(inside-out) LMITFAIFLSLIMLVEVAAAI (SEQ ID NO: 159), which is encoded by DNA

[0261] CTGATGATCACATTCGCAATCTTCCTGTCCCTCATCATGCTGGTGGAGGTGGCCGCC

[0262] GCCATC (SEQ ID NO: 160

[0263] Linker-a: GGSGPVRRYQ (SEQ ID NO: 161), which is encoded by DNA

[0264] GGCGGCAGCGGCCCTGT GAGGAG AT ACC AG (SEQ ID NO: 162)

[0265] MMP2: GGPLGVRGG (SEQ ID NO: 163), which is encoded by DNA

[0266] GGCGGTCCACTGGGCGTGCGGGGCGGC (SEQ ID NO: 164)

[0209] VL-a-CD16-118AA

[0267] MEVQLVESGGGWRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGY

[0268] ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (SEQ ID NO: 165) which is encoded by DNA

[0269] ATGGAAGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGAGACCTGGCGGGTCCCT

[0270] GCGGCTGAGCTGTGCCGCCAGCGGATTCACATTCGACGATTACGGCATGAGCTGGGT

[0271] CAGACAGGCCCCAGGAAAAGGCCTGGAATGGGTGTCTGGCATTAACTGGAACGGCG

[0272] GCAGCACCGGCTACGCCGATAGCGTTAAGGGCAGATTCACCATCAGCAGAGATAAC

[0273] GCCAAGAACTCTCTGTACCTGCAGATGAATAGCCTGCGGGCCGAAGATACCGCCGTC

[0274] TACTACTGTGCCCGGGGAAGAAGCTTGCTGTTTGACTACTGGGGACAGGGCACACTG

[0275] GTCACCGTGTCTAGG (SEQ ID NO: 166)

[0276] Linker-b and Linker-c: GGGGSGGGGSGGGGS 15AA (SEQ ID NO: 167), which is encoded by

[0277] DNA GGCGGCGGCGGCAGCGGAGGAGGCGGGTCGGGCGGCGGCGGATCC (SEQ ID NO: 168)

[0278]

[0210] VH-a-CD16-107AA

[0279] SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSS

[0280] GNTASLTITGAQAEDEADYYCNSRDSSGNHWFGGGTKLTVL (SEQ ID NO: 169) which is encoded by DNA

[0281] AGCGAGCTGACACAAGATCCTGCTGTGAGCGTCGCCCTGGGCCAGACCGTGCGGAT

[0282] CACTTGCCAGGGAGATAGCCTGAGGTCCTACTACGCCAGTTGGTATCAACAGAAGCC

[0283] TGGACAGGCCCCTGTACTGGTGATCTACGGCAAGAACAACAGACCTTCTGGCATCCC

[0284] TGATAGATTCAGCGGCTCCAGCAGCGGAAACACCGCTAGCCTGACAATCACCGGAG

[0285] CTCAGGCTGAGGACGAGGCCGACTACTACTGCAACAGCCGGGACTCTTCAGGAAAT

[0286] CACGTGGTGTTCGGCGGCGGCACCAAGCTGACAGTGCTG (SEQ ID NO: 170)

[0287] Flanking 20AA: PSGQAGAAASESLFVSNHAY (SEQ ID NO: 171), which is encoded by DNA

[0288] CCTAGCGGCCAGGCCGGCGCCGCGGCCAGCGAAAGCCTGTTCGTGTCTAACCACGC

[0289] CT AC (SEQ ID NO: 172)

[0290] 7AA flanking peptide: EASGGPE (SEQ ID NO: 173), which is encoded by DNA

[0291] GAGGCTTCCGGCGGCCCCGAG (SEQ ID NO: 174)

[0292]

[0211] VL-a-CD33-116AA

[0293] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYN QKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS (SEQ ID NO: 175) which is encoded by DNA CAGGTGCAGCTGGTGCAAAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGA AAGTGTCCTGCAAGGCCTCCGGCTACACCTTTACCGACTACAATATGCACTGGGTTC GGCAGGCCCCTGGCCAAGGTCTCGAGTGGATCGGCTACATCTATCCTTACAACGGCG GCACCGGCTACAACCAGAAGTTCAAAAGCAAGGCAACAATCACAGCCGACGAGAGC ACTAACACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCTGTTTA CTACTGCGCCAGAGGCAGACCAGCTATGGACTACTGGGGCCAAGGAACCCTGGTGA

[0294] CCGTGTCATCT (SEQ ID NO: 176)

[0295]

[0212] VH-a-CD33

[0296] DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGS

[0297] GVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO

[0298] 177), which is encoded by DNA

[0299] GATATCCAGATGACCCAGAGCCCTAGCAGCCTGTCTGCCTCTGTTGGCGACCGGGTG

[0300] ACCATCACCTGCAGAGCTTCTGAATCAGTTGACAACTACGGCATCAGCTTCATGAAC

[0301] TGGTTCCAGCAGAAACCTGGCAAGGCCCCCAAGCTGCTGATCTATGCCGCTTCTAAT

[0302] CAGGGCAGCGGCGTGCCTAGCAGATTTAGCGGCAGTGGATCGGGAACCGACTTCAC

[0303] CCTGACCATTTCTTCGCTTCAGCCCGACGATTTCGCCACATACTATTGTCAGCAGAGC

[0304] AAGGAGGTGCCATGGACATTTGGCCAGGGCACTAAGGTGGAAATCAAG (SEQ ID NO:

[0305] 178)

[0306] Linker and Myc-TAG GGSGEQKLISEEDLGG (SEQ ID NO: 179)

[0307] GGCGGCAGCGGAGAGCAGAAGCTGATCTCCGAGGAAGACCTGGGTGGA (SEQ ID NO: 180)

[0308] MMP9: PLGMTS (SEQ ID NO:181) CCCCTGGGCATGACCTCT (SEQ ID NO: 182)

[0309] CD63 -EXTRACELLULAR

[0310] PKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKA

[0311] IHKEGCVEKIGGWLRKNV (SEQ ID NO: 183)

[0312] CCTAAGAACAACCACACAGCCAGCATCCTGGACAGAATGCAGGCCGACTTCAAGTG

[0313] CTGCGGCGCCGCTAATTACACCGACTGGGAGAAAATCCCCTCCATGAGCAAAAACC

[0314] GGGTGCCCGACAGCTGTTGCATCAATGTGACCGTGGGCTGTGGCATTAACTTCAACG

[0315] AGAAGGCTATCCATAAGGAGGGCTGCGTCGAGAAGATCGGCGGCTGGCTGAGAAAG AACGTG (SEQ ID NO: 184)

[0316]

[0213] CD63-TM-HELICAL(outside-in) LVVAAAALGIAFVEVLGIVFA (SEQ ID NO: 185)

[0317] CTGGTGGTGGCCGCCGCCGCCCTGGGTATCGCCTTCGTGGAAGTGCTGGGCATCG

[0318] TGTTCGCC (SEQ ID NO: 186)

[0214] CD63 -CYTOPLASMIC CCLVKSIRSGYEVM SEQ ID NO: 187)

[0319] TGCTGCCTGGTGAAGTCTATCAGATCTGGCTATGAGGTGATG (SEQ ID NO: 189)

[0320]

[0215] Example 4: Cell Surface Expressed BIKE

[0321]

[0216] Referring to Figure 5A-B, one embodiment of the invention is a BIKE with MMP / GrzB cleavage sequences embedded in the extracellular loop between the third and fourth transmembrane region of a tetraspanin. This molecule will be expressed on the cell surface due to the lack of signaling motif GYEVM (SEQ ID NO: 337) required for sorting to extracellular vesicles.

[0322]

[0217] The construct has the amino acid sequence outlined in SEQ ID NO: 188 below:

[0323] MCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQGGPLGVRGGMEVQ LVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGY ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRG GGGSGGGGSGGGGSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVL VIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKL TVLPSGQAGAAASESLFVSNHAYEASGGPEQVQLVQSGAEVKKPGSSVKVSCKASGYTF TDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLR SEDTAVYYCARGRPAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSAS VGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGT DFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKGGSGEQKLISEEDLGGPLGMTS GGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINF NEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKS (SEQ ID NO: 188).

[0324]

[0218] The corresponding cDNA is set forth below as (SEQ ID No: 189):

[0325] ATGTGCGGCGCCTGTAAAGAAAACTACTGCCTGATGATCACATTCGCAATCTTCCTG

[0326] TCCCTCATCATGCTGGTGGAGGTGGCCGCCGCCATCGCTGGCTACGTGGGCGGCAGC

[0327] GGCCCTGTGAGGAGATACCAGGGCGGTCCACTGGGCGTGCGGGGCGGCATGGAAGT GCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGAGACCTGGCGGGTCCCTGCGGCTGA GCTGTGCCGCCAGCGGATTCACATTCGACGATTACGGCATGAGCTGGGTCAGACAG GCCCCAGGAAAAGGCCTGGAATGGGTGTCTGGCATTAACTGGAACGGCGGCAGCAC CGGCTACGCCGATAGCGTTAAGGGCAGATTCACCATCAGCAGAGATAACGCCAAGA ACTCTCTGTACCTGCAGATGAATAGCCTGCGGGCCGAAGATACCGCCGTCTACTACT GTGCCCGGGGAAGAAGCTTGCTGTTTGACTACTGGGGACAGGGCACACTGGTCACC GTGTCTAGGGGCGGCGGCGGCAGCGGAGGAGGCGGGTCGGGCGGCGGCGGATCCA GCGAGCTGACACAAGATCCTGCTGTGAGCGTCGCCCTGGGCCAGACCGTGCGGATC ACTTGCCAGGGAGATAGCCTGAGGTCCTACTACGCCAGTTGGTATCAACAGAAGCCT GGACAGGCCCCTGTACTGGTGATCTACGGCAAGAACAACAGACCTTCTGGCATCCCT GATAGATTCAGCGGCTCCAGCAGCGGAAACACCGCTAGCCTGACAATCACCGGAGC TCAGGCTGAGGACGAGGCCGACTACTACTGCAACAGCCGGGACTCTTCAGGAAATC

[0328] ACGTGGTGTTCGGCGGCGGCACCAAGCTGACAGTGCTGCCTAGCGGCCAGGCCGGC GCCGCGGCCAGCGAAAGCCTGTTCGTGTCTAACCACGCCTACGAGGCTTCCGGCGGC CCCGAGCAGGTGCAGCTGGTGCAAAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCA GCGTGAAAGTGTCCTGCAAGGCCTCCGGCTACACCTTTACCGACTACAATATGCACT GGGTTCGGCAGGCCCCTGGCCAAGGTCTCGAGTGGATCGGCTACATCTATCCTTACA ACGGCGGCACCGGCTACAACCAGAAGTTCAAAAGCAAGGCAACAATCACAGCCGAC GAGAGCACTAACACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGC TGTTTACTACTGCGCCAGAGGCAGACCAGCTATGGACTACTGGGGCCAAGGAACCCT GGTGACCGTGTCATCTGGGGGCGGCGGATCTGGCGGCGGCGGGAGCGGCGGCGGAG GAAGTGATATCCAGATGACCCAGAGCCCTAGCAGCCTGTCTGCCTCTGTTGGCGACC GGGTGACCATCACCTGCAGAGCTTCTGAATCAGTTGACAACTACGGCATCAGCTTCA TGAACTGGTTCCAGCAGAAACCTGGCAAGGCCCCCAAGCTGCTGATCTATGCCGCTT CTAATCAGGGCAGCGGCGTGCCTAGCAGATTTAGCGGCAGTGGATCGGGAACCGAC TTCACCCTGACCATTTCTTCGCTTCAGCCCGACGATTTCGCCACATACTATTGTCAGC AGAGCAAGGAGGTGCCATGGACATTTGGCCAGGGCACTAAGGTGGAAATCAAGGGC GGCAGCGGAGAGCAGAAGCTGATCTCCGAGGAAGACCTGGGTGGACCCCTGGGCAT GACCTCTGGCGGCAGCGGCCCTAAGAACAACCACACAGCCAGCATCCTGGACAGAA TGCAGGCCGACTTCAAGTGCTGCGGCGCCGCTAATTACACCGACTGGGAGAAAATC CCCTCCATGAGCAAAAACCGGGTGCCCGACAGCTGTTGCATCAATGTGACCGTGGGC TGTGGCATTAACTTCAACGAGAAGGCTATCCATAAGGAGGGCTGCGTCGAGAAGAT CGGCGGCTGGCTGAGAAAGAACGTGCTGGTGGTGGCCGCCGCCGCCCTGGGTATCG CCTTCGTGGAAGTGCTGGGCATCGTGTTCGCCTGCTGCCTGGTGAAGTCT (SEQ ID No: 189)

[0329]

[0219] The above SEQ ID NO 188 is divided into functional domains and motifs as shown in Figure

[0330] 11 and as follows:

[0331] CD63 -CYTOPLASMIC: MCGACKENYC (SEQ ID NO: 190) which is endcoded by cDNA:

[0332] ATGTGCGGCGCCTGTAAAGAAAACTACTGC (SEQ ID NO: 191)

[0333]

[0220] CD63-TM-HELICAL(inside-out) LMITFAIFLSLIMLVEVAAAI (SEQ ID NO: 192) which is encoded by cDNA:

[0334]

[0221] CTGATGATCACATTCGCAATCTTCCTGTCCCTCATCATGCTGGTGGAGGTGGCCG CCGCCATC (SEQ ID NO: 193)

[0335]

[0222] Linker-a: GGSGPVRRYQ (SEQ ID NO: 194) which is encoded by cDNA: CGGCAGCGGCCCTGTGAGGAGATACCAG (SEQ ID NO: 195)

[0223] MMP2: GGPLGVRGG (SEQ ID NO: 196) which is encoded by cDNA:

[0336] GGCGGTCCACTGGGCGTGCGGGGCGGC (SEQ ID NO: 197)

[0337]

[0224] VL-a-CD16-118AA

[0338] MEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNG GSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTL VTVSR (SEQ ID NO: 198)

[0339]

[0225] Which is encoded by cDNA:

[0340] ATGGAAGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGAGACCTGGCGGGTCCCT

[0341] GCGGCTGAGCTGTGCCGCCAGCGGATTCACATTCGACGATTACGGCATGAGCTGGGT

[0342] CAGACAGGCCCCAGGAAAAGGCCTGGAATGGGTGTCTGGCATTAACTGGAACGGCG

[0343] GCAGCACCGGCTACGCCGATAGCGTTAAGGGCAGATTCACCATCAGCAGAGATAAC

[0344] GCCAAGAACTCTCTGTACCTGCAGATGAATAGCCTGCGGGCCGAAGATACCGCCGTC

[0345] TACTACTGTGCCCGGGGAAGAAGCTTGCTGTTTGACTACTGGGGACAGGGCACACTG GTCACCGTGTCTAGG (SEQ ID NO: 199)

[0346]

[0226] Linker-b and Linker-c: GGGGSGGGGSGGGGS 15AA (SEQ ID NO:200) which is encoded by cDNA:

[0347] GGCGGCGGCGGCAGCGGAGGAGGCGGGTCGGGCGGCGGCGGATCC (SEQ ID NO: 201)

[0348]

[0227] VH-a-CD16-107AA

[0349] SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRF

[0350] SGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVZ (SEQ ID NO:202)

[0351]

[0228] which is encoded by cDNA:

[0352] AGCGAGCTGACACAAGATCCTGCTGTGAGCGTCGCCCTGGGCCAGACCGTGCGGAT

[0353] CACTTGCCAGGGAGATAGCCTGAGGTCCTACTACGCCAGTTGGTATCAACAGAAGCC

[0354] TGGACAGGCCCCTGTACTGGTGATCTACGGCAAGAACAACAGACCTTCTGGCATCCC

[0355] TGATAGATTCAGCGGCTCCAGCAGCGGAAACACCGCTAGCCTGACAATCACCGGAG

[0356] CTCAGGCTGAGGACGAGGCCGACTACTACTGCAACAGCCGGGACTCTTCAGGAAAT

[0357] CACGTGGTGTTCGGCGGCGGCACCAAGCTGACAGTGCTG (SEQ ID NO: 203)

[0358]

[0229] Flanking 20AA: PSGQAGAAASESLFVSNHAY (SEQ ID NO:204) which is encoded by cDNA:

[0359] CCTAGCGGCCAGGCCGGCGCCGCGGCCAGCGAAAGCCTGTTCGTGTCTAACCACGC

[0360] CTAC (SEQ ID NO:205)

[0230] 7AA flanking peptide: EASGGPE (SEQ ID NO:206) which is encoded by cDNA:

[0361] GAGGCTTCCGGCGGCCCCGAG (SEQ ID NO: 207)

[0362]

[0231] VL-a-CD33-116AA

[0363] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGG

[0364] TGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS (SEQ ID NO:208)

[0365]

[0232] Which is encoded by cDNA:

[0366] CAGGTGCAGCTGGTGCAAAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGA

[0367] AAGTGTCCTGCAAGGCCTCCGGCTACACCTTTACCGACTACAATATGCACTGGGTTC

[0368] GGCAGGCCCCTGGCCAAGGTCTCGAGTGGATCGGCTACATCTATCCTTACAACGGCG

[0369] GCACCGGCTACAACCAGAAGTTCAAAAGCAAGGCAACAATCACAGCCGACGAGAGC

[0370] ACTAACACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCTGTTTA

[0371] CTACTGCGCCAGAGGCAGACCAGCTATGGACTACTGGGGCCAAGGAACCCTGGTGA

[0372] CCGTGTCATCT (SEQ ID NO: 209)

[0373]

[0233] VH-a-CD33

[0374] DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQG SGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 210)

[0375]

[0234] Which is encoded by cDNA:

[0376] GATATCCAGATGACCCAGAGCCCTAGCAGCCTGTCTGCCTCTGTTGGCGACCGGGTG

[0377] ACCATCACCTGCAGAGCTTCTGAATCAGTTGACAACTACGGCATCAGCTTCATGAAC

[0378] TGGTTCCAGCAGAAACCTGGCAAGGCCCCCAAGCTGCTGATCTATGCCGCTTCTAAT

[0379] CAGGGCAGCGGCGTGCCTAGCAGATTTAGCGGCAGTGGATCGGGAACCGACTTCAC CCTGACCATTTCTTCGCTTCAGCCCGACGATTTCGCCACATACTATTGTCAGCAGAGC AAGGAGGTGCCATGGACATTTGGCCAGGGCACTAAGGTGGAAATCAAG (SEQ ID NO:

[0380] 211)

[0381]

[0235] Myc-tag ggsgeqkliseedlgg (SEQ ID NO: 380) which is encoded by cDNA:

[0382] GGCGGCAGCGGAGAGCAGAAGCTGATCTCCGAGGAAGACCTGGGTGGA (SEQ ID NO: 180)

[0383]

[0236] MMP9: ggPLGMTS (SEQ ID NO: 149) which is encoded by cDNA: CCCCTGGGCATGACCTCT (SEQ ID NO: 182)

[0384]

[0237] CD63 -EXTRACELLULAR

[0385] PKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKA

[0386] IHKEGCVEKIGGWLRKNV (SEQ ID NO : 151 )

[0238] Which is encoded by cDNA: CCTAAGAACAACCACACAGCCAGCATCCTGGACAGAATGCAGGCCGACTTCAAGTG CTGCGGCGCCGCTAATTACACCGACTGGGAGAAAATCCCCTCCATGAGCAAAAACC GGGTGCCCGACAGCTGTTGCATCAATGTGACCGTGGGCTGTGGCATTAACTTCAACG

[0387] AGAAGGCTATCCATAAGGAGGGCTGCGTCGAGAAGATCGGCGGCTGGCTGAGAAAG AACGTG (SEQ ID NO: 184)

[0388]

[0239] CD63-TM-HELICAL(outside-in) LVVAAAALGIAFVEVLGIVFA (SEQ ID NO: 185) which is encoded by cDNA:

[0389] CTGGTGGTGGCCGCCGCCGCCCTGGGTATCGCCTTCGTGGAAGTGCTGGGCATCGTG TTCGCC (SEQ ID NO: 186)

[0390]

[0240] CD63 -CYTOPLASMIC CCLVKS (SEQ ID NO: 325) which is encoded by cDNA: TGCTGCCTGGTGAAGTCT (SEQ ID NO: 379)

[0391]

[0241] Example 5: Delivery of a TRIKE with ADAM 17

[0392]

[0242] Referring to Figures 13-14, in one embodiment the invention comprises a TRIKE with

[0393] ADAM17 target sequences embedded in the Type-II TM (TNFSF14) and Type-I TM (TNR1A) domains. This molecule will be expressed on the cell surface and cleaved upon NK cell activation.

[0394]

[0243] This construct is outlined in SEQ ID NO: 212 below:

[0395] MSCSVARVGLGLLLLLMGAGLAVQGWFLLQLHWRLGEMVTRLPDGPAGSWEQLIQER RSHEVNPAAGGSGPVRRYQMEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWV RQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY YCARGRSLLFDYWGQGTLVTVSRGGGGSGGGGSGGGGSSELTQDPAVSVALGQTVRIT CQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAED EADYYCNSRDSSGNHVVFGGGTKLTVLPSGQAGAAASESLFVSNHAYEASGGPEQVQL VQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYN

[0396] QKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKA PKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTK VEIKGGSGEQKLISEEDLSGGSGSLECTKLCLPQIENVKGTEDSGGSGQVQLVQSGAEVK KPGS S VKVSCKAFGGTFS S YAISWVRQAPGQGLEWMGRIIRFLGIANYAQKFQGRVTLIA DKSTNTAYMELSSLRSEDTAVYYCAGEPGREDPDAVDIWGQGTMVTVSSGGGGSGGG GSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKARKSLIYAASS LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIKGGGGSG GGGSGGGGSSGTTVLLPLVIFFGLCLLSLLFIGLMYRYGGSGKRGRKKLLYIFKQPFMRP

[0397] VQTTQEEDG (SEQ ID NO: 212)

[0398]

[0244] The corresponding cDNA is set forth below:

[0399] ATGAGCTGCAGCGTGGCGAGAGTAGGGCTGGGCCTGCTCCTGCTGCTGATGGGCGC CGGCCTGGCCGTGCAAGGATGGTTCCTGCTGCAGCTTCATTGGAGACTGGGAGAGAT GGTGACTAGACTGCCTGACGGCCCTGCCGGCAGCTGGGAGCAGCTGATCCAGGAGA GAAGGAGCCACGAGGTGAACCCTGCTGCAGGTGGGAGCGGCCCTGTGCGGCGGTAC CAAATGGAAGTGCAGCTGGTCGAGTCTGGGGGCGGCGTGGTCCGGCCAGGCGGCAG CCTGCGTCTGTCTTGCGCCGCTAGCGGCTTCACTTTCGATGACTACGGCATGAGCTG GGTGCGGCAGGCCCCCGGCAAGGGCCTCGAGTGGGTTTCTGGCATCAACTGGAACG

[0400] GCGGCTCTACCGGCTACGCCGACAGCGTGAAGGGCCGCTTTACCATCTCCAGAGATA

[0401] ATGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGATACGGCC GTGTACTATTGTGCCCGGGGCAGAAGCCTCCTCTTCGACTACTGGGGCCAGGGCACC CTGGTTACCGTGAGCCGGGGCGGCGGCGGTTCTGGAGGCGGAGGCAGCGGCGGCGG

[0402] GGGCTCCTCTGAGCTGACCCAGGACCCCGCCGTGAGCGTGGCCCTGGGCCAGACAG TCAGAATTACATGCCAGGGAGATAGCCTGAGATCCTACTATGCCAGCTGGTACCAGC AGAAACCTGGCCAAGCTCCTGTGCTGGTCATCTACGGCAAAAACAACAGACCTAGT

[0403] GGAATCCCCGACAGATTTAGCGGATCCTCCAGCGGCAATACCGCCTCCTTGACAATC

[0404] ACCGGCGCTCAGGCCGAGGATGAAGCCGACTACTACTGCAACAGCAGAGACAGCTC TGGGAACCACGTGGTGTTCGGGGGGGGAACCAAGCTTACAGTGCTGCCTTCTGGGC AGGCGGGAGCCGCCGCTAGCGAATCTCTGTTTGTGTCTAATCACGCCTACGAGGCCA

[0405] GCGGCGGCCCCGAGCAGGTGCAGCTGGTGCAGAGTGGGGCTGAAGTGAAGAAACCT GGCAGCTCGGTGAAGGTGAGCTGCAAGGCCTCTGGCTACACCTTCACCGACTACAAC ATGCACTGGGTCAGACAGGCCCCTGGCCAGGGACTGGAATGGATCGGCTACATCTA

[0406] CCCCTACAACGGCGGCACTGGCTATAACCAAAAATTCAAGTCTAAGGCAACCATCA CAGCCGACGAGAGCACCAACACCGCCTATATGGAGCTGTCTAGCCTGAGGAGCGAG GACACCGCCGTGTACTACTGCGCCCGGGGAAGACCTGCCATGGACTACTGGGGCCA

[0407] AGGAACACTGGTGACCGTCAGCTCTGGAGGCGGCGGATCTGGCGGAGGAGGCAGCG GCGGCGGCGGTTCAGACATCCAGATGACCCAGTCACCTAGCTCTCTGAGCGCCAGCG TGGGCGACCGGGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACAACTACGGA

[0408] ATCTCATTCATGAACTGGTTCCAGCAGAAGCCCGGAAAGGCCCCGAAGCTGCTGATC TATGCTGCCAGCAACCAGGGTTCAGGGGTGCCTAGCAGATTTTCTGGGAGCGGCTCT GGCACCGACTTTACCCTGACAATCTCTAGCCTGCAGCCAGACGATTTCGCCACTTAC

[0409] TACTGTCAGCAGAGCAAGGAAGTGCCATGGACCTTCGGACAAGGAACAAAGGTTGA

[0410] AATTAAGGGAGGCAGCGGGGAACAAAAGCTGATCAGCGAAGAGGACCTGAGCGGC

[0411] GGAAGCGGCAGCCTGGAGTGCACCAAGCTGTGTCTGCCTCAGATCGAGAATGTGAA GGGCACAGAGGATTCCGGCGGCAGCGGCCAAGTGCAGCTGGTGCAGAGCGGCGCTG AGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGTAAAGCCTTCGGCGGAACC

[0412] TTCAGCTCCTACGCTATCAGCTGGGTGAGACAGGCCCCTGGCCAGGGGCTAGAATGG ATGGGCAGAATCATCCGGTTCCTGGGCATCGCCAATTACGCCCAGAAGTTTCAGGGC CGGGTGACGCTGATCGCCGATAAGAGCACCAACACCGCCTACATGGAACTGTCTTCC

[0413] CTGCGATCAGAAGACACCGCCGTGTACTACTGCGCCGGCGAACCTGGAAGAGAAGA TCCTGACGCCGTGGATATCTGGGGACAGGGCACAATGGTGACAGTGTCGAGCGGCG GAGGCGGCTCTGGTGGCGGTGGCTCTGGCGGCGGCGGCTCCGACATTCAGATGACA

[0414] CAGTCCCCTTCCTCTCTGTCCGCCTCCGTGGGCGACCGGGTGACCATCACCTGCAGA GCCAGTCAGGGCATCAGAAGCTGGCTGGCTTGGTACCAGCAGAAGCCTGAGAAGGC CAGGAAAAGCCTGATCTACGCCGCGAGCAGCCTGCAGTCTGGGGTGCCCTCTAGATT

[0415] CAGCGGTTCTGGCAGCGGCACAGATTTCACACTGACAATAAGTTCGCTGCAGCCTGA AGACTTCGCCACCTATTACTGCCAGCAATACAACAGCTACCCTCTGACCTTCGGCGG CGGAACCAAGGTTGAGATCAAGGGCGGCGGCGGTAGCGGCGGCGGCGGCTCCGGCG

[0416] GCGGAGGCAGCTCCGGCACCACCGTCCTGCTGCCTCTGGTGATCTTCTTCGGCCTGT GCCTGCTGTCACTGCTGTTCATCGGACTCATGTACAGATACGGCGGAAGCGGCC (SEQ ID NO: 213)

[0417]

[0245] Domains and motifs:

[0246] SEQ ID NO: 212 is divided into functional domains and motifs as set forth below and as shown in Figure 14.

