Peptides, nanovesicles and uses thereof for drug delivery

Abstract

The present disclosure relates to polypeptides, particularly polypeptides comprising an Eph receptor domain, i.e., Eph receptor-derived polypeptides, nanovesicles comprising such polypeptides, e.g., extracellular vesicles (EVs) and hybridosomes. The polypeptides can act as membrane-bound protein scaffolds to which molecules of interest can be attached. The polypeptides and nanovesicles can be used in targeting, therapeutic and / or diagnostic applications. Also provided are nucleic acids and expression vectors encoding such polypeptides, as well as cells expressing the polypeptides. Further provided are methods of producing nanovesicles comprising such polypeptides. Compositions comprising such polypeptides or nanovesicles and uses thereof are also described.

Description

[Technical Field]

[0001] (Priority) This application claims the benefit of priority to U.S. Patent Application No. 63 / 174,874, filed April 14, 2021, which is incorporated herein by reference in its entirety.

[0002] (Reference to electronically submitted sequence listing) This application incorporates by reference the Sequence Listing submitted herewith as a text file entitled "14497-007-228_Sequence_Listing.txt", created on April 11, 2022, and having a size of 444,261 bytes.

[0003] (1. Field) The present disclosure relates to polypeptides (particularly polypeptides comprising Eph receptor domains, i.e., Eph receptor-derived polypeptides), nanovesicles (e.g., extracellular vesicles (EVs) and hybridosomes) comprising such polypeptides. The polypeptides can serve as membrane-bound protein scaffolds onto which molecules of interest can be attached. The polypeptides and nanovesicles can be used in targeting, therapeutic, and / or diagnostic applications. Also provided are nucleic acids and expression vectors encoding such polypeptides, and cells expressing the polypeptides. Further provided are methods for producing nanovesicles comprising such polypeptides. Compositions comprising such polypeptides or nanovesicles and uses thereof are also described. [Background technology]

[0004] (2.Background) Despite major breakthroughs in identifying promising new drug candidates, translating these findings into the clinic is often hindered by challenges in delivering effective drug dosages to disease sites. Recently discovered intercellular communication pathways may provide the missing puzzle piece for more precise drug delivery. It has become clear that almost all cells in the body can establish connections with neighboring cells as well as distant cells through the release of small "balloons" called extracellular vesicles (EVs). The discovery that these EVs, particularly exosomes, are functional shuttles for signaling molecules has led to the idea that they could be ideal nanoscale candidates for drug delivery systems in modern pharmaceuticals. However, this idea faces several challenges. Therefore, suitable methods and compositions for generating, isolating, and purifying EVs are needed to improve the therapeutic and other applications of EV-based technologies.

[0005] Several strategies are under development to customize EV surfaces and cargoes to enable the loading of pharmaceuticals into EVs and the decoration of EV surfaces with tissue-targeting ligands. Bioengineering EV producer cells to enable the sorting of proteins of interest into EVs is a hot topic. In particular, utilizing EV-associated proteins endogenously involved in EV biogenesis and vesicular protein sorting has become a focus of this field. A wide range of methods have been applied to identify EV-associated proteins for many years, and mass spectrometry (MS)-based proteomics has proven extremely useful. For a long time, highly enriched endogenous proteins (so-called "EV markers") and proteins associated with such markers have been identified by combining proteomic data from purified EVs and used as sorting scaffolds for loading fusion proteins into EVs. Ubiquitous and highly expressed protein markers that have long been used as sorting proteins include tetraspanin molecules (e.g., CD63, CD81, CD9, etc.), lysosome-associated membrane protein 2 (LAMP2 and LAMP2B), platelet-derived growth factor receptor (PDGFR), GPI-anchored proteins, lactadherin, prostaglandin F2 receptor negative regulator, ubiquitin C, syntenin, syndecans, and Alix (see review by Shi et al., 2020, Methods 177:95-102 (published online September 27, 2019)).

