Extracellular vesicle comprising a fusion protein having fc binding capacity

Abstract

The present invention pertains to extracellular vesicle (EV) therapeutics, wherein the EVs are coated with proteins containing Fc domains (such as antibodies) for i.a. targeting and therapeutic applications. The coating of EVs is achieved through inventive protein engineering of EV polypeptides. The present invention thus relates to methods for coating of EVs, EVs per se, as well as pharmaceutical compositions and medical applications of such EVs coated with Fc containing proteins.

1 . An EV comprising at least one fusion protein, wherein the at least one fusion protein comprises at least one Fc binding polypeptide fused to at least one exosomal polypeptide. 
     
     
         2 . The EV according to  claim 1 , wherein the at least one Fc binding polypeptide is selected from the group comprising Protein A, Protein G, Protein A/G, Protein L, Protein LG, Z domain, ZZ domain, human FCGRI, human FCGR2A, human FCGR2B, human FCGR2C, human FCGR3A, human FCGR3B, human FCGRB, human FCAMR, human FCERA, human FCAR, mouse FCGRI, mouse FCGRIIB, mouse FCGRIII, mouse FCGRIV, mouse FCGRn, SPH peptide, SPA peptide, SPG2, SpA mimic 1, SpA mimic 2, SpA mimic 3, SpA mimic 4, SpA mimic 5, SpA mimic 6, SpA mimic 7, SpA mimic 8, SpA mimic 9, SpA mimic 10, Fcγ mimic 1, Fcγ mimic 2, and any combination thereof. 
     
     
         3 . The EV according to any one of the preceding claims, wherein the at least one Fc binding polypeptide comprises more than one Fc binding region. 
     
     
         4 . The EV according to any one of the preceding claims, wherein the at least one exosomal polypeptide is selected from the group comprising CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, ALIX, Syntenin-1, Syntenin-2, Lamp2b, TSPAN8, TSPAN14, CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d/ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18/ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, Fc receptors, interleukin receptors, immunoglobulins, CD2, CD3 epsilon, CD3 zeta, CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA, CD47, CD86, CD110, CD111, CD115, CD117, CD125, CD135, CD184, CD200, CD279, CD273, CD274, CD362, COL6A1, AGRN, EGFR, GAPDH, GLUR2, GLUR3, HLA-DM, HSPG2, L1CAM, LAMB1, LAMC1, LFA-1, LGALS3BP, Mac-1 alpha, Mac-1 beta, MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD, TCRG, VTI1A, VTI1B, other exosomal polypeptides, and any combinations thereof. 
     
     
         5 . The EV according to any one of the preceding claims, wherein the at least one Fc binding polypeptide is displayed on the outer surface of the EV. 
     
     
         6 . The EV according to any one of the preceding claims, wherein the at least one Fc binding polypeptide is bound to at least one Fc containing protein. 
     
     
         7 . The EV according to any one of the preceding claims, wherein the EV has bound to it a plurality of Fc containing proteins through interaction between the Fc binding polypeptide and the Fc domains of the plurality of Fc containing proteins, wherein the plurality of Fc containing proteins may be the same or different. 
     
     
         8 . The EV according to any one of the preceding claims, wherein the at least one Fc containing protein is an antibody and/or a protein engineered to comprise an Fc domain. 
     
     
         9 . The EV according to  claim 8 , wherein the antibody is a targeting antibody, a therapeutic antibody, an antibody-drug conjugate (ADC), or an antibody for reducing opsonization and/or immune cell-mediated clearance. 
     
     
         10 . The EV according to any one of  claims 6  to  9 , wherein the EV having bound to it at least one Fc containing protein is having bound to it at least 10, preferably at least 20, even more preferably at least 30 Fc containing proteins. 
     
     
         11 . The EV as claimed in any one of  claims 6 - 10 , wherein the at least one Fc containing protein is present inside the EV, in the lipid membrane of the EV and/or attached to the outer surface of the EV. 
     
     
         12 . A non-covalent complex between (i) a fusion protein, and (ii) an Fc containing protein, characterized in that the fusion protein comprises at least one Fc binding polypeptide fused to at least one exosomal polypeptide, wherein the Fc binding polypeptide binds to the Fc containing protein. 
     
     
         13 . The non-covalent complex according to  claim 12 , wherein the non-covalent complex is present in the lipid membrane of an EV and/or inside an EV. 
     
     
         14 . A pharmaceutical composition comprising EVs according to any one of  claims 1  to  11 , and/or the non-covalent complexes according to any one of  claims 12  to  13 , and optionally a pharmaceutically acceptable carrier. 
     
     
         15 . The EVs according to any one of  claims 1  to  11 , and/or the non-covalent complexes according to any one of  claims 12  to  13 , and/or the pharmaceutical composition according to  claim 14 , for use in medicine. 
     
     
         16 . A method for attaching to an extracellular vesicle (EV) at least one Fc containing protein, comprising the steps of:
 (i) providing an EV comprising a fusion protein comprising at least one Fc binding polypeptide and at least one exosomal polypeptide;   (ii) allowing the Fc binding polypeptide of the fusion protein to bind the Fc domain of at least one Fc containing protein.   
     
     
         17 . A polypeptide construct comprising a fusion protein comprising at least one Fc binding polypeptide and at least one exosomal polypeptide. 
     
     
         18 . A polynucleotide construct encoding for at least one polypeptide construct of  claim 17 . 
     
     
         19 . A cell comprising at least one polypeptide construct according to  claim 17 , at least one polynucleotide construct according to  claim 18 , at least one EV according to any one of  claims 1 - 11  and/or at least one non-covalent complex according to any one of  claims 12 - 13 . 
     
     
         20 . A method for delivery of at least one Fc containing protein to a target cell, comprising (i) attaching the Fc domain of the at least one Fc containing protein to an Fc binding polypeptide comprised in an EV, and (ii) contacting the EV, having attached to it the at least one Fc containing protein, with the target cell. 
     
     
         21 . The method according to  claim 20 , wherein the method is carried out in vitro, in vivo, and/or ex vivo. 
     
     
         22 . The method according to any one of  claims 20 - 21 , wherein the Fc binding polypeptide of the EV is part of a fusion protein with at least one exosomal polypeptide. 
     
     
         23 . The method according to any one of  claims 20 - 22 , wherein the Fc containing protein is delivered into a target cell. 
     
     
         24 . A method of treating a disease or disorder comprising administering to a subject a therapeutically effective amount of an EV according to  claim 1  and a pharmaceutically acceptable excipient. 
     
     
         25 . A method of treating a disease or disorder comprising administering to a subject a therapeutically effective amount of a non-covalent complex according to  claim 12  and a pharmaceutically acceptable excipient. 
     
     
         26 . A method of treating a disease or disorder comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition according to  claim 14  and a pharmaceutically acceptable excipient. 
     
     
         27 . A cell comprising at least one polynucleotide construct according to  claim 18 . 
     
     
         28 . A cell comprising at least one EV according to  claim 1 . 
     
     
         29 . A cell comprising at least one non-covalent complex according  claim 12 .