[0418]

[0247] TNFSF14 cytoplasmic MSCSVAR (SEQ ID NO:214), which is encoded by cDNA: ATGAGCTGCAGCGTGGCGAGA (SEQ ID NO: 215)

[0419]

[0248] TNFSF14 transmembrane (inside-out) VGLGLLLLLMGAGLAVQGWFL (SEQ ID NO: 216), which is encoded by cDNA:

[0420] GTGGCGAGAGTAGGGCTGGGCCTGCTCCTGCTGCTGATGGGCGCCGGCCTGGCCG

[0421] TGCAAGGATGGTTCCTG (SEQ ID NO: 217)

[0422]

[0249] TNFSF14 extracellular with cleavage seq:

[0423] GSWEQLLQLHWRLGEMVTRLPDGPAGSWEQLIQERRSHEVNPAA (SEQ ID NO:218) which is encoded by cDNA:

[0424] CTGCAGCTTCATTGGAGACTGGGAGAGATGGTGACTAGACTGCCTGACGGCCCTGCC

[0425] GGCAGCTGGGAGCAGCTGATCCAGGAGAGAAGGAGCCACGAGGTGAACCCTGCTGC

[0426] A (SEQ ID NO: 219)

[0427]

[0250] Linker-a: GGSGPVRRYQ (SEQ ID NO:220) which is encoded by cDNA:

[0428] GGTGGGAGCGGCCCTGTGCGGCGGTACCAA(SEQ ID NO: 221)

[0429]

[0251] VL-a-CD16-118AA

[0430] MEVQLVESGGGWRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGY

[0431] ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (SEQ ID NO: 222), Which is encoded by cDNA:

[0432] ATGGAAGTGCAGCTGGTCGAGTCTGGGGGCGGCGTGGTCCGGCCAGGCGGCAGCCT

[0433] GCGTCTGTCTTGCGCCGCTAGCGGCTTCACTTTCGATGACTACGGCATGAGCTGGGT

[0434] GCGGCAGGCCCCCGGCAAGGGCCTCGAGTGGGTTTCTGGCATCAACTGGAACGGCG

[0435] GCTCTACCGGCTACGCCGACAGCGTGAAGGGCCGCTTTACCATCTCCAGAGATAATG

[0436] CCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGATACGGCCGTG

[0437] TACTATTGTGCCCGGGGCAGAAGCCTCCTCTTCGACTACTGGGGCCAGGGCACCCTG GTTACCGTGAGCCGG (SEQ ID NO:223).

[0438] TRIKE -Linker: GGGGSGGGGSGGGGS (SEQ ID NO: 224) which is encoded by cDNA: GGCGGCGGCGGTTCTGGAGGCGGAGGCAGCGGCGGCGGGGGCTCC (SEQ ID NO: 225)

[0439]

[0252] VH-a-CD16-107AA

[0440] SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSS

[0441] GNTASLTITGAQAEDEADYYCNSRDSSGNHWFGGGTKLTVL (SEQ ID NO: 226)

[0442]

[0253] which is encoded by cDNA: TCTGAGCTGACCCAGGACCCCGCCGTGAGCGTGGCCCTGGGCCAGACAGTCAGAAT

[0443] TACATGCCAGGGAGATAGCCTGAGATCCTACTATGCCAGCTGGTACCAGCAGAAAC

[0444] CTGGCCAAGCTCCTGTGCTGGTCATCTACGGCAAAAACAACAGACCTAGTGGAATCC

[0445] CCGACAGATTTAGCGGATCCTCCAGCGGCAATACCGCCTCCTTGACAATCACCGGCG

[0446] CTCAGGCCGAGGATGAAGCCGACTACTACTGCAACAGCAGAGACAGCTCTGGGAAC

[0447] CACGTGGTGTTCGGGGGGGGAACCAAGCTTACAGTGCTG (SEQ ID NO: 227)

[0448]

[0254] Flanking 20AA: PSGQAGAAASESLFVSNHAY (SEQ ID NO: 228)_which is encoded by cDNA:

[0449] CCTTCTGGGCAGGCGGGAGCCGCCGCTAGCGAATCTCTGTTTGTGTCTAATCACGCC

[0450] TAC (SEQ ID NO: 229)

[0451]

[0255] 7AA flanking peptide: EASGGPE (SEQ ID NO: 230) which is encoded by cDNA:

[0452] GAGGCCAGCGGCGGCCCCGAGCAGGTG (SEQ ID NO: 231)

[0453]

[0256] VL-a-CD33-116AA

[0454] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYN QKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS (SEQ ID NO: 232)

[0455]

[0257] which is encoded by cDNA:

[0456] CAGCTGGTGCAGAGTGGGGCTGAAGTGAAGAAACCTGGCAGCTCGGTGAAGGTGAG

[0457] CTGCAAGGCCTCTGGCTACACCTTCACCGACTACAACATGCACTGGGTCAGACAGGC

[0458] CCCTGGCCAGGGACTGGAATGGATCGGCTACATCTACCCCTACAACGGCGGCACTG

[0459] GCTATAACCAAAAATTCAAGTCTAAGGCAACCATCACAGCCGACGAGAGCACCAAC

[0460] ACCGCCTATATGGAGCTGTCTAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGC

[0461] GCCCGGGGAAGACCTGCCATGGACTACTGGGGCCAAGGAACACTGGTGACCGTCAG

[0462] C (SEQ ID NO: 233)

[0463]

[0258] TRIKE-LINKER: GGGGSGGGGSGGGGS (SEQ ID NO: 234) which is encoded by cDNA: TCTGGAGGCGGCGGATCTGGCGGAGGAGGCAGCGGCGGCGGCGGTTCA (SEQ ID NO: 235)

[0464]

[0259] TRIKEVH-a-CD33

[0465] DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 236)

[0466]

[0260] Which is encoded by cDNA: GACATCCAGATGACCCAGTCACCTAGCTCTCTGAGCGCCAGCGTGGGCGACCGGGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACAACTACGGAATCTCATTCATGA ACTGGTTCCAGCAGAAGCCCGGAAAGGCCCCGAAGCTGCTGATCTATGCTGCCAGC AACCAGGGTTCAGGGGTGCCTAGCAGATTTTCTGGGAGCGGCTCTGGCACCGACTTT ACCCTGACAATCTCTAGCCTGCAGCCAGACGATTTCGCCACTTACTACTGTCAGCAG AGCAAGGAAGTGCCATGGACCTTCGGACAAGGAACAAAGGTTGAAATTAAG (SEQ ID NO: 237)

[0467]

[0261] Myc-tag GGSGEQKLISEEDLSGGSG (SEQ ID NO: 238) which is encoded by cDNA: GGAGGCAGCGGGGAACAAAAGCTGATCAGCGAAGAGGACCTG (SEQ ID NO: 239)

[0468]

[0262] TNR1 A-Extracellular with ADAM17 cleavage seq SLECTKLCLPQIENVKGTEDS (SEQ ID NO: 240) which is encoded by cDNA:

[0469] AGCCTGGAGTGCACCAAGCTGTGTCTGCCTCAGATCGAGAATGTGAAGGGCACAGA

[0470] GGAT (SEQ ID NO: 241)

[0471]

[0263] Vh-a-CD38

[0472] QVQLVQSGAEVKKPGSSVKVSCKAFGGTFSSYAISWVRQAPGQGLEWMGRIIRFLGIAN YAQKFQGRVTLIADKSTNTAYMELSSLRSEDTAVYYCAGEPGREDPDAVDIWGQGTMV TVS S (SEQ ID NO: 242)

[0473]

[0264] Which is encoded by cDNA:

[0474] CAAGTGCAGCTGGTGCAGAGCGGCGCTGAGGTGAAGAAGCCCGGCAGCAGCGTGAA GGTGAGCTGTAAAGCCTTCGGCGGAACCTTCAGCTCCTACGCTATCAGCTGGGTGAG ACAGGCCCCTGGCCAGGGGCTAGAATGGATGGGCAGAATCATCCGGTTCCTGGGCA TCGCCAATTACGCCCAGAAGTTTCAGGGCCGGGTGACGCTGATCGCCGATAAGAGC ACCAACACCGCCTACATGGAACTGTCTTCCCTGCGATCAGAAGACACCGCCGTGTAC TACTGCGCCGGCGAACCTGGAAGAGAAGATCCTGACGCCGTGGATATCTGGGGACA

[0475] GGGCACAATGGTGACAGTGTCGAGC (SEQ ID NO: 243)

[0476]

[0265] Linker GGGGSGGGGSGGGGS (SEQ ID NO: 244) which is encoded by cDNA: GGCGGAGGCGGCTCTGGTGGCGGTGGCTCTGGCGGCGGCGGCTCC (SEQ ID NO: 245)

[0477]

[0266] Vl-a-CD38

[0478] DIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKARKSLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK (SEQ ID NO: 246) which is encoded by cDNA:

[0479] GACATTCAGATGACACAGTCCCCTTCCTCTCTGTCCGCCTCCGTGGGCGACCGGGTG ACCATCACCTGCAGAGCCAGTCAGGGCATCAGAAGCTGGCTGGCTTGGTACCAGCA GAAGCCTGAGAAGGCCAGGAAAAGCCTGATCTACGCCGCGAGCAGCCTGCAGTCTG GGGTGCCCTCTAGATTCAGCGGTTCTGGCAGCGGCACAGATTTCACACTGACAATAA GTTCGCTGCAGCCTGAAGACTTCGCCACCTATTACTGCCAGCAATACAACAGCTACC CTCTGACCTTCGGCGGCGGAACCAAGGTTGAGATCAAG (SEQ ID NO: 247)

[0480]

[0267] TNRIA-Transmembrane SGTTVLLPLVIFFGLCLLSLLFIGLMYRY (SEQ ID NO: 248) which is encoded by cDNA:

[0481] TCCGGCACCACCGTCCTGCTGCCTCTGGTGATCTTCTTCGGCCTGTGCCTGCTGTCAC TGCTGTTCATCGGACTCATGTACAGATAC (SEQ ID NO: 249)

[0482]

[0268] 41BB-intracellular: KRGRKKLLYIFKQPFMRPVQTTQEEDG (SEQ ID NO: 250) which is encoded by cDNA:

[0483] AAAAGAGGCCGAAAGAAGCTGCTGTACATCTTTAAACAGCCCTTCATGAGACCCGT GCAAACAACCCAGGAGGAAGATGGC (SEQ ID NO: 251)

[0484]

[0269] Example 6: BIKE with anti-CD38 with Granzyme B Cleavage Sequences

[0485]

[0270] Referring to Figures 15 and 16, in some embodiments the invention comprises a BIKE+anti-CD38 with Granzyme-B cleavage sequences with activation domains having SEQ ID NO 71 of 252. This molecule will be membrane-bound and released upon Granzyme-B release following NK cell degranulation. Anti-CD38 will stay on NK cells following the release of the rest of the cargo and will have a function similar to a chimeric antigen receptor (CAR).

[0486] MCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQIEPDGGSGMEVQLV ESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRGGG GSGGGGSGGGGSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVI YGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLT

[0487] VLPSGQAGAAASESLFVSNHAYEASGGPEQVQLVQSGAEVKKPGSSVKVSCKASGYTFT DYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRS EDTAVYYCARGRPAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSAS VGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGT DFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKGGSGEQKLISEEDLGGIEPDGGS GQVQLVQSGAEVKKPGSSVKVSCKAFGGTFSSYAISWVRQAPGQGLEWMGRIIRFLGIA NYAQKFQGRVTLIADKSTNTAYMELSSLRSEDTAVYYCAGEPGREDPDAVDIWGQGTM VTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQK PEKARKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGG GTKVEIKAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMY PPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 252)

[0488]

[0271] The corresponding cDNA is set forth below:

[0489] ATGTGCGGCGCCTGCAAGGAAAACTACTGTCTGATGATCACTTTCGCCATCTTTCTGT CTCTGATCATGCTGGTGGAAGTGGCCGCTGCCATTGCCGGATACGTGGGCGGCTCTG GCCCTGTGCGAAGATACCAAATCGAGCCTGACGGCGGCAGCGGCATGGAAGTGCAG

[0490] CTGGTTGAGTCCGGCGGCGGAGTGGTGCGGCCTGGCGGCTCTCTGAGACTGTCTTGC

[0491] GCTGCCAGCGGTTTCACCTTCGACGATTACGGAATGAGCTGGGTGCGGCAGGCCCCA

[0492] GGCAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGAAGCACCGGCTA

[0493] CGCAGACAGCGTGAAGGGCAGATTCACCATCTCCAGAGATAACGCCAAGAATTCAC

[0494] TGTACCTGCAGATGAACAGCCTGAGAGCTGAGGACACTGCAGTTTACTACTGCGCCA

[0495] GAGGCCGGAGCCTGCTGTTCGACTACTGGGGACAGGGAACACTGGTGACCGTGAGC

[0496] AGAGGCGGCGGCGGCTCTGGCGGCGGCGGAAGCGGCGGTGGAGGCAGCTCCGAGCT

[0497] GACCCAGGACCCCGCCGTGTCCGTGGCCCTGGGCCAAACAGTGAGAATCACCTGTC

[0498] AGGGCGACTCTCTGCGCTCTTACTACGCCAGCTGGTACCAACAGAAGCCCGGCCAGG

[0499] CCCCTGTGCTGGTGATCTACGGCAAGAACAACAGACCTTCTGGTATCCCCGACAGAT

[0500] TTAGCGGCTCTTCTAGCGGCAACACCGCCTCCCTGACCATCACAGGCGCCCAGGCCG

[0501] AGGACGAGGCTGACTACTATTGCAATAGTAGAGACAGCAGCGGAAACCATGTGGTG

[0502] TTCGGCGGTGGCACAAAGCTGACAGTGCTTCCCAGCGGACAGGCCGGAGCCGCCGC

[0503] CTCTGAAAGCCTGTTCGTGAGTAATCACGCCTACGAGGCCTCCGGAGGCCCTGAGCA

[0504] GGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCAGGCAGCAGCGTTAAGG

[0505] TGAGTTGTAAAGCCTCCGGATACACCTTCACCGACTACAACATGCACTGGGTTAGAC

[0506] AGGCGCCTGGCCAGGGCCTGGAATGGATCGGCTACATCTACCCTTACAACGGCGGG

[0507] ACCGGCTATAATCAGAAGTTCAAGAGCAAGGCCACGATCACAGCCGACGAGAGCAC

[0508] CAACACAGCATACATGGAACTGAGCTCCCTGAGATCTGAGGACACCGCCGTGTACT

[0509] ACTGCGCCAGGGGTCGGCCGGCCATGGACTATTGGGGCCAGGGCACCCTGGTCACA

[0510] GTGTCTAGCGGCGGCGGCGGCTCTGGCGGCGGCGGCTCAGGCGGCGGCGGCAGCGA

[0511] TATTCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCTCTGTTGGAGATCGGGTGAC

[0512] AATCACATGTCGGGCCTCCGAGTCTGTGGACAACTACGGCATCAGCTTCATGAACTG

[0513] GTTCCAGCAGAAACCAGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCTAGCAACC

[0514] AGGGCAGCGGCGTGCCTTCCCGCTTCAGCGGCAGCGGCAGCGGAACAGATTTCACC

[0515] CTGACCATCTCTTCTCTCCAACCTGATGATTTCGCTACCTACTACTGCCAGCAGAGCA

[0516] AGGAGGTGCCTTGGACCTTCGGACAGGGCACCAAAGTGGAGATCAAGGGCGGCTCT

[0517] GGGGAACAGAAGCTGATCAGCGAGGAAGACCTGGGAGGCATCGAGCCTGATGGAG

[0518] GGAGCGGACAAGTGCAGCTGGTTCAAAGCGGCGCTGAGGTGAAAAAACCTGGGAGC

[0519] AGCGTGAAGGTGTCCTGCAAGGCTTTCGGGGGCACCTTCAGCAGCTACGCAATCAGC

[0520] TGGGTGCGGCAGGCTCCCGGCCAGGGCCTGGAATGGATGGGCAGAATCATCCGGTT

[0521] CCTCGGAATCGCCAACTACGCCCAGAAGTTCCAGGGTAGAGTGACCCTGATTGCCGA

[0522] CAAGAGCACAAACACCGCTTATATGGAACTGAGCTCTCTGCGCAGCGAAGATACCG

[0523] CCGTGTACTACTGCGCCGGCGAACCCGGCCGGGAAGATCCTGATGCCGTGGATATCT

[0524] GGGGCCAGGGCACCATGGTGACAGTGTCATCCGGTGGCGGCGGAAGCGGCGGCGGC

[0525] GGTAGTGGGGGAGGCGGCAGCGACATCCAGATGACCCAGTCACCTAGCTCTTTGTC

[0526] GGCCAGCGTCGGAGATAGAGTGACCATTACCTGTCGGGCCTCTCAGGGAATCAGAA

[0527] GCTGGCTCGCCTGGTACCAGCAAAAGCCTGAGAAGGCCAGAAAATCCCTAATCTAT

[0528] GCCGCTAGCAGCCTGCAGTCTGGCGTGCCCTCCAGATTTAGCGGAAGCGGAAGCGG

[0529] CACAGACTTCACACTGACAATCAGCTCTCTGCAACCTGAGGATTTCGCCACCTACTA

[0530] CTGCCAACAGTACAACAGCTACCCACTGACATTTGGAGGCGGTACAAAGGTGGAAA

[0531] TCAAGGCCAAGCCCACCACAACCCCTGCCCCTAGACCCCCCACCCCAGCTCCCACCA

[0532] TCGCCAGCCAGCCTCTGTCCCTGCGGCCTGAAGCCTGCAGACCCGCTGCCGGCGGCG

[0533] CCGTCCACACCCGGGGACTGGACTTCGCCCCTAGAAAGATCGAGGTGATGTACCCCC

[0534] CTCCCTACCTGGATAATGAGAAGAGCAATGGCACAATCATCCACGTGAAAGGCAAA

[0535] CACCTGTGCCCTTCTCCTCTGTTTCCTGGACCTAGCAAGCCCTTCTGGGTCCTGGTCG

[0536] TGGTCGGCGGCGTGCTGGCTTGCTACAGCCTCCTGGTGACCGTGGCCTTCATCATTTT

[0537] TTGGGTGCGCAGCAAGAGATCCAGGCTGCTGCACTCTGACTACATGAACATGACCCC

[0538] TAGACGGCCTGGTCCTACAAGAAAGCACTACCAGCCTTACGCCCCTCCCAGAGATTT CGCTGCCTACCGTAGCCGGGTGAAGTTCAGCAGATCTGCCGATGCCCCTGCTTACCA ACAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCCGGCGGGAAGAGTACG ACGTGCTGGACAAAAGAAGAGGCAGAGACCCTGAGATGGGGGGGAAGCCTCGGAG AAAGAACCCCCAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAG GCCTATTCTGAGATCGGAATGAAGGGAGAAAGACGGAGAGGCAAAGGCCACGACG GCCTGTACCAGGGTCTGTCGACCGCTACCAAGGACACCTACGACGCCCTGCACATGC AGGCTCTGCCTCCAAGA (SEQ ID NO: 253)

[0539]

[0272] SEQ ID NO: 252 is divided into functional domains and motifs as set forth in Figure 15 and as set out below.

[0540]

[0273] CD63 -CYTOPLASMIC MCGACKENYC (SEQ ID NO: 254) which is encoded by cDNA: ATGTGCGGCGCCTGCAAGGAAAACTACTGT (SEQ ID NO:255)

[0541]

[0274] CD63-TM-HELICAL(inside-out) LMITFAIFLSLIMLVEVAAAI (SEQ ID NO: 256) which is encoded by cDNA:

[0542] CTGATGATCACTTTCGCCATCTTTCTGTCTCTGATCATGCTGGTGGAAGTGGCCGCT (SEQ ID NO:257)

[0543]

[0275] Granzyme B cleavage site: IEPD (SEQ ID NO: 258) which is encoded by cDNA:

[0544] ATCGAGCCTGAC (SEQ ID NO: 259). Alternatively, Granzyme B cleavage site EEEEEVEADSEEEEEEE (SEQ ID NO: 260), Granzyme B cleavage site:

[0545] AQGVISADASNLDDFY (SEQ ID NO: 261), or Granzyme B cleavage site: LEADKGKLEYD (SEQ ID NO: 262) may be used.

[0546]

[0276] Linker-a: GGSG (SEQ ID NO: 263) GGCGGCAGCGGC (SEQ ID NO: 264)

[0547]

[0277] VL-a-CD16-118AA

[0548] MEVQLVESGGGWRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGY ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (SEQ ID NO: 265)

[0549]

[0278] Which is encoded by cDNA: ATGGAAGTGCAGCTGGTTGAGTCCGGCGGCGGAGTGGTGCGGCCTGGCGGCTCTCT GAGACTGTCTTGCGCTGCCAGCGGTTTCACCTTCGACGATTACGGAATGAGCTGGGT GCGGCAGGCCCCAGGCAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCG GAAGCACCGGCTACGCAGACAGCGTGAAGGGCAGATTCACCATCTCCAGAGATAAC GCCAAGAATTCACTGTACCTGCAGATGAACAGCCTGAGAGCTGAGGACACTGCAGT TTACTACTGCGCCAGAGGCCGGAGCCTGCTGTTCGACTACTGGGGACAGGGAACACT GGTGACCGTGAGCAGA (SEQ ID NO: 266)

[0279] VH-a-CD16-107AA

[0550] SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRF SGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVL (SEQ ID NO: 267)

[0551]

[0280] Which is encoded by cDNA:

[0552] TCCGAGCTGACCCAGGACCCCGCCGTGTCCGTGGCCCTGGGCCAAACAGTGAGAAT

[0553] CACCTGTCAGGGCGACTCTCTGCGCTCTTACTACGCCAGCTGGTACCAACAGAAGCC

[0554] CGGCCAGGCCCCTGTGCTGGTGATCTACGGCAAGAACAACAGACCTTCTGGTATCCC

[0555] CGACAGATTTAGCGGCTCTTCTAGCGGCAACACCGCCTCCCTGACCATCACAGGCGC

[0556] CCAGGCCGAGGACGAGGCTGACTACTATTGCAATAGTAGAGACAGCAGCGGAAACC

[0557] ATGTGGTGTTCGGCGGTGGCACAAAGCTGACAGTGCTT (SEQ ID NO: 268)

[0558]

[0281] Flanking 20AA: PSGQAGAAASESLFVSNHAY (SEQ ID NO: 269) which is encoded by cDNA:

[0559] CCCAGCGGACAGGCCGGAGCCGCCGCCTCTGAAAGCCTGTTCGTGAGTAATCACGC

[0560] CT AC (SEQ ID NO: 270)

[0561]

[0282] 7AA flanking peptide: EASGGPE (SEQ ID NO: 271) which is encoded by cDNA: GAGGCCTCCGGAGGCCCTGAG (SEQ ID NO: 272)

[0562]

[0283] VL-a-CD33-116AA

[0563] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYN

[0564] QKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS (SEQ ID NO: 273)

[0565]

[0284] Which is encoded by cDNA:

[0566] CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCAGGCAGCAGCGTTAA

[0567] GGTGAGTTGTAAAGCCTCCGGATACACCTTCACCGACTACAACATGCACTGGGTTAG

[0568] ACAGGCGCCTGGCCAGGGCCTGGAATGGATCGGCTACATCTACCCTTACAACGGCG

[0569] GGACCGGCTATAATCAGAAGTTCAAGAGCAAGGCCACGATCACAGCCGACGAGAGC

[0570] ACCAACACAGCATACATGGAACTGAGCTCCCTGAGATCTGAGGACACCGCCGTGTA

[0571] CTACTGCGCCAGGGGTCGGCCGGCCATGGACTATTGGGGCCAGGGCACCCTGGTCA

[0572] CAGTGTCTAGC (SEQ ID NO: 274)

[0573]

[0285] TRIKE VH-a-CD33

[0574] DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 275)

[0575]

[0286] Which is encoded by cDNA: GATATTCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCTCTGTTGGAGATCGGGTG ACAATCACATGTCGGGCCTCCGAGTCTGTGGACAACTACGGCATCAGCTTCATGAAC TGGTTCCAGCAGAAACCAGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCTAGCAA CCAGGGCAGCGGCGTGCCTTCCCGCTTCAGCGGCAGCGGCAGCGGAACAGATTTCA CCCTGACCATCTCTTCTCTCCAACCTGATGATTTCGCTACCTACTACTGCCAGCAGAG CAAGGAGGTGCCTTGGACCTTCGGACAGGGCACCAAAGTGGAGATCAAG (SEQ ID

[0576] NO: 276)

[0577]

[0287] Myc-tag: GGSGEQKLISEEDLGG (SEQ ID NO: 277), which is encoded by cDNA:

[0578] GGCGGCTCTGGGGAACAGAAGCTGATCAGCGAGGAAGACCTGGGAGGC (SEQ ID NO: 278)

[0579]

[0288] Granzyme B cleavage site. IEPD (SEQ ID NO: 279)_ATCGAGCCTGAT (SEQ ID NO: 280)

[0580]

[0289] CD38Vh:

[0581] QVQLVQSGAEVKKPGSSVKVSCKAFGGTFSSYAISWVRQAPGQGLEWMGRIIRFLGIAN YAQKFQGRVTLIADKSTNTAYMELSSLRSEDTAVYYCAGEPGREDPDAVDIWGQGTMV TVSS (SEQ ID NO: 281)

[0582]

[0290] Which is encoded by cDNA:

[0583] CAAGTGCAGCTGGTTCAAAGCGGCGCTGAGGTGAAAAAACCTGGGAGCAGCGTGAA

[0584] GGTGTCCTGCAAGGCTTTCGGGGGCACCTTCAGCAGCTACGCAATCAGCTGGGTGCG GCAGGCTCCCGGCCAGGGCCTGGAATGGATGGGCAGAATCATCCGGTTCCTCGGAA TCGCCAACTACGCCCAGAAGTTCCAGGGTAGAGTGACCCTGATTGCCGACAAGAGC ACAAACACCGCTTATATGGAACTGAGCTCTCTGCGCAGCGAAGATACCGCCGTGTAC TACTGCGCCGGCGAACCCGGCCGGGAAGATCCTGATGCCGTGGATATCTGGGGCCA GGGCACCATGGTGACAGTGTCATCC (SEQ ID NO: 282)

[0585]

[0291] Linker: GGGGSGGGGSGGGGS (SEQ ID NO: 283) which is encoded by cDNA: GGTGGCGGCGGAAGCGGCGGCGGCGGTAGTGGGGGAGGCGGCAGC (SEQ ID NO: 284)

[0586]

[0292] CD38V1:

[0587] DIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKARKSLIYAASSLQSGVPS

[0588] RFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK (SEQ ID NO: 285)

[0589]

[0293] Which is encoded by cDNA:

[0590] GACATCCAGATGACCCAGTCACCTAGCTCTTTGTCGGCCAGCGTCGGAGATAGAGTG ACCATTACCTGTCGGGCCTCTCAGGGAATCAGAAGCTGGCTCGCCTGGTACCAGCAA AAGCCTGAGAAGGCCAGAAAATCCCTAATCTATGCCGCTAGCAGCCTGCAGTCTGG CGTGCCCTCCAGATTTAGCGGAAGCGGAAGCGGCACAGACTTCACACTGACAATCA GCTCTCTGCAACCTGAGGATTTCGCCACCTACTACTGCCAACAGTACAACAGCTACC

[0591] CACTGACATTTGGAGGCGGTACAAAGGTGGAAATCAAG (SEQ ID NO: 286)

[0592]

[0294] Hinge stalk region:

[0593] AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDN EKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 287) which is encoded by cDNA:

[0594] GCCAAGCCCACCACAACCCCTGCCCCTAGACCCCCCACCCCAGCTCCCACCATCGCC AGCCAGCCTCTGTCCCTGCGGCCTGAAGCCTGCAGACCCGCTGCCGGCGGCGCCGTC CACACCCGGGGACTGGACTTCGCCCCTAGAAAGATCGAGGTGATGTACCCCCCTCCC TACCTGGATAATGAGAAGAGCAATGGCACAATCATCCACGTGAAAGGCAAACACCT

[0595] GTGCCCTTCTCCTCTGTTTCCTGGACCTAGCAAGCCC (SEQ ID NO: 288)

[0596]

[0295] CD28 transmembrane: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 289) which is encoded by cDNA:

[0597] TTCTGGGTCCTGGTCGTGGTCGGCGGCGTGCTGGCTTGCTACAGCCTCCTGGTGACC GTGGCCTTCATCATTTTTTGGGTG (SEQ ID NO: 290)

[0598]

[0296] CD28 intracellular: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:291) which is encoded by cDNA:

[0599] CGCAGCAAGAGATCCAGGCTGCTGCACTCTGACTACATGAACATGACCCCTAGACG GCCTGGTCCTACAAGAAAGCACTACCAGCCTTACGCCCCTCCCAGAGATTTCGCTGC

[0600] CTACCGTAGC(SEQ ID NO: 292)

[0601]

[0297] CD3z intracellular

[0602] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:293) which is encoded by cDNA:

[0603] CGGGTGAAGTTCAGCAGATCTGCCGATGCCCCTGCTTACCAACAGGGCCAGAACCA GCTGTACAACGAGCTGAACCTGGGCCGGCGGGAAGAGTACGACGTGCTGGACAAAA GAAGAGGCAGAGACCCTGAGATGGGGGGGAAGCCTCGGAGAAAGAACCCCCAGGA GGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCG

[0604] GAATGAAGGGAGAAAGACGGAGAGGCAAAGGCCACGACGGCCTGTACCAGGGTCT GTCGACCGCTACCAAGGACACCTACGACGCCCTGCACATGCAGGCTCTGCCTCCAAG A (SEQ ID NO: 294).