[0006] However, significant differences in the sorting of endogenous and bioengineered proteins have been reported. A recent quantitative comparison of the most commonly used sorting proteins for EV bioengineering showed that, among all EV-associated target proteins screened, fusion proteins overexpressing GFP and the tetraspanins CD9, CD81, and CD63, as well as myristoylation domains, showed the highest abundance in EVs (Corso et al., 2019, 8(1):1663043). However, not all endogenous marker proteins enriched in EVs identified by Western blotting or MS proteomics correlated with or led to efficient vesicular sorting of the corresponding GFP fusion proteins. Furthermore, GFP-EV marker fusion proteins showed highly heterogeneous sorting efficiencies into EVs despite similar overexpression levels in parental cells. A notable example is the widely used EV marker Alix, which was barely detectable in EVs when overexpressed as a fusion protein with GFP. Furthermore, although the CD63-GFP fusion protein was confirmed to be more abundant on EVs, significant differences in loading efficiency were observed depending on whether GFP was attached to the N-terminus, C-terminus, or second loop of CD63.

[0007] Thus, there is a need in the art for bioengineered proteins that are easily and efficiently incorporated into EVs, and more generally, into nanovesicles, preferably at high density.

[0008] The citation of any reference herein should not be construed as an admission that it is prior art to the present disclosure. Summary of the Invention

[0009] 3. SUMMARY OF THE DISCLOSURE In one aspect, provided herein is an extracellular vesicle (EV) comprising a polypeptide, the polypeptide comprising, from N-terminus to C-terminus: a. an ephrin receptor cysteine-rich (CR) domain; b. a first ephrin receptor fibronectin type III (FN III) domain and a second ephrin receptor FN III domain; and c. a transmembrane (TM) domain; wherein the polypeptide lacks (i) ephrin-binding activity, (ii) ephrin receptor kinase activity, or (iii) both ephrin-binding activity and ephrin receptor kinase activity. In some embodiments, the polypeptide lacks ephrin-binding activity.

[0010] In another aspect, provided herein is a hybridosome comprising a polypeptide, the polypeptide comprising, from N-terminus to C-terminus: a. an ephrin receptor CR domain; b. a first ephrin receptor FN III domain and a second ephrin receptor FN III domain; and c. a TM domain; wherein the polypeptide lacks (i) ephrin-binding activity, (ii) ephrin receptor kinase activity, or (iii) both ephrin-binding activity and ephrin receptor kinase activity. In certain embodiments, the polypeptide lacks ephrin-binding activity.

[0011] In various embodiments, the polypeptide further comprises a targeting domain at the N-terminus of the ephrin receptor CR domain. In certain embodiments, the targeting domain is selected from the group consisting of: scFv, (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Fd fragment, diabody, F(ab')3, disulfide-linked Fv, sdAb (VHH or nanobody), CDR, di-scFv, bi-scFv, tascFv (tandem scFv), triabody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, DARPin, centirin, affibody, affilin, affitin, anticalin, avimer, fynomer, Kunitz domain peptide, monobody (or adnectin), tribody, and nanophytin. In certain embodiments, the targeting domain specifically binds to a marker. In a specific embodiment, the marker is a tumor-associated antigen.In specific embodiments, the tumor-associated antigen is human epidermal growth factor receptor 2 (HER2), CD20, CD33, B-cell maturation antigen (BCMA), prostate-specific membrane antigen (PSMA), DLL3, ganglioside GD2 (GD2), CD123, anoctamin-1 (Ano1), mesothelin, carbonic ananhydrase IX (CAIX), tumor-associated calcium signaling receptor 2 (TROP2), carcinoembryonic antigen (CEA), claudin-18.2, receptor tyrosine kinase-like orphan receptor 1 (ROR1), trophoblast glycoprotein (5T4), glycoprotein non-metastatic melanoma protein B (GPNMB), folate receptor-alpha (FR-α), pregnancy-associated plasma protein A. (PAPP-A), CD37, epithelial cell adhesion molecule (EpCAM), CD2, CD19, CD30, CD38, CD40, CD52, CD70, CD79b, fms-like tyrosine kinase 3 (FLT3), glypican 3 (GPC3), B7 homolog 6 (B7H6), CC chemokine receptor type 4 (CCR4), C-X-C motif chemokine receptor 4 (CXCR4), receptor tyrosine kinase-like orphan receptor 2 (ROR2), CD133, HLA class I histocompatibility antigen, alpha chain E (HLA-E), epidermal growth factor receptor (EGFR / ERBB-1), insulin-like growth factor 1-receptor (IGF1R), and human epidermal growth factor receptor 3.