[0605]

[0298] Example 7: HEK293 cells harboring TNF-a and MMP2 or MMP9 cleavage sites

[0606]

[0299] Referring to Figures 4A-B, 5A-B, and 9-12, in some embodiments, following the teachings above, we created the constructs CD63-9-2L-TNFa-Myc-9-CD63-cyt (contains EV sorting motif) shown in Figure 4A-B, 9 and 10 , and construct CD63-9-2L-TNFa-Myc-9-CD63-D-Cyt (Lacks

[0607] EVs sorting motif) shown in Figure 5A-B, 11 and 12. These constructs differ only by the extracellular vesical sorting motif.

[0608]

[0300] Construct CD63-9-2L-TNFa-Myc-9-CD63-cyt (EVs sorting motif) LeGo-CD63-9-2L-TNFa- My C-9-CD63 -cyt

[0609]

[0301] is set out below as SEQ ID NO: 295

[0610] MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVG VFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQGGPLG MTSSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLI YSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIY LGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGPLGMTS GGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINF NEI<AIHI<EGCVEI<IGGWLRI<NVLVVAAAALGIAFVEVLGIVFACCLVI<SIRSGYEVM (SEQ ID NO: 295)

[0611]

[0302] Referring to Figures 4A-4B, 9 and 10, the domains and motifs are encoded by the following amino acid sequences.

[0612]

[0303] CD63 -CYTOPLASMIC: MAVEGGMKCVK (SEQ ID NO: 297)

[0613]

[0304] CD63-TM-HELICAL(inside-out) FLLYVLLLAFCACAVGLIAVG_(SEQ ID NO: 298)

[0614]

[0305] CD63 -EXTRACELLULAR VGAQLVLSQTIIQGATPGS (SEQ ID NO: 299)

[0615]

[0306] CD63-TM-HELICAL(outside-in) LLPWIIAVGVFLFLVAFVGC (SEQ ID NO: 300)

[0616]

[0307] CD63 -CYTOPLASMIC CGACKENYC (SEQ ID NO: 301)

[0617]

[0308] CD63-TM-HELICAL(inside-out) LMITFAIFLSLIMLVEVAAAI ((SEQ ID NO: 302)

[0618]

[0309] Linker: GGSGPVRRYQ(SEQ ID NO: 303)

[0619]

[0310] MMP9: GGPLGMTS (SEQ ID NO: 304)

[0620]

[0311] TNFa:

[0621] S S SRTP SDKP VAHV VANPQ AEGQLQ WLNRR AN ALL ANGVELRDNQLVVPSEGLYLIYS Q

[0622] VLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGG

[0623] VFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIA (SEQ ID NO: 305)

[0624]

[0312] Linker+ Myc-tag: GGSGEQKLISEEDL (SEQ ID NO: 306)

[0625]

[0313] MMP9: GGPLGMTS (SEQ ID NO: 307)

[0626]

[0314] CD63 -EXTRACELLULAR

[0627] PKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKA

[0628] IHI<EGCVEI<IGGWLRI<NVLVVAAAALGIAFVEVLGIVFACCLVI<SIRSGYEVM (SEQ ID

[0629] NO: 308)

[0630]

[0315] CD63-TM-HELICAL(outside-in) LVVAAAALGIAFVEVLGIVFA (SEQ ID NO: 309)

[0631]

[0316] CD63 -CYTOPLASMIC LVKSIRSGYEVM (SEQ ID NO: 310)

[0317] Construct CD63-9-2L-TNFa-Myc-9-CD63-D-Cyt (Lacks EVs sorting motif) is outlined in

[0632] SEQ ID NO: 311 below.

[0633] MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVG VFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQGGPLG MTSSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLI YSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIY LGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGPLGMTS GGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINF NEI<AIHI<EGCVEI<IGGWLRI<NVLVVAAAALGIAFVEVLGIVFACCLVI<S (SEQ ID NO: 311)

[0634]

[0318] Referring to Figures 5A-B, 11 and 12, the domains and motifs of this construct are encoded by the following amino acid sequences.

[0635]

[0319] CD63 -CYTOPLASMIC :

[0636] MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVG

[0637] VFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYV (SEQ ID NO: 313)

[0638]

[0320] CD63-TM-HELICAL(inside-out) FLLYVLLLAFCACAVGLIAVG.(SEQ ID NO: 314)

[0639]

[0321] CD63 -EXTRACELLULAR VGAQLVLSQTIIQGATPGS (SEQ ID NO: 315)

[0640]

[0322] CD63-TM-HELICAL(outside-in) LLPWIIAVGVFLFLVAFVGC_(SEQ ID NO: 316)

[0641]

[0323] CD63 -CYTOPLASMIC CGACKENYC (SEQ ID NO: 317)

[0642]

[0324] CD63-TM-HELICAL(inside-out) LMITFAIFLSLIMLVEVAAAI_(SEQ ID NO: 318)

[0643]

[0325] Linker: GGSGPVRRYQ(SEQ ID NO: 319)

[0644]

[0326] MMP9: GGPLGMTS (SEQ ID NO: 320)

[0645]

[0327] TNFa-

[0646] S S SRTP SDKP VAHV VANPQ AEGQLQ WLNRR AN ALL ANGVELRDNQLVVPSEGLYLIYS Q

[0647] VLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGG

[0648] VFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIA (SEQ ID NO: 321)

[0649]

[0328] Linker+ Myc-tag: GGSGEQKLISEEDL (SEQ ID NO: 322)

[0650]

[0329] MMP9: GGPLGMTS (SEQ ID NO: 323)

[0651]

[0330] CD63-EXTRC ELLUL AR

[0652] PKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKA

[0653] IHI<EGCVEI<IGGWLRI<NVLVVAAAALGIAFVEVLGIVFACCLVI<SIRSGYEVM (SEQ ID

[0654] NO: 324)

[0655]

[0331] CD63-TM-HELICAL(outside-in) LVVAAAALGIAFVEVLGIVFA (SEQ ID NO: 185)

[0656]

[0332] CD63 -CYTOPLASMIC CCLVKS (SEQ ID NO: 325).

[0657]

[0333] Functional assays

[0334] Example 8: Assay for TNFa Using HEK 293 Cells

[0658]

[0335] Referring to Figures 21 and 22, we created a functional assay to measure the presence of TNFa using HEK293 NF-KB luciferase reporter cells.

[0659]

[0336] Referring to Figures 22 and 24, to test whether the GAGE payload in this system is functional, HEK293 cells were transfected with GAGE plasmids using polyethyleneimine (PEI) in a transient transfection as follows:

[0660] - Day 0: 400,000 cells were seeded in 6 well plates.

[0661] - Day 1 : 2.5 ug of the GAGE plasmids were transfected into HEK293 wt cells.

[0662] - Day 2: HEK293+GAGE and HEK293 NF-KB Luciferase reporter cells were co-cultured for 24 hr or kept separately.

[0663] - Day 3 : The reporter cells were collected, lysed and we assayed for the functional activity of TNFa presence on HEK293 cells using NF-KB -Luciferase reporter assay. Referring to Figure 26, it is readily observed that TNFa expressed on the HEK293 cells was functional regardless of the presence or absence of the EVs sorting motif.

[0664]

[0337] Example 9: Assay For Payload Release by MMP 9 or MMP2

[0665]

[0338] Next, referring to Figure 25 and 27, to test whether the 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 a transient transfection as follows:

[0666] - Day 0: 400,000 cells were seeded in 6 well plates.

[0667] - Day 1 : 2.5 ug of the GAGE and MMP9 or MMP2 plasmids were transfected separately into HEK293 wt cells.

[0668] - Day 2: HEK293+GAGE and HEK293+MMP9 or HEK293+MMP2 cells were co-cultured for 24 hr.

[0669] - Day 3 : 200 uL of the supernatant was collected and given to HEK293 NF -KB reporter cells.

[0670]

[0339] After 6hr incubation, the reporter cells were collected, lysed with l%Triton, and we assayed for the functional activity of TNFa released from HEK293 cells using NF-KB -Luciferase reporter assay. The substrate (D-luciferin) was automatically added to the samples, and the luminescence was measured.

[0671]

[0340] Referring to Figures 25 and 27, it is readily observed that TNFa expressed on the HEK293 cells was higher for the EVs sorted plasmid, indicating that most of the GAGE payloads enriched into EVs than non-EVs sorting plasmid.

[0341] Example 10: Delivery of Payload to Extracellular Vesicles

[0672]

[0342] To verify the delivery of the GAGE payloads mainly into EVs, the experiment of Example 9 was repeated with supernatant depletion of EVs by running it through Amicon® Ultra 0.5 mL Centrifugal Filters. The luciferase reporter test was performed with HEK293 NF-KB Luciferase reporter cells. Supernatant from co-culture was either depleted from EVs or added as it is to HEK293 reporter cells expressing NF-kB in response to TNFa-payload. Figures 26 and 28 show that the Luciferase activity was remarkably reduced after EVs depletion, suggesting delivery of the GAGE presence in the supernatant containing extracellular vesicles.

[0673]

[0343] Example 11: Selection of TMD For Better Cell Surface Expression

[0674]

[0344] To evaluate alternative transmembrane (TM) protein that can mainly express on the cell surface, we exchanged the CD63 TM with C-type Lectin transmembrane CD69. Figure 31 shows higher percentage of TNFa positive cells where CD69 was used as TM protein compared to CD63 TM.

[0675]

[0345] Next, to evaluate whether the GAGE TNFa expressed on the HEK293 cells can be released in the presence of MMP9, the MMP9 cleavage was carried out by direct co-culturing of GAGE TNFa expressing cells with MMP expressing cells, supernatant from MMP9 transfected cells or providing MMP9 recombinant protein. The successful MMP9 cleavage of the MMP9 protease substrate linked to the TNFa GAGE was verified by luciferase assay using HEK293 NF-KB Luciferase reporter cells. As shown in Figures 17A-B, 18A-B, 29 and 30, the luciferase activity was only observed in the clone with the presence of MMP9 cleavage substrate, indicating that the TNFa can be released by specific protease cleavage.

[0676]

[0346] Example 12: Payload Release with Granzyme B

[0677]

[0347] We used another sheddase cleavage substrate for Granzyme B (GrzB) protein connected to TNFa GAGE as a payload to further evaluate our invention. First, both clones CD69-GrzB-TNFa- Myc-GrzB-aCD19-CAR and CD69-TNFa-Myc-aCD19-CAR were generated and transduced in K562 cells (Figure 31).

[0678]

[0348] Next, to evaluate whether the GAGE TNFa with GrzB cleavage substrate expressed on the K562 cells can be released upon co-culturing with NK92. Encountering NK92 cells against target cells usually leads to induced degranulation of NK92 with granules containing Granzyme B. The GrzB cleavage with TNFa release was carried out via co-culturing GAGE TNFa expressing K562 cells with NK92 cells. After 4hrs incubation, the cells were centrifuged, and the supernatant was collected. The successful cleavage of the GrzB protease substrate linked to the TNFa GAGE was verified by luciferase assay using HEK293 NF-KB Luciferase reporter cells. As shown in Figure 32, the luciferase activity was higher in the clone with the presence of GrzB cleavage substrate, indicating that the TNFa was specifically released by specific protease cleavage.

[0679]

[0349] Example 13: Delivery of NanoLuciferase Functional Assay as a Proof of Principle

[0680]

[0350] To increase the sensitivity of the readout, we repeated the experiment of Example 12 with another GAGE payload, in this case, we used NanoLuciferase as a payload.

[0681]

[0351] We generated three clones as follows:

[0682] CD69-GrzB-nLuc-Myc-GrzB-aCD19-CAR (GAGE 1 or GAGE original)

[0683] - CD69-nLuc-Myc -aCD 19-CAR (GAGE A)

[0684] - CD69-GrzB-nLuc-Myc-GrzB-CAR (GAGE B)

[0685]

[0352] These plasmids were transduced in K562 cells with MOK and then assayed with the Luciferase assay. Referring to Figure 33, the surface expression of GAGE nLuc in K562 cells was observed.

[0686]

[0353] Next, to evaluate whether the GAGE nLuc with GrzB cleavage substrate expressed on the K562 cells can be released upon co-culturing with NK92. The GrzB cleavage with nLuc release was carried out via co-culturing GAGE nLuc expressing K562 cells with NK92 cells. After 4hrs incubation, the cells were centrifuged, and the supernatant was collected. The successful cleavage of the GrzB protease substrate linked to the nLuc GAGE was verified by direct luciferase assay. As shown in Figure 34A, the luciferase activity was higher in the clone with the presence of GrzB cleavage substrate, indicating that the nLuc was particularly released by specific protease cleavage. Further activation of NK92 cells using PAM / Ion had no significant effect on additional cleavage of the substrate, indicated by no differences in luciferase activity assay Figure 34B.

[0687]

[0354] Example 14: Additional Clones to Evaluate Payload Cleavage and Release

[0688]

[0355] We generated the following clones CD69-GrzB-nLuc-Myc-GrzB-aCD19-CAR (GAGE 1 or GAGE original), CD69-nLuc-Myc-aCDl 9-CAR (GAGE A), and CD69-GrzB-nLuc-Myc- GrzB-CAR (GAGE B) in KHYG-1. To further evaluate the specific cleavage and payload release. We transduced the three clones mentioned above in KHYG-1 with MOI30. Referring to Figure 35, the surface expression of GAGE nLuc in KHYG1 cells was observed.

[0689]

[0356] Following the co-culturing of KHYG-1 cells expressing GAGE nLuc with Nalm-6 cells (highly expressed CD19) for three different time points indicated in Figure 36, the luciferase activity was higher in the clone with the presence of GrzB cleavage substrate with nLuc connected to sc-aCD19CAR, indicating that the nLuc was particularly released by specific protease cleavage during the period of co-culturing with highest cleavage at three hours incubation.

[0690]

[0357] Next, we sorted the cells to reduce the background activity of the NanoLuciferase. As shown in Figure 37A, KHYG-1 cells were more than 99% positive for the three clones. Interestingly, the MFI of nLuc GAGE positive cells was higher in the clone without the cleavage substrate site compared to the other two clones (Figure 37B).

[0691]

[0358] We repeated the experiment on sorted cells, and two different time points (4hr and 6hr) were selected for co-culturing sorted KHYG-1 cells expressing GAGE nLuc with K562 cells. As shown in Figure 38, the luciferase activity was higher in the clone with the presence of GrzB cleavage substrate with nLuc, indicating that the nLuc was particularly released by specific protease cleavage during the period of co-culturing with the highest cleavage at 6 hours incubation.

[0692]

[0359] To evaluate the specificity and sensitivity of the cleavage by Granzyme B, we generated three clones of CD69-GrzB-nLuc-Myc-GrzB-aCD19-CAR with different GrzB cleavage substrates, as follows (GrzB (IEPD (SEQ ID NO: 258)), GrzB (AQGVISADASNLDDFY (SEQ ID NO: 261)) and GrzB (LEADKGKLEYD (SEQ ID NO: 262) in NK92 cells. We transduced the clones mentioned above in NK92 cells with MOI30. Referring to Figures 39A and 39B, the surface expression of GAGE nLuc in NK92 cells was observed.

[0693]

[0360] Following the co-culturing of NK92 cells expressing GAGE nLuc with Raji cells for four hours, the luciferase activity was higher, with a nine-fold increase in the clone with the presence of GrzB (LEADKGKLEYD (SEQ ID NO: 262)) as cleavage substrate with nLuc connected compared to the control clone (GAGE A), indicating that this particular substrate has the higher affinity to the GrzB cleavage during the period of co-culturing (Figure 40).

[0694]

[0361] Example 15: Payload release via Caspase 3:

[0695]

[0362] We generated the following clones CD69-Casp3-nLuc-Myc-Casp3-aCD19-CAR (GAGE Casp3) and CD69-nLuc-Myc-aCD19-CAR (GAGE A), in NK92. To further evaluate the specific cleavage and payload release. We transduced the two clones mentioned above in NK92 with MOI30. Referring to Figure 41, the surface expression of GAGE nLuc inNK92 cells was observed.

[0696]

[0363] Following the co-culturing of NK92 cells expressing GAGE Casp3-nLuc against K562 cells for two different time points indicated in Figure 42, the luciferase activity was higher in the clone with the presence of Casp3 cleavage substrate with nLuc, indicating that the nLuc was particularly released by specific protease cleavage during the period of co-culturing.

[0697]

[0364] CD69cyt-TMII-TNFa-Myctag-CD28TM-cyt

[0365] The cDNA of CD69Cyt-TM-II-TNFa-Myc-CD28TM-Cyt is set forth below as SEQ ID NO: 326

[0698] GATCCGCCGCCACCATGAGTTCCGAAAACTGCTTTGTCGCCGAGAATAGTAGTCTGC ACCCCGAATCAGGACAGGAGAATGACGCAACCAGCCCCCACTTTTCAACAAGGCAC GAAGGGAGCTTCCAGGTCCCTGTGCTGTGCGCAGTGATGAACGTGGTCTTTATCACT ATTCTGATCATTGCTCTGATCGCACTGAGTGTCGGACAGTACAATGGCGGGTCAGGC CCCGTGCGGAGATATCAGAGCTCCTCTCGCACCCCTTCCGACAAGCCAGTGGCCCAT GTGGTCGCTAACCCTCAGGCAGAGGGACAGCTGCAGTGGCTGAACAGGCGAGCCAA TGCTCTGCTGGCTAACGGCGTGGAACTGCGAGATAATCAGCTGGTGGTCCCTAGCGA GGGGCTGTACCTGATCTATTCCCAGGTCCTGTTCAAAGGGCAGGGATGCCCATCTAC ACACGTGCTGCTGACCCATACAATCTCTAGAATTGCCGTCAGTTACCAGACTAAGGT GAACCTGCTGAGCGCCATCAAGTCACCATGTCAGAGGGAGACACCCGAAGGAGCAG AGGCCAAGCCCTGGTACGAGCCTATCTATCTGGGAGGCGTGTTTCAGCTGGAAAAA GGCGACCGACTGTCTGCCGAGATTAATCGGCCAGACTACCTGGATTTCGCTGAAAGT GGCCAGGTGTATTTTGGGATCATTGCACTGGGAGGCAGCGGAGAGCAGAAGCTGAT CTCTGAGGAAGATCTGGGAGGGAGCGGATGCCCATCCCCTCTGTTCCCAGGACCCAG CAAACCCTTTTGGGTCCTGGTGGTCGTGGGAGGCGTGCTGGCATGTTATAGCCTGCT GGTCACAGTGGCTTTCATTATCTTTTGGGTCCGCTCTAAACGGTCTCGGCTGCTGTAA

[0699] TGTACAA (SEQ ID NO: 326)

[0700]

[0366] The corresponding amino acid sequence is set forth as SEQ ID NO: 327 .

[0701] MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALS VGQYNGGSGPVRRYQS S SRTP SDKP V AH VV ANPQ AEGQLQ WLNRR AN ALL ANGVELR DNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRET PEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKL ISEEDLGGSGCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLL (SEQ ID NO: 327)

[0702]

[0367] SEQ ID NO: 327 is also shown in Figure 17A-B, together with a legend of domains and motifs

[0703]

[0368] CD69-CYTOPLASMIC MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVP

[0704] (SEQ ID NO: 328)

[0705]

[0369] CD69-TM-type-II(inside-out) VLCAVMNVVFITILIIALIAL (SEQ ID NO 329)

[0706]

[0370] CD69-Extracellular SVGQYNGGSGPVRRYQ (SEQ ID NO 330)

[0707]

[0371] TNFa

[0708] RTPSDKPVAHWANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVL FKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVF QLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (SEQ ID NO 331)

[0709]

[0372] linker GGSG (SEQ ID NO 332)

[0710]

[0373] Myc-tag EQKLISEEDL (SEQ ID NO: 333)

[0711]

[0374] CD28TM FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO 334)

[0712]

[0375] CD69cyt-TMII-9-TNFa-Myctag-9-CD28TM-cyt

[0713]

[0376] The cDNA of CD69cyt-TMII-9-TNFa-Myctag-9-CD28TM-cyt is set forth below as SEQ

[0714] ID NO: 335).

[0715] ATCCGCCGCCACCATGAGTTCCGAAAACTGCTTTGTCGCCGAGAATAGTAGTCTGCA

[0716] CCCCGAATCAGGACAGGAGAATGACGCAACCAGCCCCCACTTTTCAACAAGGCACG AAGGGAGCTTCCAGGTCCCTGTGCTGTGCGCAGTGATGAACGTGGTCTTTATCACTA TTCTGATCATTGCTCTGATCGCACTGAGTGTCGGACAGTACAATGGCGGGTCAGGCC CCGTGCGGAGATATCAGGGAGGGTCTGGGGGAGGACCACTGGGAATGACCTCCGGA GGGTCTGGGAGCTCCTCTCGCACCCCTTCCGACAAGCCAGTGGCCCATGTGGTCGCT AACCCTCAGGCAGAGGGACAGCTGCAGTGGCTGAACAGGCGAGCCAATGCTCTGCT GGCTAACGGCGTGGAACTGCGAGATAATCAGCTGGTGGTCCCTAGCGAGGGGCTGT ACCTGATCTATTCCCAGGTCCTGTTCAAAGGGCAGGGATGCCCATCTACACACGTGC TGCTGACCCATACAATCTCTAGAATTGCCGTCAGTTACCAGACTAAGGTGAACCTGC TGAGCGCCATCAAGTCACCATGTCAGAGGGAGACACCCGAAGGAGCAGAGGCCAAG CCCTGGTACGAGCCTATCTATCTGGGAGGCGTGTTTCAGCTGGAAAAAGGCGACCGA CTGTCTGCCGAGATTAATCGGCCAGACTACCTGGATTTCGCTGAAAGTGGCCAGGTG TATTTTGGGATCATTGCACTGGGAGGCAGCGGAGAGCAGAAGCTGATCTCTGAGGA AGATCTGGGAGGACCACTGGGAATGACCTCCGGAGGGAGCGGATGCCCATCCCCTC TGTTCCCAGGACCCAGCAAACCCTTTTGGGTCCTGGTGGTCGTGGGAGGCGTGCTGG CATGTTATAGCCTGCTGGTCACAGTGGCTTTCATTATCTTTTGGGTCCGCTCTAAACG

[0717] GTCTCGGCTGCTGTAATGTACA (SEQ ID NO:335)

[0718]

[0377] The corresponding amino acid sequence is set out below as SEQ ID NO: 336 and is also set out in Figure 18A-B together with a legend of domains and motifs.

[0719]

[0378]

[0720] MS SENCF VAENS SLHPESGQEND ATSPHF STRHEGSFQVPVLC AVMNVVFITILIIALIA LSVGQYNGGSGPVRRYQGGSGGGPLGMTSGGSGSSSRTPSDKPVAHVVANPQAEGQ LQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRI AVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDY LDFAESGQVYFGIIALGGSGEQKLISEEDLGGPLGMTSGGSGCPSPLFPGPSKPFWVLV WGGVLACYSLLVTVAFIIFWVRSKRSRLL (SEQ ID NO: 336)

[0721]

[0379] Domains and Motifs

[0722]

[0380] CD69-CYTOPLASMIC MS SENCF VAENS SLHPESGQEND ATSPHF STRHEGSFQVP (SEQ ID NO: 338)

[0723]

[0381] CD69-TM-type-II(inside-out)_VLCAVMNVVFITILIIALIAL (SEQ ID NO: 339)

[0724]

[0382] CD69-Extracellular_SVGQYNGGSGPVRRYQ (SEQ ID NO: 340)

[0725]

[0383] Linker GGSG (SEQ ID NO: 341)

[0726]

[0384] MMP9 GGPLGMTSG (SEQ ID NO: 342)

[0727]

[0385] Linker GSGSSS (SEQ ID NO: 343)

[0728]

[0386] TNFa

[0729] RTPSDKPVAHWANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQ VLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYL GGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (SEQ ID NO 344)

[0730]

[0387] Myc-tag EQKLISEEDL (SEQ ID NO 345)

[0731]

[0388] CD28TM FWVLVVVGGVLACYSLLVTVAFIIFW (SEQ ID NO: 346)

[0732]

[0389] Referring to Figure 20, some of the GAGE DNA SEQs are shown as follows:

[0390] LeGo-CD63 -9-2L-TNFa-My C-9-CD63 -cyt

[0733] MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVI IAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRR YQGGPLGMTSSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQ LVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETP EGAEAKPWYEPrYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQ KLISEEDLGGPLGMTSGGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSK NRVPDSCCINVTVGCGINFNEI<AIHI<EGCVEI<IGGWLRI<NVLVVAAAALGIAFVEVL GIVFACCLVKSIRSGYEVM (SEQ ID NO: 295)

[0734]

[0391] Which is encoded as cDNA:

[0735] GATCCGCCGCCACCATGGCCGTGGAGGGCGGGATGAAGTGCGTGAAATTCCTGC TGTACGTCCTGCTGCTGGCCTTTTGCGCATGTGCTGTGGGACTGATCGCAGTGGG AGTCGGAGCTCAGCTGGTGCTGAGTCAGACTATCATTCAGGGCGCAACCCCTGG GTCACTGCTGCCAGTGGTCATCATTGCCGTGGGCGTCTTCCTGTTTCTGGTGGCAT TCGTCGGGTGCTGTGGAGCCTGCAAGGAAAACTATTGTCTGATGATCACCTTCGC TATTTTTCTGTCTCTGATCATGCTGGTGGAGGTCGCCGCTGCAATTGCCGGCTACG TGGGAGGCAGTGGACCAGTCCGGAGATATCAGGGGGGACCCCTGGGAATGACA AGCTCCTCTAGTCGGACTCCCTCTGACAAGCCTGTGGCCCACGTGGTCGCTAATC CTCAGGCAGAAGGCCAGCTGCAGTGGCTGAACAGGCGAGCAAATGCACTGCTGG CCAACGGAGTGGAACTGAGAGATAATCAGCTGGTGGTCCCATCCGAGGGACTGT ACCTGATCTATTCTCAGGTGCTGTTCAAAGGACAGGGATGCCCAAGCACACACGT GCTGCTGACCCATACAATCTCTAGAATTGCTGTGAGTTACCAGACTAAGGTCAAC CTGCTGAGCGCCATCAAATCCCCCTGTCAGAGGGAGACCCCTGAAGGCGCTGAG GCAAAGCCATGGTACGAGCCCATCTATCTGGGCGGGGTGTTTCAGCTGGAAAAA GGGGACCGACTGAGCGCAGAGATTAATCGGCCCGACTACCTGGATTTCGCCGAA TCCGGACAGGTGTATTTTGGCATCATTGCTCTGGGAGGCTCAGGCGAGCAGAAG CTGATCAGCGAGGAAGATCTGGGAGGACCACTGGGAATGACCTCCGGAGGGTCT GGGCCTAAGAACAATCACACAGCCAGCATTCTGGACCGCATGCAGGCAGATTTC AAATGCTGTGGCGCCGCTAACTACACAGACTGGGAAAAGATCCCTTCAATGAGC AAAAACCGAGTGCCAGATTCCTGCTGTATTAATGTGACTGTCGGATGCGGCATCA ACTTTAATGAGAAGGCCATTCATAAAGAAGGCTGCGTGGAGAAGATCGGAGGCT GGCTGAGGAAAAATGTGCTGGTGGTCGCAGCCGCTGCACTGGGAATCGCCTTCG TGGAAGTCCTGGGCATCGTGTTCGCTTGCTGTCTGGTCAAAAGTATTCGCTCAGG CTATGAAGTGATGTAATGTACA (SEQ ID NO: 296)

[0736]

[0392] LeGo-CD63-9-2L-TNFa-Myc-9-CD63-D-Cyt

[0737] MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVI IAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRR YQGGPLGMTSSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQ LVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETP EGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQ KLISEEDLGGPLGMTSGGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSK NRVPDSCCINVTVGCGINFNEI<AIHI<EGCVEI<IGGWLRI<NVLVVAAAALGIAFVEVL GIVFACCLVKS (SEQ ID NO: 311)

[0738]

[0393] Which is encoded as cDNA: GGATCCGCCGCCACCATGGCCGTGGAGGGCGGGATGAAGTGCGTGAAATTCCTGCT GTACGTCCTGCTGCTGGCCTTTTGCGCATGTGCTGTGGGACTGATCGCAGTGGGAGT CGGAGCTCAGCTGGTGCTGTCCCAGACTATCATTCAGGGCGCAACCCCTGGGTCTCT GCTGCCAGTGGTCATCATTGCCGTGGGCGTCTTCCTGTTTCTGGTGGCATTCGTCGGG TGCTGTGGAGCCTGCAAGGAAAACTATTGTCTGATGATCACCTTCGCTATTTTTCTGA GCCTGATCATGCTGGTGGAGGTCGCCGCTGCAATTGCAGGATACGTGGGAGGCTCCG GACCAGTCCGGAGATATCAGGGGGGACCCCTGGGAATGACAAGCTCCTCTAGTAGG ACTCCCAGCGACAAGCCTGTGGCCCACGTGGTCGCTAATCCTCAGGCAGAAGGCCA GCTGCAGTGGCTGAACAGGCGAGCAAATGCACTGCTGGCCAACGGAGTGGAACTGC GAGATAATCAGCTGGTGGTCCCATCAGAGGGACTGTACCTGATCTATAGCCAGGTGC TGTTCAAAGGACAGGGATGCCCATCCACACACGTGCTGCTGACCCATACAATCAGCA GAATTGCCGTGAGCTACCAGACTAAGGTCAACCTGCTGAGCGCCATCAAGTCACCCT GTCAGAGGGAGACCCCTGAAGGCGCTGAGGCAAAGCCATGGTACGAGCCCATCTAT CTGGGCGGGGTGTTTCAGCTGGAAAAAGGGGACCGACTGAGTGCAGAGATTAATCG GCCCGACTACCTGGATTTCGCCGAATCAGGACAGGTGTATTTTGGCATCATTGCTCT

[0739] GGGAGGCTCTGGCGAGCAGAAGCTGATCAGTGAGGAAGATCTGGGAGGACCACTGG GAATGACCTCCGGAGGGTCTGGGCCTAAGAACAATCACACAGCTTCTATTCTGGACC GCATGCAGGCAGATTTCAAATGCTGTGGCGCCGCTAACTATACAGACTGGGAAAAG ATCCCTTCAATGAGCAAAAACCGAGTGCCAGATAGTTGCTGTATTAATGTGACTGTC GGATGCGGCATCAACTTTAATGAGAAGGCCATTCATAAAGAAGGCTGCGTGGAGAA GATCGGAGGCTGGCTGCGGAAAAATGTGCTGGTGGTCGCAGCCGCTGCACTGGGAA TCGCCTTCGTGGAGGTCCTGGGCATCGTGTTCGCTTGCTGTCTGGTCAAATCTTAATG TACA (SEQ ID NO: 347)

[0740]

[0394] LeGo-CD63 -DL 1 -2-2L-TNFa-Myc-2-CD63 -Cyt

[0741] MAVEGGMKCVKCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQG GPLGVRGGSSSRTPSDKPVAHWANPQAEGQLQWLNRRANALLANGVELRDNQLV VP SEGLYLIYSQVLFKGQGCP STHVLLTHTISRIAVS YQTKVNLLS AIKSPCQRETPEG AEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLI SEEDLGGPLGVRGGGGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNR VPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGI VFACCLVKSIRSGYEVM (SEQ ID NO: 368)

[0742]

[0395] Which is encoded by cDNA:

[0743] GGATCCGCCGCCACCATGGCAGTGGAAGGCGGGATGAAGTGCGTCAAATGTGGCGC CTGCAAGGAGAACTACTGTCTGATGATCACCTTCGCTATTTTTCTGAGCCTGATCATG CTGGTGGAAGTCGCCGCTGCAATTGCAGGATACGTGGGAGGCAGCGGACCCGTCCG GAGATATCAGGGAGGACCTCTGGGCGTGCGAGGAGGGAGCTCCTCTCGAACACCAT CCGACAAGCCTGTGGCTCACGTGGTCGCAAATCCACAGGCAGAGGGACAGCTGCAG TGGCTGAACAGGCGAGCCAATGCTCTGCTGGCCAACGGAGTGGAACTGAGGGATAA TCAGCTGGTGGTCCCCAGTGAGGGCCTGTACCTGATCTATTCACAGGTGCTGTTCAA AGGACAGGGCTGTCCTTCTACTCACGTGCTGCTGACCCATACAATCTCAAGGATTGC CGTGAGCTATCAGACTAAGGTCAACCTGCTGTCTGCTATCAAAAGTCCTTGCCAGCG AGAGACCCCAGAAGGAGCAGAGGCCAAGCCATGGTACGAGCCCATCTATCTGGGAG GCGTGTTTCAGCTGGAAAAAGGAGACAGACTGTCTGCAGAGATTAATAGGCCTGAC TACCTGGATTTCGCCGAAAGTGGCCAGGTGTATTTTGGGATCATTGCTCTGGGAGGC AGCGGAGAGCAGAAGCTGATCTCTGAGGAAGATCTGGGAGGACCTCTGGGGGTGCG CGGAGGAGGGGGAAGCGGACCAAAGAACAATCACACAGCTTCCATTCTGGACCGAA TGCAGGCAGATTTCAAATGCTGTGGCGCCGCTAACTACACTGACTGGGAAAAGATCC CAAGTATGTCAAAAAACCGGGTGCCCGATAGCTGCTGTATTAATGTGACAGTCGGGT GTGGAATCAACTTTAATGAGAAGGCCATTCATAAAGAAGGCTGCGTGGAGAAGATC GGCGGGTGGCTGCGGAAAAATGTCCTGGTGGTCGCAGCAGCTGCACTGGGAATCGC CTTCGTGGAAGTCCTGGGGATCGTGTTCGCTTGCTGTCTGGTCAAAAGCATTAGATC CGGCTATGAAGTGATGTAATGTACAA (SEQ ID NO: 348)

[0744]

[0396] LeGo-CD63 -9-2L-TNFa-My c-9-CD 19C AR

[0745] MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVI IAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRR YQGGPLGMTS S S SRTPSDKP VAHWANPQ A EGQLQ WLNRR AN ALL ANG VELRDNQ LVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETP EGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQ KLISEEDLGGPLGMTSGGSGGGSGPDIQMTQTTSSLSASLGDRVTISCRASQDISKYL NWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG NTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSG VSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMN SLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAKPTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLC PSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR PGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 369)

[0746]

[0397] Which is encoded by cDNA:

[0747] GATCCGCCGCCACCATGGCCGTGGAGGGCGGGATGAAGTGCGTCAAATTCCTGCTGT ACGTGCTGCTGCTGGCCTTTTGCGCATGTGCAGTCGGACTGATCGCAGTCGGAGTGG GAGCCCAGCTGGTGCTGTCCCAGACTATCATTCAGGGGGCAACCCCTGGATCTCTGC TGCCAGTGGTCATCATTGCTGTCGGCGTGTTCCTGTTTCTGGTCGCTTTCGTGGGCTG CTGTGGGGCATGCAAGGAGAACTACTGTCTGATGATCACATTCGCCATTTTTCTGTC CCTGATCATGCTGGTCGAAGTGGCCGCTGCAATTGCTGGCTACGTCGGAGGCTCTGG ACCTGTGCGGAGATATCAGGGGGGACCACTGGGCATGACAAGCTCCTCTAGTAGAA CTCCCAGCGACAAGCCTGTGGCCCACGTGGTCGCTAATCCACAGGCAGAGGGACAG CTGCAGTGGCTGAACAGGCGAGCTAATGCACTGCTGGCCAACGGGGTGGAGCTGAG GGATAATCAGCTGGTGGTCCCTTCCGAAGGACTGTACCTGATCTATTCTCAGGTCCT GTTCAAAGGACAGGGCTGCCCAAGCACCCACGTGCTGCTGACCCATACAATCTCCCG CATTGCCGTCTCTTACCAGACCAAGGTGAACCTGCTGAGCGCCATCAAGTCCCCATG TCAGCGGGAAACACCCGAGGGCGCCGAAGCTAAGCCATGGTACGAGCCCATCTATC TGGGCGGGGTGTTTCAGCTGGAGAAAGGCGACCGGCTGAGTGCAGAAATTAATAGA CCTGACTACCTGGATTTCGCCGAGAGCGGACAGGTGTATTTTGGCATCATTGCTCTG GGAGGCTCTGGCGAACAGAAGCTGATCAGTGAGGAAGATCTGGGAGGACCACTGGG AATGACATCTGGAGGGAGTGGAGGAGGGAGTGGACAGGTCCAGCTGGTGGAATCAG GAGGAGGACTGGTGCAGCCTGGAGGCTCTCTGAAGCTGAGTTGCGCCGCTTCAGGCT TCGACTTTAGTAGGTACTGGATGTCATGGGTGCGCCAGGCACCAGGGAAAGGACTG GAGTGGATCGGCGAAATTAACCCCACATCAAGCACTATCAATTTCACTCCTAGCCTG AAGGACAAAGTGTTTATTTCCAGAGATAACGCCAAGAATACACTGTACCTGCAGAT GAGCAAAGTGAGGTCCGAGGACACTGCTCTGTACTATTGTGCACGAGGCAACTACT ATCGGTACGGGGACGCCATGGATTATTGGGGCCAGGGGACCTCCGTCACAGTGTCA

[0748] AAGATCAGCGGGGGAGGAGGGAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCTCTG GGGGAGGCGGGAGTGGAGGCGGGGGCAGCTCTGATATCGTCCTGACACAGTCCCCA GCTTCTCTGGCAGTGTCTCTGGGCCAGCGCGCAACTATTAGTTGTCGAGCCAGTAAA TCAGTGAGCACCTCCGGATACAGCTATCTGCACTGGTATCAGCAGAAGCCCGGCCAG CCCCCTAAACTGCTGATCTATCTGGCTTCTAACCTGGAGAGTGGCGTGCCTGCAAGA TTCTCTGGAAGTGGCTCAGGGACTGACTTTACCCTGAATATTCACCCAGTGGAGGAA GAGGATGCAGCCACCTACTATTGCCAGCATTCAAGGGAGCTGCCCTTCACCTTTGGA AGCGGCACAAAGCTGGAAATCAAAGGCGGGGGAGGCTCAGGAGGCGGCGGGAGCG GCGGCGGAGGCAGCGCCAAGCCAACCACAACTCCAGCTCCACGACCACCAACACCT GCACCAACTATCGCTTCACAGCCCCTGAGCCTGAGACCTGAGGCATGTAGGCCAGCT GCAGGAGGAGCAGTCCATACTCGAGGACTGGACTTCGCCCCTAGGAAGATTGAAGT GATGTACCCTCCACCCTATCTGGATAACGAAAAAAGCAATGGAACCATCATTCACGT GAAGGGCAAACATCTGTGCCCCTCACCTCTGTTCCCAGGCCCCAGCAAGCCATTTTG GGTCCTGGTGGTCGTGGGAGGCGTGCTGGCCTGTTACAGCCTGCTGGTCACCGTGGC TTTCATCATCTTCTGGGTGCGGAGCAAACGGTCAAGACTGCTGCACAGCGACTATAT

[0749] GAACATGACCCCACGACGGCCTGGACCAACACGAAAGCATTACCAGCCATATGCAC CTCCACGAGACTTCGCAGCTTACAGGTCCCGCGTGAAGTTCAGCCGCTCCGCAGATG CACCAGCTTACCAGCAGGGACAGAATCAGCTGTATAACGAGCTGAATCTGGGGAGA AGGGAAGAGTATGACGTGCTGGATAAGCGACGAGGACGGGACCCCGAAATGGGAG GAAAGCCACGGAGAAAAAACCCTCAGGAGGGGCTTTACAATGAACTGCAGAAGGA CAAAATGGCAGAGGCCTATTCCGAAATCGGAATGAAGGGCGAGAGGCGCCGAGGG AAAGGACACGATGGCCTGTACCAGGGGCTGTCTACTGCCACCAAGGACACCTATGA TGCCCTGCATATGCAGGCTCTGCCCCCTAGATAATGTACA (SEQ ID NO: 349)

[0750]

[0398] LeGo-CD63-2-2L-TNFa-Myc-2-CD63-D-Cyt

[0751] MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVI IAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRR YQGGPLGVRGGSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDN QLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRET PEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQ KLISEEDLGGPLGVRGGGGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMS KNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEV LGIVFACCLVKS (SEQ ID NO: 371)

[0752]

[0399] Which is encoded by cDNA:

[0753] GATCCGCCGCCACCATGGCCGTGGAGGGCGGGATGAAGTGCGTGAAATTCCTGCTG TACGTCCTGCTGCTGGCCTTTTGCGCATGTGCTGTGGGACTGATCGCAGTGGGAGTC GGAGCTCAGCTGGTGCTGAGCCAGACTATCATTCAGGGAGCAACCCCTGGCAGCCT GCTGCCAGTGGTCATCATTGCCGTGGGCGTCTTCCTGTTTCTGGTGGCATTCGTCGGA TGCTGTGGCGCCTGCAAGGAAAACTATTGTCTGATGATCACTTTCGCTATTTTTCTGA GCCTGATCATGCTGGTGGAGGTCGCCGCTGCAATTGCAGGATACGTGGGAGGCAGC GGACCAGTCCGGAGATATCAGGGAGGACCACTGGGCGTGCGAGGAGGGAGCTCCTC TCGAACCCCATCCGACAAGCCTGTGGCACACGTGGTCGCTAATCCTCAGGCAGAAG GGCAGCTGCAGTGGCTGAACAGGCGCGCAAATGCCCTGCTGGCCAACGGAGTGGAA CTGAGGGATAATCAGCTGGTGGTCCCATCCGAGGGCCTGTACCTGATCTATTCTCAG GTGCTGTTCAAAGGGCAGGGATGCCCCTCTACACACGTGCTGCTGACCCATACAATC TCTAGGATTGCTGTGAGTTACCAGACAAAGGTCAACCTGCTGAGCGCCATCAAGTCC

[0754] CCCTGTCAGCGCGAGACTCCTGAAGGGGCTGAGGCAAAGCCATGGTACGAGCCCAT CTATCTGGGAGGCGTGTTTCAGCTGGAAAAAGGAGACAGACTGTCTGCAGAGATTA ATAGGCCCGACTACCTGGATTTCGCCGAAAGTGGCCAGGTGTATTTTGGGATCATTG CTCTGGGGGGATCAGGCGAGCAGAAGCTGATCAGCGAGGAAGATCTGGGAGGACCA CTGGGGGTGCGCGGAGGAGGGGGAAGTGGACCTAAGAACAATCACACCGCTTCAAT TCTGGACCGAATGCAGGCAGATTTCAAATGCTGTGGCGCCGCTAACTATACAGACTG GGAAAAGATCCCTAGTATGTCAAAAAACCGGGTGCCAGATAGCTGCTGTATTAATGT GACCGTCGGCTGCGGGATCAACTTTAATGAGAAGGCCATTCATAAAGAAGGCTGCG TGGAGAAGATCGGCGGGTGGCTGAGAAAAAATGTCCTGGTGGTCGCAGCAGCTGCA CTGGGAATCGCCTTCGTGGAGGTCCTGGGGATCGTGTTCGCTTGCTGTCTGGTCAAA AGCTAATGTA (SEQ ID NO: 350)

[0755]

[0400] LeGo_CD63 -2-2L-TNFa-Myc-2-CD 19CAR

[0756] MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIA VGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQGG PLGVRGGSSSRTPSDKPVAHWANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSE GLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPW YEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGPL GVRGGGGSGGGSGPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVK LLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT GGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKG LEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGG SYAMDYWGQGTSVTVSSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 372)

[0757]

[0401] Which is encoded by cDNA:

[0758] GGATCCGCCGCCACCATGGCCGTGGAGGGCGGGATGAAGTGCGTCAAATTCCTGCT GTACGTGCTGCTGCTGGCCTTTTGCGCATGTGCAGTCGGACTGATCGCAGTCGGAGT GGGAGCACAGCTGGTGCTGAGTCAGACTATCATTCAGGGAGCAACCCCTGGCAGCC TGCTGCCAGTGGTCATCATTGCTGTCGGCGTGTTCCTGTTTCTGGTCGCTTTCGTGGG GTGCTGTGGAGCATGCAAGGAGAACTACTGTCTGATGATCACTTTCGCCATTTTTCT GTCTCTGATCATGCTGGTCGAAGTGGCCGCTGCAATTGCTGGATACGTCGGAGGCAG CGGACCTGTGCGGAGATATCAGGGAGGACCACTGGGCGTCCGCGGAGGGAGCTCCT CTCGAACCCCAAGCGACAAGCCTGTGGCACACGTGGTCGCTAATCCTCAGGCAGAG GGACAGCTGCAGTGGCTGAACAGGCGAGCTAATGCACTGCTGGCCAACGGAGTGGA GCTGAGAGATAATCAGCTGGTGGTCCCTAGCGAAGGCCTGTACCTGATCTATTCCCA GGTCCTGTTCAAAGGACAGGGATGCCCATCCACCCACGTGCTGCTGACCCATACAAT CAGCAGAATTGCCGTCTCCTACCAGACAAAGGTGAACCTGCTGAGCGCCATCAAGA GTCCATGTCAGAGGGAAACTCCCGAGGGGGCCGAAGCTAAGCCATGGTACGAGCCC ATCTATCTGGGAGGCGTGTTTCAGCTGGAGAAAGGCGACAGGCTGAGCGCAGAAAT TAATCGCCCTGACTACCTGGATTTCGCCGAGTCCGGCCAGGTGTATTTTGGGATCATT GCTCTGGGGGGCAGCGGCGAACAGAAGCTGATCTCTGAGGAAGATCTGGGAGGACC ACTGGGGGTGAGGGGAGGAGGGGGATCAGGAGGAGGAAGCGGACAGGTCCAGCTG GTGGAAAGCGGAGGAGGACTGGTGCAGCCAGGAGGGTCCCTGAAGCTGTCTTGCGC CGCTAGTGGCTTCGACTTTTCTCGATACTGGATGAGTTGGGTGAGGCAGGCCCCTGG AAAAGGCCTGGAGTGGATCGGCGAAATTAACCCCACAAGTTCAACTATCAATTTCAC CCCTTCTCTGAAGGACAAAGTGTTTATTAGTCGCGATAACGCCAAGAATACTCTGTA

[0759] CCTGCAGATGTCAAAAGTGCGGAGCGAGGACACCGCTCTGTACTATTGTGCACGAG GGAACTACTATCGGTACGGAGACGCCATGGATTATTGGGGGCAGGGAACCTCTGTC ACAGTGTCCAAGATCTCTGGCGGAGGAGGATCAGGAGGAGGAGGCAGCGGCGGAG GAGGGTCCGGAGGCGGGGGATCTGGCGGGGGAGGCAGCTCCGATATCGTCCTGACA CAGAGTCCCGCTTCACTGGCAGTGTCACTGGGCCAGCGGGCAACCATTAGCTGTAGA GCCTCTAAAAGCGTGAGCACCAGCGGCTACAGCTATCTGCACTGGTATCAGCAGAA GCCTGGGCAGCCCCCTAAACTGCTGATCTATCTGGCTTCCAACCTGGAGTCTGGCGT GCCTGCACGCTTCTCCGGCTCTGGGAGTGGAACTGACTTTACCCTGAATATTCACCC AGTGGAGGAAGAGGATGCAGCCACTTACTATTGCCAGCATTCCCGGGAGCTGCCCTT CACCTTTGGCTCTGGGACAAAGCTGGAAATCAAAGGGGGAGGCGGGAGTGGCGGCG GCGGATCAGGCGGAGGAGGCAGCGCCAAGCCAACCACAACTCCAGCTCCACGACCA CCAACACCTGCACCAACTATCGCTAGTCAGCCCCTGTCACTGAGACCTGAGGCATGT AGGCCAGCTGCAGGAGGAGCAGTCCATACCCGAGGACTGGACTTCGCACCACGAAA GATTGAAGTGATGTACCCTCCACCCTATCTGGATAACGAAAAAAGCAATGGCACAA TCATTCACGTGAAGGGGAAACATCTGTGCCCCAGTCCTCTGTTCCCAGGCCCCTCAA AGCCCTTTTGGGTCCTGGTGGTCGTGGGAGGGGTGCTGGCCTGTTACAGCCTGCTGG TCACAGTGGCTTTCATCATCTTCTGGGTGCGGAGCAAACGGTCCAGACTGCTGCACT CCGACTATATGAACATGACACCACGACGGCCTGGCCCAACTAGAAAGCATTACCAG CCCTATGCCCCTCCAAGGGACTTCGCCGCTTACAGGTCCCGCGTGAAATTTTCAAGA AGCGCAGATGCCCCAGCTTACCAGCAGGGACAGAATCAGCTGTATAACGAGCTGAA TCTGGGCAGAAGGGAAGAGTATGACGTGCTGGATAAGCGACGAGGACGGGACCCCG AAATGGGAGGCAAGCCACGGAGAAAAAACCCTCAGGAGGGACTTTACAATGAACTG CAGAAGGACAAAATGGCAGAGGCCTATTCTGAAATCGGCATGAAGGGGGAGAGGC GCCGAGGAAAAGGCCACGATGGGCTGTACCAGGGACTGAGCACTGCCACCAAGGAC ACCTATGATGCCCTGCATATGCAGGCTCTGCCCCCTCGGTAATGTACA (SEQ ID NO: 351)

[0760]

[0402] LeGo-CD63-2L-TNFa-Myc-CD63-Cyt

[0761] MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIA VGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQGG PLGVRGGSSSRTPSDKPVAHWANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSE GLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPW YEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLISEEDLGGPL GVRGGGGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTV GCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGY EVM (SEQ ID NO: 364)

[0762]

[0403] Which is encoded by cDNA:

[0763] GGATCCGCCGCCACCATGGCCGTGGAGGGCGGGATGAAGTGCGTGAAATTCCTGCT GTACGTCCTGCTGCTGGCCTTTTGCGCATGTGCTGTGGGACTGATCGCAGTGGGAGT CGGAGCTCAGCTGGTGCTGTCTCAGACTATCATTCAGGGGGCAACCCCTGGAAGTCT GCTGCCAGTGGTCATCATTGCCGTGGGCGTCTTCCTGTTTCTGGTGGCATTCGTCGGA TGCTGTGGCGCCTGCAAGGAAAACTATTGTCTGATGATCACTTTCGCTATTTTTCTGT CCCTGATCATGCTGGTGGAGGTCGCCGCTGCAATTGCCGGATACGTGGGAGGCTCTG GCCCAGTCCGGAGATATCAGGGAGGACCACTGGGCGTGCGAGGAGGGAGCTCCTCT CGAACCCCAAGTGACAAGCCTGTGGCCCACGTGGTCGCTAATCCTCAGGCAGAAGG GCAGCTGCAGTGGCTGAACAGGCGCGCAAATGCCCTGCTGGCCAACGGAGTGGAAC TGAGGGATAATCAGCTGGTGGTCCCAAGTGAGGGCCTGTACCTGATCTATTCACAGG TGCTGTTCAAAGGGCAGGGATGCCCCAGCACACACGTGCTGCTGACCCATACAATCT CAAGGATTGCTGTGAGCTACCAGACAAAGGTCAACCTGCTGTCTGCCATCAAAAGTC CCTGTCAGCGCGAGACTCCTGAAGGGGCTGAGGCAAAGCCATGGTACGAGCCCATC TATCTGGGAGGCGTGTTTCAGCTGGAAAAAGGAGACAGACTGTCAGCAGAGATTAA TAGGCCCGACTACCTGGATTTCGCCGAAAGCGGCCAGGTGTATTTTGGGATCATTGC TCTGGGAGGCAGCGGAGAGCAGAAGCTGATCTCTGAGGAAGATCTGGGAGGACCAC TGGGGGTGCGCGGAGGAGGGGGAAGCGGACCTAAGAACAATCACACCGCTTCCATT CTGGACCGAATGCAGGCAGATTTCAAATGCTGTGGCGCCGCTAACTACACAGACTG GGAAAAGATCCCTAGTATGTCAAAAAACCGGGTGCCAGATTCCTGCTGTATTAATGT GACCGTCGGCTGCGGGATCAACTTTAATGAGAAGGCCATTCATAAAGAAGGCTGCG TGGAGAAGATCGGCGGGTGGCTGCGGAAAAATGTCCTGGTGGTCGCAGCAGCTGCA CTGGGAATCGCCTTCGTGGAAGTCCTGGGGATCGTGTTCGCTTGCTGTCTGGTCAAA AGCATTAGATCCGGCTATGAAGTGATGTAATGTACAA (SEQ ID NO: 352)

[0764]

[0404] LeGo-CD63 -DL 1 -2L-TNFa-Myc-CD63 -Cyt

[0765] MAVEGGMKCVKCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVGGSGPVRRYQG GPLGVRGGSSSRTPSDKPVAHWANPQAEGQLQWLNRRANALLANGVELRDNQLV VP SEGLYLIYSQVLFKGQGCP STHVLLTHTISRIAVS YQTKVNLLS AIKSPCQRETPEG AEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGSGEQKLI SEEDLGGPLGVRGGGGSGPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNR VPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGI VFACCLVKSIRSGYEVM (SEQ ID NO: 368)

[0766]

[0405] Which is encoded by cDNA:

[0767] GGATCCGCCGCCACCATGGCAGTGGAAGGCGGGATGAAGTGCGTCAAATGTGGCGC CTGCAAGGAGAACTACTGTCTGATGATCACCTTCGCTATTTTTCTGAGCCTGATCATG CTGGTGGAAGTCGCCGCTGCAATTGCAGGATACGTGGGAGGCAGCGGACCCGTCCG GAGATATCAGAGCTCCTCTCGAACACCATCCGACAAGCCTGTGGCTCACGTGGTCGC AAATCCACAGGCAGAGGGACAGCTGCAGTGGCTGAACAGGCGAGCCAATGCTCTGC TGGCCAACGGAGTGGAACTGAGGGATAATCAGCTGGTGGTCCCCAGTGAGGGCCTG TACCTGATCTATTCACAGGTGCTGTTCAAAGGACAGGGCTGTCCTTCTACTCACGTG CTGCTGACCCATACAATCTCAAGGATTGCCGTGAGCTATCAGACTAAGGTCAACCTG CTGTCTGCTATCAAAAGTCCTTGCCAGCGAGAGACCCCAGAAGGAGCAGAGGCCAA GCCATGGTACGAGCCCATCTATCTGGGAGGCGTGTTTCAGCTGGAAAAAGGAGACA GACTGTCTGCAGAGATTAATAGGCCTGACTACCTGGATTTCGCCGAAAGTGGCCAGG TGTATTTTGGGATCATTGCTCTGGGAGGCAGCGGAGAGCAGAAGCTGATCTCTGAGG AAGATCTGGGGGGAAGCGGACCAAAGAACAATCACACAGCTTCCATTCTGGACCGA ATGCAGGCAGATTTCAAATGCTGTGGCGCCGCTAACTACACTGACTGGGAAAAGAT CCCAAGTATGTCAAAAAACCGGGTGCCCGATAGCTGCTGTATTAATGTGACAGTCGG GTGTGGAATCAACTTTAATGAGAAGGCCATTCATAAAGAAGGCTGCGTGGAGAAGA TCGGCGGGTGGCTGCGGAAAAATGTCCTGGTGGTCGCAGCAGCTGCACTGGGAATC GCCTTCGTGGAAGTCCTGGGGATCGTGTTCGCTTGCTGTCTGGTCAAAAGCATTAGA TCCGGCTATGAAGTGATGTAATGTACAA (SEQ ID NO: 353)

[0768]

[0406] LeGO-CD69Cyt-TM-GrnzB-TNFa-Myc-GmzB-a-CD19sc-Stalk-CD28-CD3Z

[0769] MS SENCF VAENS SLHPESGQEND ATSPHF STRHEGSFQVPVLC AVMNVVFITILIIALI ALS VGQYNGGSGPVRRYQGGSGGGEEEEEVEAD SEEEEEEESGGSGS S SRTPSDKP V AHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQG CPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLE KGDRLSAEINRPDYLDFAESGQVYFGIIALEQKLISEEDLGGSGEEEEEVEADSEEEEE EEGGSGGGSGGGSGPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDG TVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGG TKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSW IRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYY CAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSAKPTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLC PSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR PGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV

[0770] LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 373)

[0771]

[0407] Which is encoded by cDNA:

[0772] GGATCCATGTCATCCGAGAACTGCTTTGTCGCCGAAAACTCATCACTGCACCCCGAA TCAGGCCAGGAAAACGACGCAACCTCCCCCCACTTCTCAACTAGACACGAGGGCAG CTTCCAGGTGCCAGTGCTGTGCGCCGTGATGAACGTGGTGTTTATCACCATTCTGATC ATTGCTCTGATCGCCCTGAGCGTGGGGCAGTACAATGGCGGGTCCGGACCCGTGCGG AGATATCAGGGAGGCTCCGGAGGAGGAGAGGAAGAGGAAGAGGTGGAGGCCGACT CTGAAGAGGAAGAGGAAGAGGAATCCGGGGGATCTGGCAGCTCCTCTAGAACCCCT TCCGATAAGCCAGTGGCTCATGTGGTGGCCAACCCTCAGGCTGAGGGACAGCTGCA GTGGCTGAACAGGAGGGCTAATGCTCTGCTGGCTAACGGAGTGGAGCTGAGGGACA

[0773] ATCAGCTGGTGGTGCCTTCTGAAGGGCTGTACCTGATCTATAGTCAGGTGCTGTTCA AAGGGCAGGGATGCCCATCTACACACGTGCTGCTGACCCATACAATCAGTCGCATTG CCGTGAGCTACCAGACCAAGGTGAACCTGCTGAGCGCCATCAAGAGCCCTTGTCAG CGGGAAACACCAGAGGGGGCCGAAGCTAAGCCCTGGTACGAGCCTATCTATCTGGG

[0774] CGGGGTGTTTCAGCTGGAGAAAGGCGATAGACTGAGCGCCGAAATTAATAGGCCCG ACTACCTGGATTTCGCTGAGAGCGGCCAGGTGTATTTTGGGATCATTGCCCTGGAAC AGAAGCTGATCTCCGAGGAAGACCTGGGAGGCTCTGGCGAAGAGGAAGAGGAGGT GGAGGCCGATAGCGAGGAAGAAGAGGAAGAGGAAGGAGGCAGCGGAGGAGGATCT GGAGGAGGAAGTGGACAGGTGCAGCTGGTGGAGAGCGGCGGAGGACTGGTGCAGC CAGGAGGGTCACTGAAGCTGAGCTGCGCCGCTTCCGGCTTCGACTTTAGCCGCTACT GGATGTCCTGGGTGCGGCAGGCTCCTGGCAAAGGGCTGGAGTGGATCGGCGAAATT AACCCCACTAGTTCAACCATCAATTTCACACCTTCACTGAAGGACAAAGTGTTTATT

[0775] AGCAGAGATAACGCCAAGAATACACTGTACCTGCAGATGTCTAAAGTGAGGAGTGA GGACACTGCCCTGTACTATTGTGCTCGCGGAAATTACTATCGGTACGGCGACGCCAT GGATTATTGGGGACAGGGCACAAGCGTGACTGTGAGTAAGATCTCAGGAGGCGGCG GATCTGGAGGAGGAGGCAGTGGGGGAGGAGGGTCAGGAGGAGGAGGAAGCGGCGG AGGAGGCAGCTCCGATATCGTGCTGACTCAGAGCCCAGCTTCCCTGGCCGTGTCCCT

[0776] GGGACAGCGCGCCACTATTTCTTGTCGGGCTTCTAAGAGCGTGAGCACCAGCGGGTA

[0777] CTCCTATCTGCACTGGTACCAGCAGAAGCCTGGACAGCCCCCTAAACTGCTGATCTA TCTGGCCTCAAACCTGGAGAGCGGCGTGCCTGCTAGATTCTCCGGGTCTGGAAGTGG CACCGACTTTACACTGAATATTCACCCAGTGGAGGAAGAGGATGCCGCTACCTACTA TTGCCAGCATAGTAGGGAGCTGCCCTTCACATTTGGGAGCGGAACTAAGCTGGAGAT CAAGGGAGGAGGAGGGTCCGGAGGCGGGGGATCTGGCGGCGGAGGCAGTGCCAAG CCAACCACAACTCCAGCTCCTCGCCCACCAACTCCAGCTCCAACCATCGCTTCCCAG CCACTGTCTCTGCGGCCCGAGGCTTGTAGGCCTGCTGCTGGGGGAGCTGTGCATACA AGAGGCCTGGACTTCGCCCCAAGGAAGATTGAAGTGATGTACCCTCCACCCTATCTG GATAACGAAAAATCCAATGGCACTATCATTCACGTGAAGGGGAAACATCTGTGCCC TAGTCCACTGTTCCCCGGCCCTTCAAAGCCCTTTTGGGTGCTGGTGGTGGTGGGAGG

[0778] GGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCTTTCATCATCTTCTGGGTGCGG AGCAAACGCAGTCGGCTGCTGCACTCTGACTATATGAACATGACCCCAAGACGGCC CGGACCAACAAGGAAGCATTACCAGCCATATGCTCCTCCAAGGGACTTCGCTGCTTA CAGAAGCAGGGTGAAATTTTCACGCAGCGCCGATGCTCCTGCCTACCAGCAGGGAC AGAATCAGCTGTATAACGAGCTGAATCTGGGCAGGCGCGAAGAGTATGACGTGCTG GATAAGCGGAGAGGACGGGACCCCGAAATGGGAGGCAAGCCAAGGCGCAAAAACC CCCAGGAGGGCCTGTATAACGAACTGCAGAAGGACAAAATGGCTGAGGCCTATAGC GAAATCGGAATGAAGGGCGAGCGGAGAAGGGGCAAAGGGCACGATGGGCTGTACC AGGGACTGTCCACCGCCACTAAAGATACCTATGATGCTCTGCACATGCAGGCTCTGC CACCCCGATAATGTACAA (SEQ ID NO: 354)

[0779]

[0408] LeGO-CD69Cyt-TM-n-9-TNFa-Myc-9-CD28TM-Cyt

[0780] MS SENCF VAENS SLHPESGQEND ATSPHF STRHEGSFQVPVLC AVMNVVFITILIIALI ALS VGQYNGGSGPVRRYQGGSGGGPLGMTSGGSGS S SRTPSDKP VAHVVANPQAEG QLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTIS RIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRP DYLDF AESGQ VYFGIIALGGSGEQKLISEEDLGGPLGMTS GGS GCPSPLFPGP SKPFW VLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLL (SEQ ID NO: 377)

[0781]

[0409] Which is encoded by cDNA:

[0782] GGATCCGCCGCCACCATGAGTTCCGAAAACTGCTTTGTCGCCGAGAATAGTAGTCTG CACCCCGAATCAGGACAGGAGAATGACGCAACCAGCCCCCACTTTTCAACAAGGCA CGAAGGGAGCTTCCAGGTCCCTGTGCTGTGCGCAGTGATGAACGTGGTCTTTATCAC TATTCTGATCATTGCTCTGATCGCACTGAGTGTCGGACAGTACAATGGCGGGTCAGG CCCCGTGCGGAGATATCAGGGAGGGTCTGGGGGAGGACCACTGGGAATGACCTCCG GAGGGTCTGGGAGCTCCTCTCGCACCCCTTCCGACAAGCCAGTGGCCCATGTGGTCG CTAACCCTCAGGCAGAGGGACAGCTGCAGTGGCTGAACAGGCGAGCCAATGCTCTG CTGGCTAACGGCGTGGAACTGCGAGATAATCAGCTGGTGGTCCCTAGCGAGGGGCT GTACCTGATCTATTCCCAGGTCCTGTTCAAAGGGCAGGGATGCCCATCTACACACGT GCTGCTGACCCATACAATCTCTAGAATTGCCGTCAGTTACCAGACTAAGGTGAACCT GCTGAGCGCCATCAAGTCACCATGTCAGAGGGAGACACCCGAAGGAGCAGAGGCCA AGCCCTGGTACGAGCCTATCTATCTGGGAGGCGTGTTTCAGCTGGAAAAAGGCGACC GACTGTCTGCCGAGATTAATCGGCCAGACTACCTGGATTTCGCTGAAAGTGGCCAGG TGTATTTTGGGATCATTGCACTGGGAGGCAGCGGAGAGCAGAAGCTGATCTCTGAG GAAGATCTGGGAGGACCACTGGGAATGACCTCCGGAGGGAGCGGATGCCCATCCCC TCTGTTCCCAGGACCCAGCAAACCCTTTTGGGTCCTGGTGGTCGTGGGAGGCGTGCT GGCATGTTATAGCCTGCTGGTCACAGTGGCTTTCATTATCTTTTGGGTCCGCTCTAAA CGGTCTCGGCTGCTGTAATGTACAA (SEQ ID NO: 355)

[0783]

[0410] LeGO-CD69Cyt-TM-n-TNFa-Myc-CD28TM-Cyt

[0784] MS SENCF VAENS SLHPESGQEND ATSPHF STRHEGSFQVPVLC AVMNVVFITILIIALI ALSVGQYNGGSGPVRRYQSSSRTPSDKPVAHWANPQAEGQLQWLNRRAN ALLAN GVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAI KSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDF AESGQ VYFGII ALGGSGEQKLISEEDLGGSGCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFW VRSKRSRLL (SEQ ID NO: 370)

[0785]

[0411] Which is encoded by cDNA:

[0786] GGATCCGCCGCCACCATGAGTTCCGAAAACTGCTTTGTCGCCGAGAATAGTAGTCTG CACCCCGAATCAGGACAGGAGAATGACGCAACCAGCCCCCACTTTTCAACAAGGCA CGAAGGGAGCTTCCAGGTCCCTGTGCTGTGCGCAGTGATGAACGTGGTCTTTATCAC TATTCTGATCATTGCTCTGATCGCACTGAGTGTCGGACAGTACAATGGCGGGTCAGG CCCCGTGCGGAGATATCAGAGCTCCTCTCGCACCCCTTCCGACAAGCCAGTGGCCCA TGTGGTCGCTAACCCTCAGGCAGAGGGACAGCTGCAGTGGCTGAACAGGCGAGCCA ATGCTCTGCTGGCTAACGGCGTGGAACTGCGAGATAATCAGCTGGTGGTCCCTAGCG AGGGGCTGTACCTGATCTATTCCCAGGTCCTGTTCAAAGGGCAGGGATGCCCATCTA CACACGTGCTGCTGACCCATACAATCTCTAGAATTGCCGTCAGTTACCAGACTAAGG TGAACCTGCTGAGCGCCATCAAGTCACCATGTCAGAGGGAGACACCCGAAGGAGCA GAGGCCAAGCCCTGGTACGAGCCTATCTATCTGGGAGGCGTGTTTCAGCTGGAAAA AGGCGACCGACTGTCTGCCGAGATTAATCGGCCAGACTACCTGGATTTCGCTGAAAG TGGCCAGGTGTATTTTGGGATCATTGCACTGGGAGGCAGCGGAGAGCAGAAGCTGA TCTCTGAGGAAGATCTGGGAGGGAGCGGATGCCCATCCCCTCTGTTCCCAGGACCCA GCAAACCCTTTTGGGTCCTGGTGGTCGTGGGAGGCGTGCTGGCATGTTATAGCCTGC TGGTCACAGTGGCTTTCATTATCTTTTGGGTCCGCTCTAAACGGTCTCGGCTGCTGTA ATGTACAA (SEQ ID NO: 356)

[0787]

[0412] LeGO-CD69Cyt-TM-GrzB-TNFa-Myc-GrzB-a-CD19sc-Stalk-CD28-CD3z

[0788] MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALS VGQYNGGSGPVRRYQGGSGGGEEEEEVEADSEEEEEEESGGSGSSSRTPSDKPVAHVVA NPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLL THTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEIN RPDYLDFAESGQVYFGIIALEQKLISEEDLGGSGEEEEEVEADSEEEEEEEGGSGGGSGGG SGPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSG VPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSSGGGGSGGGGSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:373)

[0789]

[0413] Which is encoded by cDNA:

[0790] GGATCCATGTCATCCGAGAACTGCTTTGTCGCCGAAAACTCATCACTGCACCCCGAA TCAGGCCAGGAAAACGACGCAACCTCCCCCCACTTCTCAACTAGACACGAGGGCAG CTTCCAGGTGCCAGTGCTGTGCGCCGTGATGAACGTGGTGTTTATCACCATTCTGATC ATTGCTCTGATCGCCCTGAGCGTGGGGCAGTACAATGGCGGGTCCGGACCCGTGCGG AGATATCAGGGAGGCTCCGGAGGAGGAGAGGAAGAGGAAGAGGTGGAGGCCGACT CTGAAGAGGAAGAGGAAGAGGAATCCGGGGGATCTGGCAGCTCCTCTAGAACCCCT TCCGATAAGCCAGTGGCTCATGTGGTGGCCAACCCTCAGGCTGAGGGACAGCTGCA GTGGCTGAACAGGAGGGCTAATGCTCTGCTGGCTAACGGAGTGGAGCTGAGGGACA ATCAGCTGGTGGTGCCTTCTGAAGGGCTGTACCTGATCTATAGTCAGGTGCTGTTCA AAGGGCAGGGATGCCCATCTACACACGTGCTGCTGACCCATACAATCAGTCGCATTG CCGTGAGCTACCAGACCAAGGTGAACCTGCTGAGCGCCATCAAGAGCCCTTGTCAG CGGGAAACACCAGAGGGGGCCGAAGCTAAGCCCTGGTACGAGCCTATCTATCTGGG CGGGGTGTTTCAGCTGGAGAAAGGCGATAGACTGAGCGCCGAAATTAATAGGCCCG ACTACCTGGATTTCGCTGAGAGCGGCCAGGTGTATTTTGGGATCATTGCCCTGGAAC AGAAGCTGATCTCCGAGGAAGACCTGGGAGGCTCTGGCGAAGAGGAAGAGGAGGT GGAGGCCGATAGCGAGGAAGAAGAGGAAGAGGAAGGAGGCAGCGGAGGAGGATCT GGAGGAGGAAGTGGACAGGTGCAGCTGGTGGAGAGCGGCGGAGGACTGGTGCAGC CAGGAGGGTCACTGAAGCTGAGCTGCGCCGCTTCCGGCTTCGACTTTAGCCGCTACT GGATGTCCTGGGTGCGGCAGGCTCCTGGCAAAGGGCTGGAGTGGATCGGCGAAATT AACCCCACTAGTTCAACCATCAATTTCACACCTTCACTGAAGGACAAAGTGTTTATT AGCAGAGATAACGCCAAGAATACACTGTACCTGCAGATGTCTAAAGTGAGGAGTGA

[0791] GGACACTGCCCTGTACTATTGTGCTCGCGGAAATTACTATCGGTACGGCGACGCCAT

[0792] GGATTATTGGGGACAGGGCACAAGCGTGACTGTGAGTAAGATCTCAGGAGGCGGCG

[0793] GATCTGGAGGAGGAGGCAGTGGGGGAGGAGGGTCAGGAGGAGGAGGAAGCGGCGG

[0794] AGGAGGCAGCTCCGATATCGTGCTGACTCAGAGCCCAGCTTCCCTGGCCGTGTCCCT

[0795] GGGACAGCGCGCCACTATTTCTTGTCGGGCTTCTAAGAGCGTGAGCACCAGCGGGTA

[0796] CTCCTATCTGCACTGGTACCAGCAGAAGCCTGGACAGCCCCCTAAACTGCTGATCTA

[0797] TCTGGCCTCAAACCTGGAGAGCGGCGTGCCTGCTAGATTCTCCGGGTCTGGAAGTGG

[0798] CACCGACTTTACACTGAATATTCACCCAGTGGAGGAAGAGGATGCCGCTACCTACTA

[0799] TTGCCAGCATAGTAGGGAGCTGCCCTTCACATTTGGGAGCGGAACTAAGCTGGAGAT

[0800] CAAGGGAGGAGGAGGGTCCGGAGGCGGGGGATCTGGCGGCGGAGGCAGTGCCAAG

[0801] CCAACCACAACTCCAGCTCCTCGCCCACCAACTCCAGCTCCAACCATCGCTTCCCAG

[0802] CCACTGTCTCTGCGGCCCGAGGCTTGTAGGCCTGCTGCTGGGGGAGCTGTGCATACA

[0803] AGAGGCCTGGACTTCGCCCCAAGGAAGATTGAAGTGATGTACCCTCCACCCTATCTG

[0804] GATAACGAAAAATCCAATGGCACTATCATTCACGTGAAGGGGAAACATCTGTGCCC

[0805] TAGTCCACTGTTCCCCGGCCCTTCAAAGCCCTTTTGGGTGCTGGTGGTGGTGGGAGG

[0806] GGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCTTTCATCATCTTCTGGGTGCGG

[0807] AGCAAACGCAGTCGGCTGCTGCACTCTGACTATATGAACATGACCCCAAGACGGCC

[0808] CGGACCAACAAGGAAGCATTACCAGCCATATGCTCCTCCAAGGGACTTCGCTGCTTA

[0809] CAGAAGCAGGGTGAAATTTTCACGCAGCGCCGATGCTCCTGCCTACCAGCAGGGAC

[0810] AGAATCAGCTGTATAACGAGCTGAATCTGGGCAGGCGCGAAGAGTATGACGTGCTG

[0811] GATAAGCGGAGAGGACGGGACCCCGAAATGGGAGGCAAGCCAAGGCGCAAAAACC

[0812] CCCAGGAGGGCCTGTATAACGAACTGCAGAAGGACAAAATGGCTGAGGCCTATAGC

[0813] GAAATCGGAATGAAGGGCGAGCGGAGAAGGGGCAAAGGGCACGATGGGCTGTACC

[0814] AGGGACTGTCCACCGCCACTAAAGATACCTATGATGCTCTGCACATGCAGGCTCTGC

[0815] CACCCCGATAATGTACA (SEQ ID NO: 357)

[0816]

[0414] LeGO-CD69Cyt-TM-GrzB-nLuc-Myc-GrzB-a-CD19sc-Stalk CD28-CD3z

[0817] MS SENCF VAENS SLHPESGQEND ATSPHF STRHEGSFQVPVLC AVMNVVFITILIIALI ALSVGQYNGGSGPVRRYSRQGGSGGGEEEEEVEADSEEEEEEESGGSGSSSSRVFTL EDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIP YEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIA VFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAEQKLISEE DLLEGGSGEEEEEVEADSEEEEEEEGGSGGGSGLEPRGGSGVNPDIQMTQTTSSLSAS LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYS LTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESG PGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKS RLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSV NSGGGGSGGGGSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA PRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSL LVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 358)

[0818]

[0415] Which is encoded by cDNA:

[0819] GCCAGTCCTCCGATTGACTGAGTCGCCCGGATCCCGCCACCATGAGCTCTGAGAA CTGCTTCGTGGCCGAGAACAGCAGCCTCCACCCCGAGAGTGGTCAGGAGAACGA CGCCACCAGCCCCCACTTCAGCACAAGACACGAGGGCTCATTCCAGGTGCCTGT GCTGTGCGCCGTGATGAACGTGGTGTTCATCACCATCCTGATCATCGCCCTGATT GCCCTTTCTGTGGGCCAATACAATGGCGGCTCCGGTCCTGTGCGCAGATACTCTA GACAGGGCGGCTCTGGCGGAGGCGAAGAGGAAGAAGAGGTGGAAGCCGATAGC GAGGAAGAAGAAGAAGAGGAGAGCGGCGGCTCCGGCAGCAGCAGCTCTAGAGT GTTCACCCTCGAAGATTTCGTGGGCGACTGGAGACAGACCGCCGGCTACAACCT GGACCAGGTGCTGGAACAGGGCGGCGTGAGCTCTCTGTTTCAGAATCTGGGGGT TAGCGTGACACCAATCCAGAGGATCGTGCTGTCCGGCGAGAACGGGCTGAAGAT CGACATCCACGTGATCATTCCATACGAGGGCCTGTCTGGCGATCAGATGGGCCA GATCGAGAAGATCTTCAAGGTGGTGTACCCTGTAGACGACCACCATTTTAAGGTG ATCCTGCACTACGGCACCCTGGTCATCGACGGCGTGACACCCAACATGATCGACT ACTTCGGCAGACCATATGAGGGTATTGCCGTGTTTGATGGAAAGAAGATCACAG TCACCGGCACCCTGTGGAACGGCAACAAGATTATCGATGAACGGCTGATCAACC CCGACGGCTCTCTGCTGTTTAGAGTGACCATCAATGGCGTGACAGGCTGGAGACT GTGCGAGAGAATCCTGGCTGAGCAGAAACTGATCAGCGAGGAAGACCTGCTCGA GGGCGGTAGCGGCGAGGAGGAAGAAGAAGTGGAAGCTGACAGCGAAGAAGAG GAAGAGGAGGAGGGCGGGAGCGGAGGCGGCAGTGGCCTCGAGCCTAGGGGCGG CAGCGGAGTTAACCCCGATATTCAGATGACTCAGACTACCAGCTCCCTGAGCGC ATCCCTGGGCGACCGAGTGACAATCAGTTGCCGAGCCAGCCAGGACATCAGCAA GTATCTGAACTGGTACCAGCAGAAGCCTGACGGAACCGTCAAACTGCTGATCTA TCACACATCCCGCCTGCATTCTGGCGTGCCAAGTCGGTTCTCTGGAAGTGGCTCA GGGACCGACTACTCACTGACAATCAGCAACCTGGAGCAGGAAGATATTGCCACC TATTTCTGCCAGCAGGGCAATACTCTGCCTTACACCTTTGGCGGGGGAACTAAGC TGGAGATTACCGGCGGAGGAGGCAGCGGAGGAGGAGGGTCCGGAGGCGGGGGA TCTGAGGTGAAACTGCAGGAAAGCGGACCTGGCCTGGTCGCTCCAAGCCAGTCC CTGTCTGTGACTTGTACCGTCAGCGGAGTGTCCCTGCCCGATTACGGCGTCTCTT GGATCAGACAGCCCCCTAGAAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGAA GTGAAACCACATACTATAATTCCGCCCTGAAAAGCCGGCTGACAATCATTAAGG ACAACAGTAAATCACAGGTGTTCCTGAAGATGAATTCCCTGCAGACAGACGATA CTGCAATCTACTATTGCGCCAAACACTACTATTACGGCGGGAGCTATGCTATGGA TTACTGGGGGCAGGGAACCTCCGTCACAGTGTCTAGTGTTAACTCAGGCGGCGG CGGCAGCGGAGGCGGAGGCAGTGCCAAGCCTACAACCACCCCCGCCCCTAGACC TCCTACACCTGCCCCTACCATCGCTAGCCAGCCACTCTCTCTCAGACCTGAAGCC TGTAGACCCGCCGCTGGCGGCGCCGTGCACACCAGAGGCCTGGACTTCGCGCCT CGGAAGATCGAGGTGATGTACCCCCCGCCTTACCTGGACAACGAGAAGTCTAAT GGAACAATCATCCACGTGAAGGGAAAACACCTGTGTCCTTCTCCTCTGTTCCCTG GCCCCAGCAAGCCTTTCTGGGTCCTGGTGGTGGTCGGCGGCGTGCTGGCGTGCTA CAGCCTGCTCGTGACCGTGGCCTTCATCATTTTCTGGGTGCGGAGCAAACGGAGC AGACTGCTGCATAGCGATTACATGAACATGACCCCAAGAAGACCTGGGCCAACC AGAAAGCACTACCAGCCTTACGCCCCCCCCAGAGATTTCGCCGCTTATAGAAGCC GGGTGAAGTTCTCCAGATCCGCCGACGCCCCTGCTTACCAACAGGGCCAGAACC AGCTGTATAACGAGCTGAATCTGGGCAGAAGAGAGGAGTACGACGTGCTGGATA AGCGGAGAGGAAGAGACCCTGAGATGGGCGGCAAGCCCCGGCGGAAGAACCCT CAGGAGGGCCTGTATAACGAGCTGCAAAAAGATAAGATGGCCGAGGCTTATAGC GAGATCGGAATGAAGGGCGAGCGGCGGAGGGGCAAAGGCCACGACGGCCTGTA CCAGGGACTGAGCACAGCCACAAAGGACACCTACGACGCTCTGCACATGCAGGC CCTGCCTCCTAGATAATGTACAAGTAAAGCGGCCGGCC (SEQ ID NO: 382)

[0820]

[0416] LeGO-CD69Cyt-TM-GrzB-nLuc-Myc-GrzB-CD28-CD3z, (Delta-sc, GAGE B) MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALS VGQYNGGSGPVRRYSRQGGSGGGEEEEEVEADSEEEEEEESGGSGSSSSRVFTLEDFVG DWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGD QMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVT GTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAEQKLISEEDLLEGGSGEEEE EVEADSEEEEEEEGGSGGGSGLEPRGGSGVNSGGGGSGGGGSAKPTTTPAPRPPTPAPTI ASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCP SPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPT

[0821] RKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR (SEQ ID NO: 374)

[0822]

[0417] Which is encoded by cDNA:

[0823] ATGAGCTCTGAGAACTGCTTCGTGGCCGAGAACAGCAGCCTCCACCCCGAGAGT GGTCAGGAGAACGACGCCACCAGCCCCCACTTCAGCACAAGACACGAGGGCTCA TTCCAGGTGCCTGTGCTGTGCGCCGTGATGAACGTGGTGTTCATCACCATCCTGA TCATCGCCCTGATTGCCCTTTCTGTGGGCCAATACAATGGCGGCTCCGGTCCTGT GCGCAGATACTCTAGACAGGGCGGCTCTGGCGGAGGCGAAGAGGAAGAAGAGG TGGAAGCCGATAGCGAGGAAGAAGAAGAAGAGGAGAGCGGCGGCTCCGGCAGC AGCAGCTCTAGAGTGTTCACCCTCGAAGATTTCGTGGGCGACTGGAGACAGACC GCCGGCTACAACCTGGACCAGGTGCTGGAACAGGGCGGCGTGAGCTCTCTGTTT CAGAATCTGGGGGTTAGCGTGACACCAATCCAGAGGATCGTGCTGTCCGGCGAG AACGGGCTGAAGATCGACATCCACGTGATCATTCCATACGAGGGCCTGTCTGGC GATCAGATGGGCCAGATCGAGAAGATCTTCAAGGTGGTGTACCCTGTAGACGAC CACCATTTTAAGGTGATCCTGCACTACGGCACCCTGGTCATCGACGGCGTGACAC CCAACATGATCGACTACTTCGGCAGACCATATGAGGGTATTGCCGTGTTTGATGG AAAGAAGATCACAGTCACCGGCACCCTGTGGAACGGCAACAAGATTATCGATGA ACGGCTGATCAACCCCGACGGCTCTCTGCTGTTTAGAGTGACCATCAATGGCGTG ACAGGCTGGAGACTGTGCGAGAGAATCCTGGCTGAGCAGAAACTGATCAGCGAG GAAGACCTGCTCGAGGGCGGTAGCGGCGAGGAGGAAGAAGAAGTGGAAGCTGA CAGCGAAGAAGAGGAAGAGGAGGAGGGCGGGAGCGGAGGCGGCAGTGGCCTCG AGCCTAGGGGCGGCAGCGGAGTTAACTCAGGCGGCGGCGGCAGCGGAGGCGGA GGCAGTGCCAAGCCTACAACCACCCCCGCCCCTAGACCTCCTACACCTGCCCCTA

[0824] CCATCGCTAGCCAGCCACTCTCTCTCAGACCTGAAGCCTGTAGACCCGCCGCTGG CGGCGCCGTGCACACCAGAGGCCTGGACTTCGCGCCTCGGAAGATCGAGGTGAT GTACCCCCCGCCTTACCTGGACAACGAGAAGTCTAATGGAACAATCATCCACGT GAAGGGAAAACACCTGTGTCCTTCTCCTCTGTTCCCTGGCCCCAGCAAGCCTTTC TGGGTCCTGGTGGTGGTCGGCGGCGTGCTGGCGTGCTACAGCCTGCTCGTGACCG TGGCCTTCATCATTTTCTGGGTGCGGAGCAAACGGAGCAGACTGCTGCATAGCGA TTACATGAACATGACCCCAAGAAGACCTGGGCCAACCAGAAAGCACTACCAGCC TTACGCCCCCCCCAGAGATTTCGCCGCTTATAGAAGCCGGGTGAAGTTCTCCAGA TCCGCCGACGCCCCTGCTTACCAACAGGGCCAGAACCAGCTGTATAACGAGCTG AATCTGGGCAGAAGAGAGGAGTACGACGTGCTGGATAAGCGGAGAGGAAGAGA CCCTGAGATGGGCGGCAAGCCCCGGCGGAAGAACCCTCAGGAGGGCCTGTATAA CGAGCTGCAAAAAGATAAGATGGCCGAGGCTTATAGCGAGATCGGAATGAAGG GCGAGCGGCGGAGGGGCAAAGGCCACGACGGCCTGTACCAGGGACTGAGCACA GCCACAAAGGACACCTACGACGCTCTGCACATGCAGGCCCTGCCTCCTAGAtaaTG

[0825] TACA (SEQ ID NO: 359)

[0826]

[0418] CD69Cyt-TM-nLuc-Myc-a-CD19sc-Stalk-CD28-CD3z, , (Delta-GrzB, GAGE A)

[0827] MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALS

[0828] VGQYNGGSGPVRRYSRVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPI QRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR LCERILAEQKLISEEDLLEPRGGSGVNPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLN WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

[0829] YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDT AIYYCAKHYYYGGSYAMDYWGQGTSVTVSSVNSGGGGSGGGGSAKPTTTPAPRPPTPA PTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKH LCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRP GPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK

[0830] RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR (SEQ ID NO: 375)

[0831]

[0419] Which is encoded by cDNA:

[0832] ATGAGCTCTGAGAACTGCTTCGTGGCCGAGAACAGCAGCCTCCACCCCGAGAGTGG TCAGGAGAACGACGCCACCAGCCCCCACTTCAGCACAAGACACGAGGGCTCATTCC AGGTGCCTGTGCTGTGCGCCGTGATGAACGTGGTGTTCATCACCATCCTGATCATCG CCCTGATTGCCCTTTCTGTGGGCCAATACAATGGCGGCTCCGGTCCTGTGCGCAGAT ACTCTAGAGTGTTCACCCTCGAAGATTTCGTGGGCGACTGGAGACAGACCGCCGGCT

[0833] ACAACCTGGACCAGGTGCTGGAACAGGGCGGCGTGAGCTCTCTGTTTCAGAATCTGG GGGTTAGCGTGACACCAATCCAGAGGATCGTGCTGTCCGGCGAGAACGGGCTGAAG ATCGACATCCACGTGATCATTCCATACGAGGGCCTGTCTGGCGATCAGATGGGCCAG ATCGAGAAGATCTTCAAGGTGGTGTACCCTGTAGACGACCACCATTTTAAGGTGATC CTGCACTACGGCACCCTGGTCATCGACGGCGTGACACCCAACATGATCGACTACTTC

[0834] GGCAGACCATATGAGGGTATTGCCGTGTTTGATGGAAAGAAGATCACAGTCACCGG CACCCTGTGGAACGGCAACAAGATTATCGATGAACGGCTGATCAACCCCGACGGCT CTCTGCTGTTTAGAGTGACCATCAATGGCGTGACAGGCTGGAGACTGTGCGAGAGA ATCCTGGCTGAGCAGAAACTGATCAGCGAGGAAGACCTGCTCGAGCCTAGGGGCGG CAGCGGAGTTAACCCCGATATTCAGATGACTCAGACTACCAGCTCCCTGAGCGCATC

[0835] CCTGGGCGACCGAGTGACAATCAGTTGCCGAGCCAGCCAGGACATCAGCAAGTATC TGAACTGGTACCAGCAGAAGCCTGACGGAACCGTCAAACTGCTGATCTATCACACAT CCCGCCTGCATTCTGGCGTGCCAAGTCGGTTCTCTGGAAGTGGCTCAGGGACCGACT ACTCACTGACAATCAGCAACCTGGAGCAGGAAGATATTGCCACCTATTTCTGCCAGC AGGGCAATACTCTGCCTTACACCTTTGGCGGGGGAACTAAGCTGGAGATTACCGGCG

[0836] GAGGAGGCAGCGGAGGAGGAGGGTCCGGAGGCGGGGGATCTGAGGTGAAACTGCA GGAAAGCGGACCTGGCCTGGTCGCTCCAAGCCAGTCCCTGTCTGTGACTTGTACCGT CAGCGGAGTGTCCCTGCCCGATTACGGCGTCTCTTGGATCAGACAGCCCCCTAGAAA GGGCCTGGAGTGGCTGGGCGTGATCTGGGGAAGTGAAACCACATACTATAATTCCG CCCTGAAAAGCCGGCTGACAATCATTAAGGACAACAGTAAATCACAGGTGTTCCTG

[0837] AAGATGAATTCCCTGCAGACAGACGATACTGCAATCTACTATTGCGCCAAACACTAC TATTACGGCGGGAGCTATGCTATGGATTACTGGGGGCAGGGAACCTCCGTCACAGTG TCTAGTGTTAACTCAGGCGGCGGCGGCAGCGGAGGCGGAGGCAGTGCCAAGCCTAC AACCACCCCCGCCCCTAGACCTCCTACACCTGCCCCTACCATCGCTAGCCAGCCACT CTCTCTCAGACCTGAAGCCTGTAGACCCGCCGCTGGCGGCGCCGTGCACACCAGAGG

[0838] CCTGGACTTCGCGCCTCGGAAGATCGAGGTGATGTACCCCCCGCCTTACCTGGACAA CGAGAAGTCTAATGGAACAATCATCCACGTGAAGGGAAAACACCTGTGTCCTTCTCC TCTGTTCCCTGGCCCCAGCAAGCCTTTCTGGGTCCTGGTGGTGGTCGGCGGCGTGCT GGCGTGCTACAGCCTGCTCGTGACCGTGGCCTTCATCATTTTCTGGGTGCGGAGCAA ACGGAGCAGACTGCTGCATAGCGATTACATGAACATGACCCCAAGAAGACCTGGGC

[0839] CAACCAGAAAGCACTACCAGCCTTACGCCCCCCCCAGAGATTTCGCCGCTTATAGAA GCCGGGTGAAGTTCTCCAGATCCGCCGACGCCCCTGCTTACCAACAGGGCCAGAACC AGCTGTATAACGAGCTGAATCTGGGCAGAAGAGAGGAGTACGACGTGCTGGATAAG

[0840] CGGAGAGGAAGAGACCCTGAGATGGGCGGCAAGCCCCGGCGGAAGAACCCTCAGG

[0841] AGGGCCTGTATAACGAGCTGCAAAAAGATAAGATGGCCGAGGCTTATAGCGAGATC

[0842] GGAATGAAGGGCGAGCGGCGGAGGGGCAAAGGCCACGACGGCCTGTACCAGGGAC

[0843] TGAGCACAGCCACAAAGGACACCTACGACGCTCTGCACATGCAGGCCCTGCCTCCTA

[0844] GAtaaTGTACA (SEQ ID NO: 360)

[0845]

[0420] CD69Cyt-TM-GrzB(IEPD)-nLuc-Myc-GrzB(IEPD)-a-CD19sc-Stalk-CD28-CD3z

[0846] MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALS VGQYNGGSGPVRRYSRQGGSGGGIEPDSGGSGSRVFTLEDFVGDWRQTAGYNLDQVLE QGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVD DHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINP DGSLLFRVTINGVTGWRLCERILAEQKLISEEDLLEGGSGIEPDGGSGGGSGLEPRGGSGV NPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGV PSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSET TYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGT SVTVSSVNSGGGGSGGGGSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR GLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLAC YSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKF SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 383)

[0847]

[0421] Which is encoded by cDNA:

[0848] GCCAGTCCTCCGATTGACTGAGTCGCCCGGATCCCGCCACCATGAGCTCTGAGAA CTGCTTCGTGGCCGAGAACAGCAGCCTCCACCCCGAGAGTGGTCAGGAGAACGA CGCCACCAGCCCCCACTTCAGCACAAGACACGAGGGCTCATTCCAGGTGCCTGT GCTGTGCGCCGTGATGAACGTGGTGTTCATCACCATCCTGATCATCGCCCTGATT GCCCTTTCTGTGGGCCAATACAATGGCGGCTCCGGTCCTGTGCGCAGATACTCTA GACAGGGCGGCTCTGGCGGAGGCATCGAGCCTGACAGCGGCGGCTCCGGCTCTA GAGTGTTCACCCTCGAAGATTTCGTGGGCGACTGGAGACAGACCGCCGGCTACA ACCTGGACCAGGTGCTGGAACAGGGCGGCGTGAGCTCTCTGTTTCAGAATCTGG GGGTTAGCGTGACACCAATCCAGAGGATCGTGCTGTCCGGCGAGAACGGGCTGA AGATCGACATCCACGTGATCATTCCATACGAGGGCCTGTCTGGCGATCAGATGG GCCAGATCGAGAAGATCTTCAAGGTGGTGTACCCTGTAGACGACCACCATTTTAA GGTGATCCTGCACTACGGCACCCTGGTCATCGACGGCGTGACACCCAACATGATC GACTACTTCGGCAGACCATATGAGGGTATTGCCGTGTTTGATGGAAAGAAGATC ACAGTCACCGGCACCCTGTGGAACGGCAACAAGATTATCGATGAACGGCTGATC AACCCCGACGGCTCTCTGCTGTTTAGAGTGACCATCAATGGCGTGACAGGCTGGA GACTGTGCGAGAGAATCCTGGCTGAGCAGAAACTGATCAGCGAGGAAGACCTGC TCGAGGGCGGTAGCGGCATCGAGCCTGACGGCGGGAGCGGAGGCGGCAGTGGC CTCGAGCCTAGGGGCGGCAGCGGAGTTAACCCCGATATTCAGATGACTCAGACT ACCAGCTCCCTGAGCGCATCCCTGGGCGACCGAGTGACAATCAGTTGCCGAGCC AGCCAGGACATCAGCAAGTATCTGAACTGGTACCAGCAGAAGCCTGACGGAACC GTCAAACTGCTGATCTATCACACATCCCGCCTGCATTCTGGCGTGCCAAGTCGGT TCTCTGGAAGTGGCTCAGGGACCGACTACTCACTGACAATCAGCAACCTGGAGC AGGAAGATATTGCCACCTATTTCTGCCAGCAGGGCAATACTCTGCCTTACACCTT TGGCGGGGGAACTAAGCTGGAGATTACCGGCGGAGGAGGCAGCGGAGGAGGAG GGTCCGGAGGCGGGGGATCTGAGGTGAAACTGCAGGAAAGCGGACCTGGCCTG GTCGCTCCAAGCCAGTCCCTGTCTGTGACTTGTACCGTCAGCGGAGTGTCCCTGC CCGATTACGGCGTCTCTTGGATCAGACAGCCCCCTAGAAAGGGCCTGGAGTGGC TGGGCGTGATCTGGGGAAGTGAAACCACATACTATAATTCCGCCCTGAAAAGCC GGCTGACAATCATTAAGGACAACAGTAAATCACAGGTGTTCCTGAAGATGAATT CCCTGCAGACAGACGATACTGCAATCTACTATTGCGCCAAACACTACTATTACGG CGGGAGCTATGCTATGGATTACTGGGGGCAGGGAACCTCCGTCACAGTGTCTAG TGTTAACTCAGGCGGCGGCGGCAGCGGAGGCGGAGGCAGTGCCAAGCCTACAAC CACCCCCGCCCCTAGACCTCCTACACCTGCCCCTACCATCGCTAGCCAGCCACTC TCTCTCAGACCTGAAGCCTGTAGACCCGCCGCTGGCGGCGCCGTGCACACCAGA GGCCTGGACTTCGCGCCTCGGAAGATCGAGGTGATGTACCCCCCGCCTTACCTGG ACAACGAGAAGTCTAATGGAACAATCATCCACGTGAAGGGAAAACACCTGTGTC CTTCTCCTCTGTTCCCTGGCCCCAGCAAGCCTTTCTGGGTCCTGGTGGTGGTCGGC GGCGTGCTGGCGTGCTACAGCCTGCTCGTGACCGTGGCCTTCATCATTTTCTGGG TGCGGAGCAAACGGAGCAGACTGCTGCATAGCGATTACATGAACATGACCCCAA GAAGACCTGGGCCAACCAGAAAGCACTACCAGCCTTACGCCCCCCCCAGAGATT TCGCCGCTTATAGAAGCCGGGTGAAGTTCTCCAGATCCGCCGACGCCCCTGCTTA CCAACAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCAGAAGAGAGG AGTACGACGTGCTGGATAAGCGGAGAGGAAGAGACCCTGAGATGGGCGGCAAG CCCCGGCGGAAGAACCCTCAGGAGGGCCTGTATAACGAGCTGCAAAAAGATAAG ATGGCCGAGGCTTATAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGGGGCAA AGGCCACGACGGCCTGTACCAGGGACTGAGCACAGCCACAAAGGACACCTACGA CGCTCTGCACATGCAGGCCCTGCCTCCTAGATAATGTACAAGTAAAGCGGCCGG

[0849] CC (SEQ ID NO: 361)

[0850]

[0422] CD69Cyt-TM-GrzB(SASA)-nLuc-Myc-GrzB(SASA)-a-CD19sc-Stalk-CD28-CD3z

[0851] MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALS VGQYNGGSGPVRRYSRQGGSGGGAQGVISASASNLDDFYSGGSGSRVFTLEDFVGDWR QTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMG QIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTL WNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAEQKLISEEDLLEGGSGAQGVISA SASNLDDFYGGSGGGSGLEPRGGSGVNPDIQMTQTTSSLSASLGDRVTISCRASQDISKY LNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGN TLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLP DYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSVNSGGGGSGGGGSAKPTTTPAPRPPTP APTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGK HLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR RPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR (SEQ ID NO: 376)

[0852]

[0423] Which is encoded by cDNA:

[0853] GCCAGTCCTCCGATTGACTGAGTCGCCCGGATCCCGCCACCATGAGCTCTGAGAACT GCTTCGTGGCCGAGAACAGCAGCCTCCACCCCGAGAGTGGTCAGGAGAACGACGCC ACCAGCCCCCACTTCAGCACAAGACACGAGGGCTCATTCCAGGTGCCTGTGCTGTGC GCCGTGATGAACGTGGTGTTCATCACCATCCTGATCATCGCCCTGATTGCCCTTTCTG TGGGCCAATACAATGGCGGCTCCGGTCCTGTGCGCAGATACTCTAGACAGGGCGGCT CTGGCGGAGGCGCCCAGGGCGTGATTAGCGCCAGCGCCAGCAATCTGGACGACTTT TACAGCGGCGGCTCCGGCTCTAGAGTGTTCACCCTCGAAGATTTCGTGGGCGACTGG AGACAGACCGCCGGCTACAACCTGGACCAGGTGCTGGAACAGGGCGGCGTGAGCTC TCTGTTTCAGAATCTGGGGGTTAGCGTGACACCAATCCAGAGGATCGTGCTGTCCGG CGAGAACGGGCTGAAGATCGACATCCACGTGATCATTCCATACGAGGGCCTGTCTG GCGATCAGATGGGCCAGATCGAGAAGATCTTCAAGGTGGTGTACCCTGTAGACGAC CACCATTTTAAGGTGATCCTGCACTACGGCACCCTGGTCATCGACGGCGTGACACCC AACATGATCGACTACTTCGGCAGACCATATGAGGGTATTGCCGTGTTTGATGGAAAG AAGATCACAGTCACCGGCACCCTGTGGAACGGCAACAAGATTATCGATGAACGGCT GATCAACCCCGACGGCTCTCTGCTGTTTAGAGTGACCATCAATGGCGTGACAGGCTG GAGACTGTGCGAGAGAATCCTGGCTGAGCAGAAACTGATCAGCGAGGAAGACCTGC TCGAGGGCGGTAGCGGCGCCCAGGGCGTGATTAGCGCCAGCGCCAGCAATCTGGAC

[0854] GACTTTTACGGCGGGAGCGGAGGCGGCAGTGGCCTCGAGCCTAGGGGCGGCAGCGG AGTTAACCCCGATATTCAGATGACTCAGACTACCAGCTCCCTGAGCGCATCCCTGGG CGACCGAGTGACAATCAGTTGCCGAGCCAGCCAGGACATCAGCAAGTATCTGAACT GGTACCAGCAGAAGCCTGACGGAACCGTCAAACTGCTGATCTATCACACATCCCGCC TGCATTCTGGCGTGCCAAGTCGGTTCTCTGGAAGTGGCTCAGGGACCGACTACTCAC TGACAATCAGCAACCTGGAGCAGGAAGATATTGCCACCTATTTCTGCCAGCAGGGC AATACTCTGCCTTACACCTTTGGCGGGGGAACTAAGCTGGAGATTACCGGCGGAGG AGGCAGCGGAGGAGGAGGGTCCGGAGGCGGGGGATCTGAGGTGAAACTGCAGGAA

[0855] AGCGGACCTGGCCTGGTCGCTCCAAGCCAGTCCCTGTCTGTGACTTGTACCGTCAGC GGAGTGTCCCTGCCCGATTACGGCGTCTCTTGGATCAGACAGCCCCCTAGAAAGGGC CTGGAGTGGCTGGGCGTGATCTGGGGAAGTGAAACCACATACTATAATTCCGCCCTG AAAAGCCGGCTGACAATCATTAAGGACAACAGTAAATCACAGGTGTTCCTGAAGAT GAATTCCCTGCAGACAGACGATACTGCAATCTACTATTGCGCCAAACACTACTATTA CGGCGGGAGCTATGCTATGGATTACTGGGGGCAGGGAACCTCCGTCACAGTGTCTA GTGTTAACTCAGGCGGCGGCGGCAGCGGAGGCGGAGGCAGTGCCAAGCCTACAACC ACCCCCGCCCCTAGACCTCCTACACCTGCCCCTACCATCGCTAGCCAGCCACTCTCTC TCAGACCTGAAGCCTGTAGACCCGCCGCTGGCGGCGCCGTGCACACCAGAGGCCTG

[0856] GACTTCGCGCCTCGGAAGATCGAGGTGATGTACCCCCCGCCTTACCTGGACAACGAG AAGTCTAATGGAACAATCATCCACGTGAAGGGAAAACACCTGTGTCCTTCTCCTCTG TTCCCTGGCCCCAGCAAGCCTTTCTGGGTCCTGGTGGTGGTCGGCGGCGTGCTGGCG TGCTACAGCCTGCTCGTGACCGTGGCCTTCATCATTTTCTGGGTGCGGAGCAAACGG AGCAGACTGCTGCATAGCGATTACATGAACATGACCCCAAGAAGACCTGGGCCAAC CAGAAAGCACTACCAGCCTTACGCCCCCCCCAGAGATTTCGCCGCTTATAGAAGCCG GGTGAAGTTCTCCAGATCCGCCGACGCCCCTGCTTACCAACAGGGCCAGAACCAGCT GTATAACGAGCTGAATCTGGGCAGAAGAGAGGAGTACGACGTGCTGGATAAGCGGA GAGGAAGAGACCCTGAGATGGGCGGCAAGCCCCGGCGGAAGAACCCTCAGGAGGG

[0857] CCTGTATAACGAGCTGCAAAAAGATAAGATGGCCGAGGCTTATAGCGAGATCGGAA TGAAGGGCGAGCGGCGGAGGGGCAAAGGCCACGACGGCCTGTACCAGGGACTGAG CACAGCCACAAAGGACACCTACGACGCTCTGCACATGCAGGCCCTGCCTCCTAGATA ATGTACAAGTAAAGCGGCCGGCC (SEQ ID NO: 362)

[0858]

[0424] CD69Cyt-TM-GrzB(ADKG)-TNFa-Myc-GrzB(ADKG)-a-CD19sc-Stalk-CD28-CD3z

[0859] MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALS VGQYNGGSGPVRRYSRQGGSGGGLEADKGKLEYDSGGSGSRVFTLEDFVGDWRQTAG YNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIF KVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK RDERLINPDGSLLFRVTINGVTGWRLCERILAEQKLISEEDLLEGGSGLEADKGKLEYDG GSGGGSGLEPRGGSGVNPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT KLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQ

[0860] PPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKH YYYGGSYAMDYWGQGTSVTVSSVNSGGGGSGGGGSAKPTTTPAPRPPTPAPTIASQPLS LRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPG PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR (SEQ ID NO: 384)

[0861]

[0425] Which is encoded by cDNA:

[0862] GCCAGTCCTCCGATTGACTGAGTCGCCCGGATCCCGCCACCATGAGCTCTGAGAA CTGCTTCGTGGCCGAGAACAGCAGCCTCCACCCCGAGAGTGGTCAGGAGAACGA CGCCACCAGCCCCCACTTCAGCACAAGACACGAGGGCTCATTCCAGGTGCCTGT GCTGTGCGCCGTGATGAACGTGGTGTTCATCACCATCCTGATCATCGCCCTGATT GCCCTTTCTGTGGGCCAATACAATGGCGGCTCCGGTCCTGTGCGCAGATACTCTA GACAGGGCGGCTCTGGCGGAGGCCTGGAGGCCGACAAGGGCAAGCTGGAGTAC GATAGCGGCGGCTCCGGCTCTAGAGTGTTCACCCTCGAAGATTTCGTGGGCGACT GGAGACAGACCGCCGGCTACAACCTGGACCAGGTGCTGGAACAGGGCGGCGTG AGCTCTCTGTTTCAGAATCTGGGGGTTAGCGTGACACCAATCCAGAGGATCGTGC TGTCCGGCGAGAACGGGCTGAAGATCGACATCCACGTGATCATTCCATACGAGG GCCTGTCTGGCGATCAGATGGGCCAGATCGAGAAGATCTTCAAGGTGGTGTACC CTGTAGACGACCACCATTTTAAGGTGATCCTGCACTACGGCACCCTGGTCATCGA CGGCGTGACACCCAACATGATCGACTACTTCGGCAGACCATATGAGGGTATTGC CGTGTTTGATGGAAAGAAGATCACAGTCACCGGCACCCTGTGGAACGGCAACAA GATTATCGATGAACGGCTGATCAACCCCGACGGCTCTCTGCTGTTTAGAGTGACC ATCAATGGCGTGACAGGCTGGAGACTGTGCGAGAGAATCCTGGCTGAGCAGAAA CTGATCAGCGAGGAAGACCTGCTCGAGGGCGGTAGCGGCCTGGAGGCCGACAAG GGCAAGCTGGAGTACGATGGCGGGAGCGGAGGCGGCAGTGGCCTCGAGCCTAG GGGCGGCAGCGGAGTTAACCCCGATATTCAGATGACTCAGACTACCAGCTCCCT GAGCGCATCCCTGGGCGACCGAGTGACAATCAGTTGCCGAGCCAGCCAGGACAT CAGCAAGTATCTGAACTGGTACCAGCAGAAGCCTGACGGAACCGTCAAACTGCT GATCTATCACACATCCCGCCTGCATTCTGGCGTGCCAAGTCGGTTCTCTGGAAGT GGCTCAGGGACCGACTACTCACTGACAATCAGCAACCTGGAGCAGGAAGATATT GCCACCTATTTCTGCCAGCAGGGCAATACTCTGCCTTACACCTTTGGCGGGGGAA CTAAGCTGGAGATTACCGGCGGAGGAGGCAGCGGAGGAGGAGGGTCCGGAGGC GGGGGATCTGAGGTGAAACTGCAGGAAAGCGGACCTGGCCTGGTCGCTCCAAGC CAGTCCCTGTCTGTGACTTGTACCGTCAGCGGAGTGTCCCTGCCCGATTACGGCG TCTCTTGGATCAGACAGCCCCCTAGAAAGGGCCTGGAGTGGCTGGGCGTGATCT GGGGAAGTGAAACCACATACTATAATTCCGCCCTGAAAAGCCGGCTGACAATCA TTAAGGACAACAGTAAATCACAGGTGTTCCTGAAGATGAATTCCCTGCAGACAG ACGATACTGCAATCTACTATTGCGCCAAACACTACTATTACGGCGGGAGCTATGC TATGGATTACTGGGGGCAGGGAACCTCCGTCACAGTGTCTAGTGTTAACTCAGGC GGCGGCGGCAGCGGAGGCGGAGGCAGTGCCAAGCCTACAACCACCCCCGCCCCT AGACCTCCTACACCTGCCCCTACCATCGCTAGCCAGCCACTCTCTCTCAGACCTG AAGCCTGTAGACCCGCCGCTGGCGGCGCCGTGCACACCAGAGGCCTGGACTTCG CGCCTCGGAAGATCGAGGTGATGTACCCCCCGCCTTACCTGGACAACGAGAAGT CTAATGGAACAATCATCCACGTGAAGGGAAAACACCTGTGTCCTTCTCCTCTGTT CCCTGGCCCCAGCAAGCCTTTCTGGGTCCTGGTGGTGGTCGGCGGCGTGCTGGCG TGCTACAGCCTGCTCGTGACCGTGGCCTTCATCATTTTCTGGGTGCGGAGCAAAC GGAGCAGACTGCTGCATAGCGATTACATGAACATGACCCCAAGAAGACCTGGGC CAACCAGAAAGCACTACCAGCCTTACGCCCCCCCCAGAGATTTCGCCGCTTATAG AAGCCGGGTGAAGTTCTCCAGATCCGCCGACGCCCCTGCTTACCAACAGGGCCA GAACCAGCTGTATAACGAGCTGAATCTGGGCAGAAGAGAGGAGTACGACGTGCT

[0863] GGATAAGCGGAGAGGAAGAGACCCTGAGATGGGCGGCAAGCCCCGGCGGAAGA ACCCTCAGGAGGGCCTGTATAACGAGCTGCAAAAAGATAAGATGGCCGAGGCTT ATAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGGGGCAAAGGCCACGACGGC CTGTACCAGGGACTGAGCACAGCCACAAAGGACACCTACGACGCTCTGCACATG

[0864] CAGGCCCTGCCTCCTAGATAATGTACAAGTAAAGCGGCCGGCC (SEQ ID NO: 363)

[0865]

[0426] Example 19: Creation of Lentiviral vectors

[0866]

[0427] The transfer vector containing the gene of interest was transfected into 293 T cells along with the lentiviral packaging vectors (pMDLg / pRRE and pRSV-Rev), and an envelope vector (phCMV-VSV-G) and the viral supernatant was harvested.

[0867]

[0428] Small-scale production of VSV-G pseudotyped lentiviral vectors

[0868]

[0429] This method describes the production of VSV-G pseudotyped lentiviral vectors in 6-well plates using a calcium phosphate transfection kit. The amount of virus supernatant per well was 4 ml, and the virus concentration depended on the vector used.

[0869]

[0430] Materials Table 6.

[0431] General materials:

[0870]

[0432] T75 flasks, cell culture pipettes, micropipettes and tips, 1.5 ml microcentrifuge tubes, 5ml syringes, Trypan Blue, Virkon, and M-ytdes.

[0871]

[0433] Plasmids

[0872] Vector: depending on the choice

[0873] Gag-pol plasmid: pMDLg / pRRE

[0874] Rev plasmid: pRSV-Rev

[0875] Envelope plasmid: phCMV-VSV-G

[0876]

[0434] Procedure

[0877]

[0435] Maintaining HEK293FT Cells

[0878]

[0436] Maintain cells by splitting them every other day 1 :5 - 1 :4 and keep them in a T75 or T150 flask. After thawing the cells, culture them for at least 3 passages before using them for virus production so that they recover and start exponential growth. Using cells with high passage numbers is not recommended, as this negatively affects virus production.

[0879]

[0437] Day 1: Plating & Transfection

[0880] - Plate 500.000 cells / well in complete growth medium.

[0881] - Pipette off the medium from the flask.

[0882] - Wash the cells with 10ml of room temperature PBS

[0883] - Add 1 ml (T75) or 2 ml (T150) of TrypLE Express and incubate at 37°C for 5 minutes

[0884] - After incubation, add 10-20 ml of the complete medium into the flask, resuspend thoroughly and dissociate all the clumps by pipetting up and down several times

[0885] - Count the cells using Trypan Blue and make a cell suspension of 250.000 cells / ml

[0886] - Put 2ml of this suspension into a well (Poly-L-Lysine coated 6-well plate). Prepare one well for each vector.

[0887] - Put the plate in the incubator for at least 7-8 hours

[0888]

[0438] After incubation, transfect the cells. Confirm that the cells have been attached, and a confluence of optimally 80% is obtained. If the cells are viable and at an appropriate density, put the plate back in the incubator and prepare the transfection mix. Abort the transfection if confluency is below 60%.

[0889] - Prepare 1ml of complete growth medium with the addition of Chloroquine at a final concentration of 25pM. Put it into the incubator for pre-warming. It is important that the components of the calcium phosphate precipitation kit are brought to room temperature before starting the transfection.

[0439] - Prepare the plasmid mixture into a microcentrifuge tube as follows (4pg DNA in total):

[0890] - 2 pg of LeGO-iG2 vector (including transgene)

[0891] - 1 pg of pMDLg / pRRE (Gag / Pol)

[0892] - 0.75 pg of pRSV-REV (Rev)

[0893] - 0.25 pg of phCMV-VSV-G (Envelope)

[0894] - Mix the plasmids, and adjust the volume up to 54pl with ddEEO

[0895] - Add 6pl of 2.5 M CaCh solution to the DNA mixture

[0896] - Into a separate microtube, put 60pl of 2X HeBS buffer. Add the CaCh / DNA mixture and vortex

[0897] - Let this mixture sit at room temperature for 15 minutes. Do not allow it to sit for more than 30 minutes, as this might decrease the transfection efficiency.

[0898] - During these 15 minutes, take your dish out, discard the medium and add 1ml prewarmed fullgrowth medium containing 25 pM Chloroquine.

[0899] - After the 15-minute incubation, add the 120pl mixture into the well dropwise, while gently swirling the dish in a circular motion.

[0900] - Close the lid of the dish and put it back into the incubator for 10-12 hours.

[0901]

[0440] Day 2: Medium change

[0902] - 10-12 hours post-transfection:

[0903] - Aspirate the medium containing the transfection mix and chloroquine from the wells and discard.

[0904] - Add 2ml of complete growth medium per well. Ensure the medium is prewarmed at least to room temperature, preferably 37°C. Put the cells into the incubator.

[0905]

[0441] Day 3: Collection of supernatant 1

[0906] - 24 hours post medium change:

[0907] - Check the cells under a UV microscope for GFP expression. The transfection efficiency should be above 90%.

[0908] - Prepare a 0.45 pm filter, a 5ml syringe, a 5ml microtube and a 1.5 tube per well.

[0909] - Harvest the medium from the dish using the 5ml syringe. Apply the filter and filter the supernatant into the 5ml microtube. Be gentle while filtering, do not create air bubbles, and do not apply extreme force. When you are done, drop the syringe and filter into the Virkon solution.

[0910] - Take a 100 pl aliquot from the filtered supernatant into the 1.5 ml microcentrifuge tube (virus titration). Aliquot the rest of the supernatant as you wish and freeze at -80°C for long-term storage.

[0911] - Add 2 ml of full-growth medium per dish. Ensure the medium is prewarmed at least to room temperature, preferably 37°C. Put the cells into the incubator.

[0912]

[0442] Day 4: Collection of supernatant 2 - 48 hours post medium change:

[0913] - Collect virus supernatants in the same manner as the previous day.

[0914] - Discard the plates.

[0915]

[0443] Generation of Cell lines

[0916]

[0444] The lentiviral particles harvested in the procedure mentioned above were transduced into K562 and KHYG-1 cells, sorted, and expanded. The resulting cells were tested for the expression of the target genes either using the corresponding antibodies or fluorescent-labeled proteins.

[0917] 1. Prepare medium RPMI 10%FBS =400ul / well

[0918] 2. Add lentiviral vector and transduction medium.

[0919] 3. Mark a 24-well plate with the date, name, cell type and what virus you use for transduction.

[0920] 4. Set the temperature of the centrifuge to 32°C.

[0921] 5. Count the cells using trypan blue and make a cell suspension with 106 cells / ml in medium + 10% FBS.

[0922] 6. Distribute 250 pl of 50,000 cells to 24 well plate.

[0923] 7. Take the required protamine sulfate stock for a final concentration of 8 pg / ml.

[0924] 8. Avoid repeated freeze / thaw of the stock.

[0925] 9. Add the medium according to the calculations.

[0926] 10. Keep the virus on dry ice until usage. Thaw the required amount of virus quickly.

[0927] 11. Mix the virus carefully so that contact with air is minimal.

[0928] 12. Add the calculated amount of virus to each well.

[0929] 13. Pipette protamine sulfate (8 pg / ml) and IL-2 (100 lU / ml) to each well in the case of KHYG-1 cells.

[0930] 14. Mix the cells carefully by pipetting up and down.

[0931] 15. Centrifuge the plate with 1000 x g for 1 hr at 32°C without a break.

[0932] 16. Take out the plate and incubate between 4 hr and overnight (depending on your construct and virus titer; should be tested) in the incubator.

[0933] 17. At the end of the incubation, centrifuge the plate again with 1000 x g for Ihr at 32°C.

[0934] 18. Remove 80% of the medium carefully from all the wells and fill with 500 ul fresh pre-warmed medium with serum.

[0935] 19. Put the plate back into the incubator.

[0936]

[0445] Day 1 & 2: Check the cells under the microscope and search for colonies.

[0937]

[0446] Day 3 : Analyze the cells with flow cytometry.

[0938]

[0447] HEK293 NF-KB Luciferase Assay Procedure 1. The day before the assay, harvest NF-KB reporter (Luc)-HEK293 cells and seed cells at a density of 20,000 cells per well into a 96-well microplate in lOOpl of assay medium (DMEM + 10% FBS + 1% P / S). Incubate cells at 37C with 5% CO2 overnight for approximately 24 hours.

[0939] 2. The next day, aspirate the medium on 96 wells microplate and add lOOpL of the supernatant from GAGE expressing cells alone or co-cultured with the target cells into corresponding well and incubate at 37°C with 5% CO2 for 6 hours.

[0940] 3. 6 hours after, aspirate the medium from the cells and add lOOpL of 0.1% Triton buffer to each sample well.

[0941] 4. Rock the plate slowly several times to ensure complete coverage of the cells with Lysis Buffer.

[0942] 5. Incubate for 10 minutes at room temperature to allow lysis to occur.

[0943] 6. Transfer 30pL of lysate to a new white opaque 96-well plate for the Luciferase assay system and set the GLOMAX machine automatically to add 50pL of D- Luciferine or nanoGLOW assay system (Promega) per well and measure luminescence using a luminometer following the manufacturer’s instructions. The luciferase activity data presented in the results are normalized to the percentages and MFI of the GAGE expression in the cells.

[0944]

[0448] Flow cytometry

[0945]

[0449] The staining and washes were performed in a flow cytometry acquisition buffer. A singlecell suspension of the cells was incubated with a blocking reagent for 10 min on ice and then stained with antibodies and viability staining for 30 min on ice. Samples are analyzed on a Cytoflex flow cytometer (BD Biosciences), and the data are analyzed using FlowJo software (TreeStar, Ashland, OR). For sorting AriaFusion (BD Biosciences) machine was used. Sorted cells were cultured in a medium with antibiotics for two weeks. Onwards, cells were cultured without antibiotics.

[0946]

[0450] Example 20

[0947]

[0451] Experiments were performed relating to the following Caspase 3 sequences and constructs:

[0948]

[0452] CD69Cyt-TM-Cas3(DEVD)-TNFa-Myc-Cas3(DEVD)-a-CD19sc-Stalk-CD28-CD3z

[0949]

[0453] Cas3 (DEVDG) sequences are given as BOLD UNDERLINED CAPITALIZED

[0454] DNA sequence

[0950] ATGAGCTCTGAGAACTGCTTCGTGGCCGAGAACAGCAGCCTCCACCCCGAGAGTG GTCAGGAGAACGACGCCACCAGCCCCCACTTCAGCACAAGACACGAGGGCTCAT TCCAGGTGCCTGTGCTGTGCGCCGTGATGAACGTGGTGTTCATCACCATCCTGATC

[0951] \ I ( XKXXTGATTGCCCTTTC 'TGTGGGC X^AAILACAATGGCGGCI'CCGGI'CC'rGlXiCG CAGATACTCTAGACAGGGCGGCTCTGGCGGAGGCGATGAAGTGGACGGCAGCG GCGGCTCCGGCAGCAGCAGCTCTAGAGTGTTCACCCTCGAAGATTTCGTGGGCGA CTGGAGAC AGACCGCCGGC TAG AACC TGGACCAGGTGCTGGAACAGGGCGGCGT GAGCTCTCTGTTTCAGAATCTGGGGGTTAGCGTGACACCAATCCAGAGGATCGTG CTGTCCGGCGAGAACGGGCTGAAGATCGACATCCACGTGATCATTCCATACGAGG GCCTGTCTGGCGATCAGATGGGCCAGATCGAGAAGATCTTCAAGGTGGTGTACCC TGTAGACGACCACCATTTTAAGGTGATCCTGCACTACGGCACCCTGGTCATCGAC GGCGTGACACCCAACATGATCGACTACTTCGGCAGACCATATGAGGGTATTGCCG TGTTTGATGGAAAGAAGATCACAGTCACCGGCACCC TGTGGAACGGCAACAAGA TTATCGATGAACGGCTGATCAACCCCGACGGCTCTCTGCTGTTTAGAGTGACCATC AA TGGCGTGACAGGCTGGAGACTGTGCGAGAGAATCCTGGCTGAGCAGAAACTG ATC AGC G AGG A AG AC C I GC T C G AGGGC GGT AGC GGC €yATGAAGTGGA€JGG€-GG CGGGAGCGGAGGCGGCAGTGGCCTCGAGCCTAGGGGCGGCAGCGGAGTTAACCC CGATATTCAGATGACTCAGACTACCAGCTCCCTGAGCGCATCCCTGGGCGACCGA GTGACAATCAGTTGCCGAGCCAGCCAGGACATCAGCAAGTATCTGAACTGGTACC AGCAGAAGCCTGACGGAACCGTCAAACTGCTGATCTATCACACATCCCGCCTGCA TTCTGGCGTGCCAAGTCGGTTCTCTGGAAGTGGCTCAGGGACCGACTACTCACTG ACAATCAGCAACCTGGAGCAGGAAGATATTGCCACCTATTTCTGCCAGCAGGGCA ATACTCTGCCTTACACCTTTGGCGGGGGAACTAAGCTGGAGATTACCGGCGGAGG

[0952] AGGCAGCGGAGGAGGAGGGTCCGGAGGCGGGGGATCTGAGGTGAAACTGCAGG AAAGCGGACCTGGCCTGGTCGCTCCAAGCCAGTCCCTGTCTGTGACTTGTACCGT CAGCGGAGTGTCCCTGCCCGATTACGGCGTCTCTTGGATCAGACAGCCCCCTAGA AAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGAAGTGAAACCACATACTATAATT CCGCCCTGAAAAGCCGGCTGACAATCATTAAGGACAACAGTAAATCACAGGTGTT CCTG / XAGATGAATTCCCTGCAGACAGACGATACTGCAATCTACTATTGCGCCAAA CACTACTATTACGGCGGGAGCTATGCTATGGATTACTGGGGGCAGGGAACCTCCG TC AC AGTGTCTAGTGTTAACTC AGGCGGCXKKXJGC AGCGGAGGCGGAGGCAGTG CCAAGCCTACAACCACCCCCGCCCCTAGACCTCCTACACCTGCCCCTACCATCGCT AGCCAGCCACTCTCTCTCAGACCTGAAGCCTGTAGACCCGCCGCTGGCGGCGCCG TGC A ( AC CAGAGC JC CT GG A C T T( GCGCC TO ( JG A A( J AT C ( J A( JGTG AT G I AC C C C (. ’ C GCCTTACCTGGACAACGAGAAGTCTAATGGAACAATCATCCACGTGAAGGGAAA ACACCTGTGTCCTTCTCCTCTGTTCCCTGGCCCCAGCAAGCCTTTCTGGGTCCTGG TGGTGGTCGGCGGCGTGCTGGCGTGCTACAGCCTGCTCGTGACCGTGGCCTTCAT CATTTTCTGGGTGCGGAGCAAACGGAGCAGACTGCTGCATAGCGATTACATGAAC ATGACCCCAAGAAGACCTGGGCCAACCAGAAAGCACTACCAGCCTTACGCCCCCC CCAGAGATTTCGCCGCTTATAGAAGCCGGGTGAAGTTCTCCAGATCCGCCGACGC CCCTGCTTACCAACAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCAGA AGAGAGGAGTACGACGTGCTGGATAAGCGGAGAGGAAGAGACCCTGAGATGGGC GGCAAGCCCCGGCGGAAGAACCCTCAGGAGGGCCTGTATAACGAGCTGCAAAAA GATAAGATGGCCGAGGCTTATAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGG GGCAAAGGCCACGACGGCCTGTACCAGGGACTGAGCACAGCCACAAAGGACACC TACGACGCTCTGCACATGCAGGCCCTGCCTCCTAGAtaa (SEQ ID NO: 386)

[0953]

[0455] CD69Cyt-TM-Cas3(DEVD)-TNFa-Myc-Cas3(DEVD)-a-CD19sc-Stalk-CD28-CD3z

[0954]

[0456] Protein sequence

[0955] MS SENCF VAENS SLHPESGQEND ATSPHF STRHEGSFQVPVLC AVMNVVFITILIIALIA LSVGOYNGGSGPVRRYSROGGSGGGDEVDGSGGSGSSSSRVFTLEDFVGDWROTAG YNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIE KIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLW NGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAEOKLISEEDLLEGGSGDEVDGG GSGGGSGLEPRGGSGVNPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKP DGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFG GGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV SWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTnKDNSKSQVFLKMNSLQTDDTAI YYCAKHYYYGGSYAMDYWGQGTSVTVSSVNSGGGGSGGGGSAKPTTTPAPRPPTPA PTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGK HLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTP RRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 387)

[0457] CD69Cyt-TM-Cas3(EAALVDMVNDG)-TNFa-Myc-Cas3(EAALVDMVNDG)-a-

[0956] CD19sc-Stalk-CD28-CD3z

[0957]

[0458] Cas3 (EAALVDMVNDG (SEQ ID NO: 388)) sequences are given as

[0958] BOLD UNDERLINED CAPITALIZED

[0959]

[0459] DNA sequence

[0960] ATGAGCTCTGAGAACTGCTTCGTGGCCGAGAACAGCAGCCTCCACCCCGAGAGTG GTCAGGAGAACGACGCCACCAGCCCCCACTTCAGCACAAGACACGAGGGCTCAT

[0961] TCCAGGTGCCTGTGCTGTGCGCCGTGATGAACGTGGTGTTCATCACCATCCTGATC

[0962] ATCGCCCTGATTGCCCTTTCTGTGGGCCAATACAATGGCGGCTCCGGTCCTGTGCG CAGATACTCTAGAGGCGGCTCTGGCGGAGGCGAGGCCGCCCTGGTGGATATGG TGAACGACGGCAGCGGCGGCTCCGGCGGATCTAGAGTGTTCACCCTCGAAGATT

[0963] TCGTGGGCGACTGGAGACAGACCGCCGGCTACAACCTGGACCAGGTGCTGGAAC

[0964] AGGGCGGCGTGAGCTCTCTGTTTCAGAATCTGGGGGTTAGCGTGACACCAATCCA

[0965] GAGGATCGTGCTGTCCGGCGAGAACGGGCTGAAGATCGACATCCACGTGATCATT CCATACGAGGGCCTGTCTGGCGATCAGATGGGCCAGATCGAGAAGATCTTCAAGG TGGTGTACCCTGTAGACGACCACCATTTTAAGGTGATCCTGCACTACGGCACCCT GGTCATCGACGGCGTGACACCCAACATGATCGACTACTTCGGCAGACCATATGAG GG TATTGCCGTGTTTGA TGGAAAGAAGA TCACAGTCACCGGCACCCTGTGGAACG

[0966] GCAACAAGATTATCGATGAACGGCTGATCAACCCCGACGGCTCTCTGCTGTTTAG AGTGACCATCAATGGCGTGACAGGCTGGAGACTGTGCGAGAGAATCCTGGCTG / \ GCAGAAACTGATCAGCGAGGAAGACCTGCTCGAGGGCGGCTCTGGCGGAGGCGA

[0967] GGCCGCCCTGGTGGATATGGTGAACGACGGCAGCGGCGGCTCCGGCGGACTC

[0968] GAGCCTAGGGGCGGCAGCGGAGTTAACCCCGATATTCAGATGACTCAGACTACCA GCTCCCTGAGCGCATCCCTGGGCGACCGAGTGACAATCAGTTGCCGAGCCAGCCA GGACATCAGCAAGTATCTGAAC TGG TACCAGCAGAAGCCTGACGGAACCGTCAA ACTGCTGATCTATCACACATCCCGCCTGCATTCTGGCGTGCCAAGTCGGTTCTCTG GAAGTGGCTCAGGGACCGACTACTCACTGACAATCAGCAACCTGGAGCAGGAAG

[0969] ATATTGCCACCTATTTCTGCCAGCAGGGCAATACTCTGCCTTACACCTTTGGCGGG GGAA.CTAAGCTGGAGATTACCGGCGGAGGAGGCAGCGGAGGAGGAGGGTCCGG AGGCGGGGGATCTGAGGTGAAACTGCAGGAAAGCGGACCTGGCCTGGTCGCTCC

[0970] AAGCCAGTCCCTGTCTGTGACTTGTACCGTCAGCGGAGTGTCCCTGCCCGATTACG

[0971] CKX]4X:4X24GXK}A4X2AGACAGCCCCCIAGAAAGGGCC'rGGAGI'GGCI'GGGCGI'GArr CTGGGGAAGTGAAACCACATACTATAATTCCGCCCTGAAAAGCCGGCTGACAATC

[0972] ATTAAGGACAACAGTAAATCACAGGTGTTCCTGAAGATGAATTCCCTGCAGACAG

[0973] ACGATACTGCAATCTACTATTGCGCCAAACACTACTATTACGGCGGGAGCTATGC

[0974] TATGGATTACTGGGGGCAGGGAACCTCCGTCACAGTGTCTAGTGTTAACTCAGGC

[0975] GGCGGCGGCAGCGGAGGCGGAGGCAGTGCCAAGCCTACAACCACCCCCGCCCCT

[0976] AGACCTCCTACACCTGCCCCTACCATCGCTAGCCAGCCACTCTCTCTCAGACCTGA

[0977] AGCCTGTAGACCCGCCGCTGGCGGCGCCGTGCACACCAGAGGCCTGGACTTCGCG

[0978] CCTCGGAAGATCGAGGTGATGTACCCCCCGCCTTACCTGGACAACGAGAAGTCTA

[0979] ATGGAACAATCATCCACGTGAAGGGAAAACACCTGTGTCCTTCTCCTCTGTTCCCT

[0980] GGCCCCAGCAAGCCTTTCTGGGTCCTGGTGGTGGTCGGCGGCGTGCTGGCGTGCT

[0981] ACAGCXTGCTCGTGACCGTGGCCTTCATCATTTTCTGGGTGCGGAGCAAACGGAG

[0982] CAGACTGCTGCATAGCGATTACATGAACATGACCCCAAGAAGACCTGGGCCAACC

[0983] AGAAAGCACTACCAGCCTTACGCCCCCCCCAGAGATTTCGCCGCTTATAGAAGCC

[0984] GGGTGAAGTTCTCCAGATCCGCCGACGCCCCTGCTTACCAACAGGGCCAGAACCA

[0985] GCTGTATAACGAGCTGAATCTGGGCAGAAGAGAGGAGTACGACGTGCTGGATAA

[0986] GCGGAGAGGAAGAGACCCTGAGATGGGCGGCAAGCCCCGGCGGAAGAACCCTCA

[0987] GGAGGGCCTGTATAACGAGCTGCAAAAAGATAAGATGGCCGAGGCTTATAGCGA

[0988] GATCGGAATGAAGGGCGAGCGGCGGAGGGGCAAAGGCCACGACGGC’CTGTACCA

[0989] GGGACTGAGCACAGCCACAAAGGACACCTACGACGCTCTGCACATGCAGGCCCT

[0990] GCCTCCTAGAtaa (SEQ ID NO: 389)

[0991]

[0460] > CD69Cyt-TM-Cas3(EAALVDMVNDG)-TNFa-Myc-Cas3(EAALVDMVNDG)-a-

[0992] CD19sc-Stalk-CD28-CD3z

[0993]

[0461] Protein sequence

[0994] MS SENCF VAENS SLHPESGQEND ATSPHF STRHEGSFQVPVLC AVMNVVFITILIIALIA

[0995] LSVGOYNGGSGPVRRYSRGGSGGGEAALVDMVNDGSGGSGGSRVFTLEDFVGDWR

[0996] QTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQM GQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVT GTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAEQKLISEEDLLEGGSGGG EAALVDMVNDGSGGSGGLEPRGGSGVNPDIOMTQTTSSLSASLGDRVTISCRASODI

[0997] SKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC

[0998] QQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCT

[0999] VSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLK

[1000] MNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSVNSGGGGSGGGGSAKP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNE KSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSR LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLY NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM

[1001] KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 390)

[1002]

[0462] CD69Cyt-TM-Cas3(EDYGRDSGPP)-TNFa-Myc-Cas3(EDYGRDSGPP)-a-CD19sc-

[1003] Stalk-CD28-CD3z

[1004]

[0463] Cas3 (EDYGRDSGPP (SEO ID NO: 391)) sequences are given as

[1005] BOLD UNDERLINED CAPITALIZED

[1006]

[0464] DNA sequence

[1007] ATGAGCTCTGAGAACTGCTTCGTGGCCGAGAACAGCAGCCTCCACCCCGAGAGTG

[1008] GTCAGGAGAACGACGCCACCAGCCCCCACTTCAGCACAAGACACGAGGGCTCAT

[1009] TCCAGGTGCCTGTGCTGTGCGCCGTGATGAACGTGGTGTTCATCACCATCCTGATC

[1010] ATCGCCCTGATTGCCCTTTCTGTGGGCCAATACAATGGCGGCTCCGGTCCTGTGCG

[1011] CAGATACTCTAGAGGCGGCTCTGGCGGAGGCGAGGACTACGGCAGAGATAGCG

[1012] GCCCTCCCAGCGGCGGCTCCGGCAGCAGCAGCTCTAGAGTGTTCACCCTCGAAG

[1013] ATTTCGTGGGCGACTGGAGACAGACCGCCGGCTACAACCTGGACCAGGTGCTGGA

[1014] ACAGGGCGGCGTGAGCTCTCTGTTTCAGAATCTGGGGGTTAGCGTGACACCAATC

[1015] CAGAGGATCGTGCTG TCCGGCGAGAACGGGCTGAAGA TCGACATCCACGTGA TC

[1016] ATTCCATACGAGGGCCTGTCTGGCGATCAGATGGGCCAGATCGAGAAGATCTTCA

[1017] AGGTGGTGTACCCTGTAGACGACCACCATTTTAAGGTGATCCTGCACTACGGCAC

[1018] CCTGGTCATCGACGGCGTGACACCCAACATGATCGACTACTTCGGCAGACCATAT

[1019] GAGGGTATTGCCGTGTTTGATGGAAAGAAGATCACAGTCACCGGCACCCTGTGGA

[1020] A(XKTCA / \CAAGATl\\lXXMlXiAA(XTGCFrGA4X’’AAC'CCCGACGGCrrCrrCrrGCI'G'ri'

[1021] TAGAGTGACCATCAATGGCGTGACAGGCTGGAGACTGTGCGAGAGAATCCTGGCT

[1022] GAGCAGAAACTGATCAGC GAGGAAGACCTGCTCGAGGGCGGCTCTGGCGGAGGC

[1023] GAGGACTACGGCAGAGATAGCGGCCCTCCCAGCGGCGGCTCCGGCAGCAGCA

[1024] GCCTCGAGCCTAGGGGCGGCAGCGGAGTTAACCCCGATATTCAGATGACTCAGAC

[1025] TACCAGCTCCCTGAGCGCATCCCTGGGCGACCGAGTGACAATCAGTTGCCGAGCC

[1026] AGCCAGGACATCAGCAAGTATCTGAACTGGTACCAGCAGAAGCCTGACGGAACC GTCAAACTGCTGATCTATCACACATCCCGCCTGCATTCTGGCGTGCCAAGTCGGTT CTCTGGAAGTGGCTCAGGGACCGACTACTCACTGACAATCAGCAACCTGGAGCAG

[1027] GAAGATATTGCCACCTATTTCTGCCAGCAGGGCAATACTCTGCCTTACACCTTTGG

[1028] CGGGGGAACTAAGCTGGAGATTACCGGCGGAGGAGGCAGCGGAGGAGGAGGGT

[1029] CCGGAGGCGGGGGATCTGAGGTGAAACTGCAGGAAAGCGGACCTGGCCTGGTCG

[1030] CTCCAAGCCAGTCCCTGTCTGTGACTTGTACCGTCAGCGGAGTGTCCCTGCCCGAT

[1031] TACGGCGTCTCTTGGATCAGACAGCCCCCTAGAAAGGGCCTGGAGTGGCTGGGCG

[1032] TGATCTGGGGAAGTGAAACCACATACTATAATTCCGCCCTGAAAAGCCGGCTGAC

[1033] AATCATTAAGGACAACAGTAAATCACAGGTGTTCCTGAAGATGAATTCCCTGCAG

[1034] ACAGACGATACTGCAATCTACTATTGCGCCAAACACTACTATTACGGCGGGAGCT

[1035] ATGCTATGGATTACTGGGGGCAGGGAACCTCCGTCACAGTGTCTAGTGTTAACTC

[1036] AGGCGGCGGCGGCAGC:GGAGG(:GGAGGCAGrFGC:CAAGCC:TACAACCACCCCCGC

[1037] CCCTAGACCTCCTACACCTGCCCCTACCATCGCTAGCCAGCCACTCTCTCTCAGAC

[1038] CTGAAGCCTGTAGACCCGCCGCTGGCGGCGCCGTGCACACCAGAGGCCTGGACTT

[1039] CGCGCCTCGGAAGATCGAGGTGATGTACCCCCCGCCTTACCTGGACAACGAGAAG

[1040] TCTAATGGAACAATCATCCACGTGAAGGGAAAACACCTGTGTCCTTCTCCTCTGTT

[1041] CCCTGGCCCCAGCAAGCCTTTCTGGGTCCTGGTGGTGGTCGGCGGCGTGCTGGCG

[1042] TGCTACAGCCTGCTCGTGACCGTGGCCTTCATCATTTTCTGGGTGCGGAGCAAACG

[1043] GAGCAGACTGCTGCATAGCGATTACATGAACATGACCCCAAGAAGACCTGGGCC

[1044] AACCAGAAAGCACTACCAGCCTTACGCCCCCCCCAGAGATTTCGCCGCTTATAGA

[1045] AGCCGGGTGAAGTTCTCCAGATCCGCCGACGCCCCTGCTTACCAACAGGGCCAGA

[1046] ACCAGCTGTATAACGAGCTGAATCTGGGCAGAAGAGAGGAGTACGACGTGCTGG

[1047] ATAAGCGGAGAGGAAGAGACCCTGAGATGGGCGGCAAGCCCCGGCGGAAGAAC

[1048] CC I C AGGAGGGCCT G I ATAACGAGC TGC A AA AAGA TAAGA I GGCCGAGGCT TAT

[1049] AGCGAGATCGGAATGAAGGGCGAGCGGCGGAGGGGCAAAGGCCACGACGGCCT

[1050] GTACCAGGGACTGAGCACAGCCACAAAGGACACCTACGACGCTCTGCACATGCA

[1051] GGCCCTGCCTCCTAGAtaa (SEQ ED NO: 392)

[1052]

[0465] CD69Cyt-TM-Cas3(EDYGRDSGPP)-TNFa-Myc-Cas3(EDYGRDSGPP)-a-CD19sc-

[1053] Stalk-CD28-CD3z

[1054]

[0466] Protein sequence

[1055] MS SENCF VAENS SLHPESGQEND ATSPHF STRHEGSFQVPVLC AVMNVVFITILIIALIA

[1056] LSVGOYNGGSGPVRRYSRGGSGGGEDYGRDSGPPSGGS GS S S SRVFTLEDF VGDWR QTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQM

[1057] GQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVT

[1058] GTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAEQKLISEEDLLEGGSGGG

[1059] EDYGRDSGPPSGGSGSSSLEPRGGSGVNPDIOMTQTTSSLSASLGDRVTISCRASODIS

[1060] KYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC

[1061] QQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCT

[1062] VSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLK

[1063] MNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSVNSGGGGSGGGGSAKP

[1064] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNE KSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSR LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLY

[1065] NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 393)

[1066]

[0467] CD69Cyt-TM-Cas3(TPSEPDSGQGPPQ)-TNFa-Myc-Cas3(TPSEPDSGQGPPQ)-a-

[1067] CD19sc-Stalk-CD28-CD3z

[1068]

[0468] Cas3 (TPSEPDSGQGPPQ (SEQ ID NO: 394)) sequences are given as

[1069] BOLD UNDERLINED CAPITALIZED

[1070]

[0469] DNA sequence

[1071] ATGAGCTCTGAGAACTGCTTCGTGGCCGAGAACAGCAGCCTCCACCCCGAGAGTG

[1072] GTCAGGAGAACGACGCCACCAGCCCCCACTTCAGCACAAGACACGAGGGCTCAT

[1073] TCCAGGTGCCTGTGCTGTGC:GCCGTGATGAACGTGGTGTTCATCACCATCCTGATC

[1074] ATCGCCCTGATTGCCCTTTCTGTGGGCCAATACAATGGCGGCTCCGGTCCTGTGCG

[1075] CAGATACTCTAGAGGCGGCTCTGGCGGAGGCACCCCTAGCGAGCCCGGATCTG

[1076] GCAGAGGCCCTCCACAGAGCGGCGGCTCCGGCGGATCTAGAGTGTTCACCCTCG

[1077] AAGATTTCGTGGGCGACTGGAGACAGACCGCCGGCTACAACCTGGACCAGGTGCT

[1078] GGAACAGGGCGGCGTGAGCTCTCTGTTTCAGAATCTGGGGGTTAGCGTGACACCA

[1079] ATCCAGAGGATCGTGCTGTCCGGCGAGAACGGGCTGAAGATCGACATCCACGTG

[1080] ATCATTCCATACGAGGGCCTGTCTGGCGATCAGATGGGCCAGATCGAGAAGATCT

[1081] TCAAGGTGGTGTACCCTGTAGACGACCACCATTTTAAGGTGATCCTGCACTACGG

[1082] CACCCTGGTCATCGACGGCGTGACACCCAACATGATCGACTACTTCGGCAGACCA

[1083] TATGAGGGTATTGCCGTGTTTGATGGAAAGAAGATCACAGTCACCGGCACCCTGT GGAACGGCAACAAGATTATCGATGAACGGCTGATCAACCCCGACGGCTCTCTGCT

[1084] GTTTAGAGTGACCATCAATGGCGTGACAGGCTGGAGACTGTGCGAGAGAATCCTG

[1085] GCTGAGCAGAAACTGATCAGCGAGGAAGACCTGCTCGAGGGCGGCTCTGGCGGA

[1086] GGCACCCCTAGCGAGCCCGGATCTGGCAGAGGCCCTCCACAGAGCGGCGGC l

[1087] CCGGCGGACTCGAGCCTAGGGGCGGCAGCGGAGTTAACCCCGATATTCAGATGA

[1088] CTCAGACTACCAGCTCCCTGAGCGCATCCCTGGGCGACCGAGTGACAATCAGTTG

[1089] CCGAGCCAGCCAGGACATCAGCAAGTATCTGAACTGGTACCAGCAGAAGCCTGA

[1090] CGGAACCGTCAAACTGCTGATCTATCACACATCCCGCCTGCATTCTGGCGTGCCA

[1091] AGTCGGTTCTCTGGAAGTGGCTCAGGGACCGACTACTCACTGACAATCAGCAACC

[1092] TGGAGCAGGAAGATATTGCCACCTATTTCTGCCAGCAGGGCAATACTCTGCCTTA

[1093] CACCTTTGGCGGGGGAACTAAGCTGGAGATTACCGGCGGAGGAGGCAGCGGAGG

[1094] AGGAGGGTCCGGAGGCGGGGGATCTGAGGTGAAACTGCAGGAAAGCGGACCTGG

[1095] (X:iXK}TCGC:iX:CAAG(X:ACHXXX:i<}IX:iXjlX}AClG,GrrACCGTCAGCGGAGrrG’rC(2C

[1096] TGCCCGATTACGGCGTCTCTTGGATCAGACAGCCCCCTAGAAAGGGCCTGGAGTG

[1097] GCTGGGCGTGATCTGGGGAAGTGAAACCACATACTATAATTCCGCCCTGAAAAGC

[1098] CGGCTGACAATCATTAAGGACAACAGTAAATCACAGGTGTTCCTGAAGATGAATT

[1099] CCCTGCAGACAGACGATACTGCAATCTACTATTGCGCCAAACACTACTATTACGG

[1100] CGGGAGCTATGCTATGGATTACTGGGGGCAGGGAACCTCCGTCACAGTGTCTAGT

[1101] GTTAACTCAGGCGGCGGCGGCAGCGGAGGCGGAGGCAGTGCCAAGCCTACAACC

[1102] ACCCCCGCCCCTAGACCTCCTACACCTGCCCCTACCATCGCTAGCCAGCCACTCTC

[1103] TCTCAGACCTGAAGCCTGTAGACCCGCCGCTGGCGGCGCCGTGCACACCAGAGGC

[1104] CTGGACTTCGCGCCTCGGAAGATCGAGGTGATGTACCCCCCGCCTTACCTGGAC / \

[1105] ACGAGAAGTCTAATGGAACAATCATCCACGTGAAGGGAAAACACCTGTGTCCTTC

[1106] TCCTCTGTTCCCTGGCCCCAGCAAGCCTTTCTGGGTCCTGGTGGTGGTCGGCGGCG

[1107] TGCTGGCGTGCTACAGCCTGCTCGTGACCGTGGCCTTCATCATTTTCTGGGTGCGG

[1108] AGCAAACGGAGCAGACTGCTGCATAGCGATTACATGAACATGACCCCAAGAAGA

[1109] CCTGGGCCAACCAGAAAGCACTACCAGCCTTACGCCCCCCCCAGAGATTTCGCCG

[1110] CTTATAGAAGCCGGGTGAAGTTCTCCAGATCCGCCGACGCCCCTGCTTACCAACA

[1111] GGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCAGAAGAGAGGAGTACGA

[1112] CGTGCTGGATAAGCGGAGAGGAAGAGACCCTGAGATGGGCGGCAAGCCCCGGCG

[1113] GAAGAACCCTCAGGAGGGCCTGTATAACGAGCTGCAAAAAGATAAGATGGC,CGA

[1114] GGCTTATAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGGGGCAAAGGCCACGA

[1115] CGGCCTGTACCAGGGACTGAGCACAGCCACAAAGGACACCTACGACGCTCTGCA

[1116] CATGCAGGCCCTGCCTCCTAGAtaa (SEQ ID NO: 395)

[0470] CD69Cyt-TM-Cas3(TPSEPDSGQGPPQ)-TNFa-Myc-Cas3(TPSEPDSGQGPPQ)-a-

[1117] CD19sc-Stalk-CD28-CD3z

[1118]

[0471] Cas3 (TPSEPDSGQGPPQ (SEQ ID NO: 394)) sequences are given as

[1119] BOLD UNDERLINED CAPITALIZED

[1120]

[0472] Protein sequence

[1121] MS SENCF VAENS SLHPESGQEND ATSPHF STRHEGSFQVPVLC AVMNVVFITILIIALIA LSVGOYNGGSGPVRRYSRGGSGGGTPSEPGSGRGPPOSGGSGGSRVFTLEDFVGDW RQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQ MGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITV TGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAEQKLISEEDLLEGGSGG GTPSEPGSGRGPPOSGGSGGLEPRGGSGVNPDIOMTOTTSSLSASLGDRVTISCRASO DISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATY FCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVT CTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSVNSGGGGSGGGGSA KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLD NEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSK RSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 396)

Claims

CLAIMSWe claim:

1. A peptide delivery system for delivering a peptide payload to a target cell or target locus, the peptide delivery system comprising: a cell, extracellular vesicle, or lipid-containing particle into which an exogenous polypeptide has been anchored, which exogenous polypeptide comprises: a first and a second transmembrane domain connected by a loop region, which loop region comprises a first cleavage site, a peptide payload, and a second cleavage site; and optionally, a targeting moiety for targeting the peptide delivery system to the target cell or the target locus.

2. The peptide delivery system of claim 1 wherein the first and / or second cleavage sites comprise proteolytic cleavage substrates, preferably sheddase cleavage sites MMP cleavage sites, GrzB cleavage sites, and Casp3 cleavage sites.

3. The peptide delivery system of claim 2, which is configured to release the peptide payload upon cleavage by a protease, preferably aby a sheddase, which may be endogenous or exogenous (e.g., included as a component of the peptide delivery system).

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

5. The peptide delivery system of claim 3 or 4, wherein the sheddase is a full-time sheddase selected from ADAM proteases (metalloproteases), BACE proteases, serine-protease Granzyme-B and Site-1 proteases.

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

7. The peptide delivery system of claim 3 or 4, wherein the sheddase is a part-time sheddase selected from Meprin B, MT-MMPs (Membrane Type Matrix Metalloproteases), Proprotein convertases, transmembrane serine proteases, Matrix Metalloproteases, Legumain and Cathepsin S and L.

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

9. The peptide delivery system of claim 7, wherein the Pro-protein convertases are selected from PCSK1 / 3, KCSK2, Furin, PCSK4, PSCK5 / 6, PACE4, PCSK7, and PCSK9.

10. The peptide delivery system of claim 7, wherein the transmembrane serine proteases are selected from membrane-anchored type II proteases including Matriptase, Matriptase-2, Matriptase-3, Polymerase-1, Corin, Hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, Spinesin, Enteropeptidase, HAT, DESCL1, TMPRSS 11A, HAT -like 4 and HAT -like 5.

11. The peptide delivery system of claim 7, wherein the Matrix Metalloproteases are 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, MMP21, MMP 23A-B, MMP24, MMP25, MMP26, MMP27 and MMP28.

12. The peptide delivery system of claim 11, wherein the Matrix Metalloproteases are selected from MMP2, MMP9 and / or MMP25.

13. The peptide delivery system of claim 12, wherein the MMP2 is selected from SEQ ID NOS: 2-6.

14. The peptide delivery system of claiml2, wherein the MMP9 is selected from SEQ ID NOS: 7-8.

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

16. The peptide delivery system of any of the preceding claims, wherein the transmembrane domain comprises a transmembrance domain from one or more of the following proteins: CD63, CD9, CD81, CD28, CD4, CD8, CD34, CD69, CD19, CD20, Integrins, TNFSF14, TNR1A, Aquaporins, NOTCH, NgRl, NRG1, GPi anchor, EGFR, or Rhodopsin.

17. The peptide delivery system of claim 1, which further comprises an intracellular or extracellular domain selected from or derived from one or more of the following proteins: OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1, Programmed Death-I (PD- 1), Inducible T cell Co-Stimulator (ICOS), lymphocyte Function-associated Antigen-I (LFA-1 (CDI la / CD 18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, 1g alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), The costimulatory ligand ( PD-L2, 4-1 BBL, OX40L, Inducible Co-Stimulatory Ligand (ICOS-L), Inter-Cellular Adhesion Molecule (ICAM, CD30L, CD70, CD83, HLA- G, MICA, Ml CB, HVEM, lymphotoxin b receptor, 3 / TR6, ILT3, ILT4), activating NK cellreceptors, BTLA, a 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, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, IT GAM, CD1 lb, ITGAX, CD1 le, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, a ligand that specifically binds with CD83, or any combination thereof.

18. The peptide delivery system of claim 1, which comprises a targeting moiety and the targeting moiety binds to BCMA, MUC16 (also known as CA125), EGFR, EGFRvIII, MUCI, Flt-3, WT-1, CD38, CD70, CD90, CD133, MHC-WTI , TSPANIO, MHC-PRAME, MHC-NY-ESOI, HER2 (ERBB2), CA-IX (Carbonic anhydrase IX), LIVI, ADAMIO, CHRNA2, LeY, NKG2D, CSI, CD44v6, CD24, LGR5, ALDH, ALDH1, CD54, Seal, CD271, CD 123, CD36, CD 109, CD 110, CD71 -negative, CCA, ABCG2, Claudin-18.2 (Claudin-18A2, or Claudinl8 isoform 2), PSCA, DLL3 (Delta-like protein 3, Drosophila Delta homolog 3, Delta3 ), Mud 7 (Mucinl 7, Muc3, Muc3), FAP alpha (Fibroblast Activation 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 scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGI, LLCAM, integrin a5pi, integrin avP3, legumain, MORAb-009, MS4A1, MUCI, mucin CanAg, C-MET, CCR4, CD152, CD10, CD19, CD20, CD200, N-glycolylneuraminic acid, NPC-IC, PDGF-R a, PDL192, phosphatidylserine, tumor antigen CTAA16.88, VEGF- A, VEGFR-1, VEGFR2, vimentin, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF-P, TRAIL-R1, TRAIL-R2, folic acid receptor, transferrin receptors, or any combination thereof.

19. The peptide delivery system of claim 1, wherein the second transmembrane domain is connected to an intracellular region comprising an intracellular domain from one or more of 41BB, ICOS, and CD3^20. The peptide delivery system of claim 19, wherein phosphorylation by a kinase within the intracellular domain of 41BB, ICOS, or CD3(^ activates GrzB and allows GrzB to be transported out of the cell and cleave the the first and / or second cleavage site.

21. The peptide delivery system of claim 1, which comprises a third and fourth transmembrane domain connected by a second loop region, wherein the third transmembrane domain is connected to an intracellular region comprising an intracellular domain from 4 IBB, ICOS, or CD3z.

22. The peptide delivery system of claim 21, wherein phosphorylation by a kinases within the intracellular domain of 41BB, ICOS or CD3(^ activates the cell to release endogenous GrzB allowing the GrzB to be transported out of the cell and cleave GrzB cleavage present in the first or second loop region thereby releasing the peptide payload.

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

24. The peptide delivery system of any of the preceding claims, wherein the payload comprises: an antibody, an fragment of an antibody, a VHH, a cytokine or chemokine consisting a fraction or derivative of a sulfated xylan, which is an antagonist of a ligand selected from the list comprising IL-la, IL-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL- 11, IL-12, IL-3, IL-14, IL-15 or IL-17 family of cytokines including IL-25, interferon, G- CSF, M-CSF, GM-CSF, BDNF, CNTF, EGF, EPO, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, LIF, PDGF, SCF, TGFa, TGFB, TNFa, TNFB, TPO, VEGF, GH, NGF, NT3, NT4, NT5, NT6, NT7, Oncostatin M (OSM), insulin, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, a member of the MIP-1 family including MIP-la, MIP-2, eotaxin (eotaxin-1, -2 or -3), PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase; an enzyme of the cathepsin family, a cell adhesion molecules such as PEC AM-1, a soluble or cell- or virus-bound receptor, cytokine-induced neutrophil chemoattractant (KC), TNF-a and IFN-g), 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, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16,CXCL17, XCL1, XCL2, CX3CL1, thrombin, urokinase, a vaccine or any combination thereof.

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

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

27. The peptide delivery system of claim 25, wherein the linker is selected from the linkers set forth in Table 4.

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

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

30. The peptide delivery system of claim 1, which comprises two or more sheddase cleavage sites, and each cleavage site can be the same or different from any of the other cleavage sites.

31. The peptide delivery system of claim 1, which comprises an intracellular domain and the intracellular domain comprises a stalk domain of CD45 phosphatase.

32. The peptide delivery system of claim 1, comprising a single domain antibody or single chain of an antibody which binds the self-associated antigen (S AA) to bring the SAA in proximity, e.g., in cis or in trans.

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

34. The peptide delivery system of claim 1, wherein the peptide payload is any peptide or protein that serves a function within the human body, including hormones, cytokines, chemokines, pro-drugs, and antibodies in various formats, such as chimeric antigen receptors (CARs), bi-specific killer cell engager (BIKE), tri-specific killer cell engager (TRIKE), etc.

35. The peptide delivery system of claim 1, wherein the peptide delivery system is selected from: CD63-MMP2-a-CD16-a-CD33-Myc-MMP9-CD63-GPi (SEQ ID NO: 124), CD63- MMP2-a-CD16-a-CD33-Myc-MMP9-CytCD63-EV Motif (SEQ ID NO: 155), CD63- MMP2-a-CD16-a-CD33-Myc-MMP9-CytCD63 (SEQ ID NO: 36 or 188), TNFSF14-a- CD16-a-CD33-Myc-TNRlA-ADAM17-a-CD38-41BB (SEQ ID NO: 54 or 366 or 367),CD63-GrzB-a-CD16-a-CD33-Myc-GrzB-a-CD38-CD28-CD3z (SEQ ID NO: 71 or 252), CD63-MMP9-2L-TNFa-Myc-MMP9-CD63-Cyt (SEQ ID NO: 88 or 295), CD63-MMP9- 2L-TNFa-Myc-MMP9-CD63-D-Cyt (Lacks EVs sorting motif) (SEQ ID NO: 103 or 311), CD63-DLl-MMP2-2L-TNFa-Myc-MMP2-CD63-Cyt (SEQ ID NO: 104 or 368), CD63- MMP9-2L-TNFa-Myc-MMP9-aCD19CAR (SEQ ID NO: 105 or 369), CD63-MMP2-2L- TNFa-Myc-MMP2-CD63-D-Cyt (SEQ ID NO: 106)or 370 or 371, CD63-MMP2-2L- TNFa-Myc-MMP2-aCD19CAR (SEQ ID NO: 107 or 372 o4 373), CD69cyt-TMII-TNFa- Myctag-CD28TM-cyt (SEQ ID NO: 119 or 370), CD69cyt-TMII-MMP9-TNFa-Myc- MMP9-CD28TM-cyt (SEQ ID NO: 120 or 377), CD69Cyt-TM-GrzB-TNFa-Myc-GrzB-a- CD19sc-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, GAGE A) (SEQ ID NO: 109 or 375), CD69Cyt-TM-GrzB (Original)-nLuc-Myc-GrzB(Original)-a-CD19sc-Stalk-CD28-CD3z (SEQ ID NO: 358 or 382), CD69Cyt-TM-GrzB(IEPD)-nLuc-Myc-GrzB(IEPD)-a-CD19sc- Stalk-CD28-CD3z (SEQ ID NO: 110 or 383), CD69Cyt-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 of any of the preceding claims, which comprises a cell into which the exogenous polypeptide has been anchored.

37. The peptide delivery system of claim 36, wherein the cell is selected from a Hematopoietic Stem Cells (HSC), an induced Pluripotent Stem Cell (iPSC) and their derived cell products, an Adoptive T cell, a Dendritic cell (DC), Natural killer (NK) cells, or any therapeutic immune or non-immune cells.

38. An adoptive cell or engineered immune cell comprising the peptide payload of claim 1, wherein the immune cells are autologous or allogeneic.

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

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

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

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

43. A method of delivering a peptide to a target locus in a patient in need thereof, the method comprising: administering to the patient a peptide delivery system comprising a lipid- comprising vescicle (e.g., cell, extracellular vesicle, lipid nanoparticle) into which an exogenous polypeptide has been anchored, the exogenous polypeptide comprising: a peptide payload; a first and a second transmembrane region; the first transmembrane region comprising a transmembrane domain connected to at least one sheddase or other proteolytic cleavage site; the second transmembrane region comprising a transmembrane domain connected to at least one sheddase or other proteolytic cleavage site; and optionally, a targeting moiety for targeting the exogenous polypeptide to a target locus, wherein when the lipid-comprising vescicle arrives at the target locus, a sheddase and / or other protease cleaves the cleavage sites of the first and second transmembrane regions releasing the peptide payload.

44. The method of claim 43, wherein the cleaveage sites are derived from proteolytic cleavage substrates naturally present in Glycosylphosphatidylinositol (GPi) anchored proteins, membrane proteins with a single, two, three, four, or more transmembrane domains, intracellular domains, or extracellular domains in the extracellular matrix or in neighboring cells.

45. The method of claim 43, wherein the sheddases are selected from full- or part-time sheddases.

46. The method of claim 45, wherein the full-time sheddases are selected from ADAM proteases, BACE proteases, serine-protease Granzyme-B, and Site-1 proteases.

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

48. The method of claim 45, wherein part-time sheddases are selected from: Meprin B, MT- MMPs (Membrane Type Matrix Metalloproteases), Pro-protein convertases, transmembrane serine proteases, Matrix Metalloproteases, Legumain and Cathepsin S and L, wherein the Meprin B is a membrane-anchored type 1 metalloprotease, and wherein the MT-MMPs are membrane-anchored type 1 or GPI-anchored, and include MT1 -MMP, MT2- MMP, MT3-MMP, MT4-MMP, MT5-MMP, and MT6-MMP.

49. The method of claim 48, wherein the Pro-protein convertases are selected from PCSK1 / 3, KCSK2, Furin, PCSK4, PSCK5 / 6, PACE4, PCSK7, and PCSK9.

50. The method of claim 48, wherein the transmembrane serine proteases are selected from membrane-anchored type II proteases, including, for example, Matriptase, Matriptase-2, Matriptase-3, Polymerase-1, Corin, Hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, Spinesin, Enteropeptidase, HAT, DESCL1, TMPRSS11 A, HAT -like 4 and HAT -like 5.

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

52. The method of claim 51, wherein the Matrix Metalloproteases are selected from MMP2, MMP9, and / or MMP25.

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

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

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

56. The method of claim 43, wherein the transmembrane 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, Integrins, TNFSF14, TNR1A, Aquaporins, NOTCH, NgRl, NRG1, GPi anchor, EGFR, and Rhodopsin.

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

58. The method of claim 43, wherein peptide delivery system further comprises a sheddase, and cleavage occurs at one or more of the cleavage sites by the peptide delivery system sheddase.

59. The method of claim 43, wherein an intracellular or extracellular domain of the exogenous polypeptide is selected from an intracellular or extracllular domain from one or more of the following: OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, B7-1 (CD80), B7-2 (CD86), PDL-1 , Programmed Death-I (PD- 1), Inducible T cell Co-Stimulator (ICOS), lymphocyte Function-associated Antigen-I (LFA-1 (CDI la / CD 18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, 1g alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), The costimulatory ligand ( PD-L2, 4-1 BBL, OX40L, Inducible Co-StimulatoryLigand (ICOS-L), Inter-Cellular Adhesion Molecule (ICAM, CD30L, CD70, CD83, HLA- G, MICA, Ml CB, HVEM, lymphotoxin b receptor, 3 / TR6, ILT3, ILT4), activating NK cell receptors, BTLA, a 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, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, IT GAM, CD1 lb, ITGAX, CD1 le, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, a ligand that specifically binds with CD83, or any combination thereof.

60. The method of claim 43, wherein the targeting moiety binds BCMA, MUC16 (also known as CA125), EGFR, EGFRvIII, MUCI, Flt-3, WT-1, CD38, CD70, CD90, CD133, MHC- WTI , TSPANIO, MHC-PRAME, MHC-NY-ESOI, HER2 (ERBB2), CA-IX (Carbonic anhydrase IX), LIVI, ADAMIO, CHRNA2, LeY, NKG2D, CSI, CD44v6, CD24, LGR5, ALDH, ALDH1, CD54, Seal, CD271, CD123, CD36, CD109, CD110, CD71 -negative, CCA, ABCG2, Claudin-18.2 (Claudin-18A2, or Claudinl8 isoform 2), PSCA, DLL3 (Deltalike protein 3, Drosophila Delta homolog 3, Delta3 ), Mud 7 (Mucinl 7, Muc3, Muc3), FAP alpha (Fibroblast Activation 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 scatter factor receptor kinase, IGF-1 receptor, IGF- I, IgGI, LLCAM, integrin a5pi, integrin avP3, legumain, MORAb-009, MS4A1, MUCI, mucin CanAg, C-MET, CCR4, CD 152, CD 10, CD 19, CD20, CD200, N-glycolylneuraminic acid, NPC-IC, PDGF-R a, PDL192, phosphatidylserine, tumor antigen CTAA16.88, VEGF- A, VEGFR-1, VEGFR2, vimentin, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF-P, TRAIL-R1, TRAIL-R2, folic acid receptor, transferrin receptors, and any combination thereof.

61. The method of claim 43, wherein the first and / or second transmembrane region comprises (i) an intracellular region comprising an intracellular region sequence from 4 IBB, ICOS, orCD3^, and (ii) a GrzB cleavage site; and wherein the action of kinases on the intracellular region sequence from 41BB, ICOS, or CD3(^ induces the release of the GrzB allowing it to be transported out of the cell and cleave GrzB cleavage sites, thereby releasing the peptide payload, the peptide payload optionally comprising a bispecific antibody.

62. The method of claim 61, wherein the second transmembrane region comprises a transmembrane 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 milieu.

63. The method of claim 43, wherein the peptide payload comprises: a cytokine or chemokine consisting a fraction or derivative of a sulfated xylan, which is an antagonist of a ligand selected from the list comprising IL-la, IL-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-11, IL-12, IL-3, IL-14, IL-15 or IL-17 family of cytokines including IL-25, interferon, G-CSF, M-CSF, GM-CSF, BDNF, CNTF, EGF, EPO, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, LIF, PDGF, SCF, TGFa, TGFB, TNFa, TNFB, TPO, VEGF, GH, NGF, NT3, NT4, NT5, NT6, NT7, Oncostatin M (OSM), insulin, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, a member of the MIP-1 family including MIP-la, MIP-2, eotaxin ( eotaxin-1, -2 or -3), , PBP (platelet basic protein), SDF-1, PBSF, PF4, RANTES, elastase; an enzyme of the cathepsin family, a cell adhesion molecules such as PECAM-1, a soluble or cell- or virus-bound receptor, cytokine- induced neutrophil chemoattractant (KC), TNF-a and IFN-g), 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, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, thrombin, urokinase, a vaccine or any combination thereof.

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

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

66. The method of Claim 64, wherein the linker is selected from Table 4.

67. The method of claim 43, wherein the exogenous polypeptide can also be configure for sorting specifically into extracellular vesicles (EVs).

68. The method of claim 43, wherein the peptide payload comprises two or more bispecific engagers, and the bispecific engagers are released intracellularly or extracell ularly.

69. The method of claim 43, wherein peptide delivery system comprises sheddase cleavage sites, and each cleavage site can be the same or different from any of the other cleavage sites.

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

71. The method of claim 43, wherein the peptide delivery system comprising a single domain antibody or single chain of an antibody which binds the self-associated antigen (SAA) to bring the SAA in proximity, e.g., in cis or in trans.

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

73. The method of claim 43, wherein the peptide payload is any peptide that serves a function within the body is a potential therapeutic, including hormones, cytokines, chemokines, prodrugs, and antibodies in various formats, such as chimeric antigen receptors (CARs), bispecific killer cell engager (BIKE), tri-specific killer cell engager (TRIKE), etc.

74. The method of claim 43, wherein the peptide delivery system is selected from: CD63- MMP2-a-CD16-a-CD33-Myc-MMP9-CD63-GPi (SEQ ID NO: 124), CD63-MMP2-a- CD16-a-CD33-Myc-MMP9-CytCD63-EV Motif (SEQ ID NO: 155), CD63-MMP2-a- CD16-a-CD33-Myc-MMP9-CytCD63 (SEQ ID NO: 36 or 188), TNFSF14-a-CD16-a- CD33-Myc-TNRlA-ADAM17-a-CD38-41BB (SEQ ID NO: 54 or 366 or 367), CD63- GrzB-a-CD16-a-CD33-Myc-GrzB-a-CD38-CD28-CD3z (SEQ ID NO: 71 or 252), CD63- MMP9-2L-TNFa-Myc-MMP9-CD63-Cyt (SEQ ID NO: 88 or 295), CD63-MMP9-2L- TNFa-Myc-MMP9-CD63-D-Cyt (Lacks EVs sorting motif) (SEQ ID NO: 103 or 311), CD63-DLl-MMP2-2L-TNFa-Myc-MMP2-CD63-Cyt (SEQ ID NO: 104 or 368), CD63- MMP9-2L-TNFa-Myc-MMP9-aCD19CAR (SEQ ID NO: 105 or 369), CD63-MMP2-2L- TNFa-Myc-MMP2-CD63-D-Cyt (SEQ ID NO: 106 or 370 or 371), CD63-MMP2-2L-TNFa-Myc-MMP2-aCD19CAR (SEQ ID NO: 107 or 372 or 373), CD69cyt-TMII-TNFa- Myctag-CD28TM-cyt (SEQ ID NO: 119 or 370), CD69cyt-TMII-MMP9-TNFa-Myc- MMP9-CD28TM-cyt (SEQ ID NO: 120 or 377), CD69Cyt-TM-GrzB-TNFa-Myc-GrzB-a- CD19sc-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, GAGE A) (SEQ ID NO: 109 or 375), CD69Cyt-TM-GrzB, (Original)-nLuc-Myc-GrzB(Original)-a-CD19sc-Stalk-CD28-CD3z (SEQ ID NO: 358 or 382), CD69Cyt-TM-GrzB(IEPD)-nLuc-Myc-GrzB(IEPD)-a-CD19sc- Stalk-CD28-CD3z (SEQ ID NO: 110 or 383), CD69Cyt-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).

75. The method of claim 43, wherein the lipid-comprising vesicle comprises a cell, and the cell is selected from a Hematopoietic Stem Cells (HSC), induced Pluripotent Stem Cell (iPSC) and a derived cell product, an Adoptive T cell, a Dendritic cell (DC), a Natural killer (NK) cell, or any therapeutic immune cell or non-immune cell.

76. The method of Claim 75, wherein the immune cell is autologous or allogeneic.

77. The method of Claim 43, wherein the peptide delivery system is introduced into a cell individually in the form of viral or nonviral vectors, mRNAs, peptides, proteins, antibodies, nanobodies, oligonucleotides, or extracellular vesicles (EVs).

78. The method of Claim 43, wherein the peptide delivery system is delivered using CPPs, micelle, liposomes, nanoparticles, dendrimer, nanotube, electroporation, viral transduction, nucleofection, transfection, cell fusion or microinjection.

Images