[0012] In various embodiments, the polypeptide further comprises a cargo protein or cargo-binding domain C-terminal to the TM domain. In certain embodiments, the cargo protein or cargo-binding domain is fused to the remainder of the polypeptide via a linker. In certain embodiments, the cargo protein or cargo-binding domain is covalently fused to the remainder of the polypeptide via a linker. In specific embodiments, the linker is a peptide linker. In a specific embodiment, the peptide linker comprises the amino acid sequence of (GGGS)n (SEQ ID NO: 226), where n is an integer from 1 to 10. In another specific embodiment, the peptide linker comprises the amino acid sequence GGGS.

[0013] In various embodiments, the polypeptide comprises a cargo-binding domain that can bind to a cargo protein directly or indirectly via a scaffold-binding domain (SBD) linked to the cargo protein. In specific embodiments, the bond between the cargo-binding domain and the cargo protein is a non-covalent bond. In specific embodiments, the bond between the cargo-binding domain and the cargo protein is a reversible bond. In specific embodiments, the bond between the cargo-binding domain and the cargo protein can be controlled. In specific embodiments, the bond between the cargo-binding domain and the cargo protein can be controlled by pH. In another specific embodiment, the bond between the cargo-binding domain and the cargo protein can be controlled by ionic strength. In some embodiments, the bond between the cargo-binding domain and the cargo protein can be controlled so that the cargo protein binds to the cargo-binding domain in vitro but is released from the cargo-binding domain in vivo. In other embodiments, the bond between the cargo-binding domain and the cargo protein can be controlled so that the cargo protein is released from the cargo-binding domain in a manner that depends on the intracellular compartment in which it is located. In a specific embodiment, the cargo-binding domain comprises a phosphotyrosine, the cargo protein or SBD comprises a domain capable of binding to phosphotyrosine, and the bond between the cargo-binding domain and the cargo protein is a bond between the phosphotyrosine and the domain capable of binding to phosphotyrosine. In a specific embodiment, the domain capable of binding to phosphotyrosine is a phosphotyrosine-binding (PTB) domain. In another specific embodiment, the domain capable of binding to phosphotyrosine is a Src homology 2 (SH2) domain. In a specific embodiment, the cargo-binding domain comprises a first sterile alpha-motif (SAM) domain, the cargo protein or SBD comprises a second SAM domain, and the bond between the cargo-binding domain and the cargo protein is a bond between the first SAM domain and the second SAM domain.In a specific embodiment, the cargo-binding domain comprises a PDZ-binding motif (PBM) domain, the cargo protein or the SBD comprises a PDZ domain, and the bond between the cargo-binding domain and the cargo protein is the bond between the PBM domain and the PDZ domain. In a specific embodiment, the cargo-binding domain comprises a PDZ domain, the cargo protein or the SBD comprises a PBM domain, and the bond between the cargo-binding domain and the cargo protein is the bond between the PDZ domain and the PBM domain.

[0014] In various embodiments, the polypeptide is a cargo protein.

[0015] In various embodiments, the polypeptide can bind to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises an ephrin receptor JM domain C-terminal to the TM domain. In certain embodiments, the binding between the ephrin receptor JM domain and the cargo protein is a non-covalent bond. In certain embodiments, the binding between the ephrin receptor JM domain and the cargo protein is a reversible bond. In certain embodiments, the binding between the ephrin receptor JM domain and the cargo protein can be controlled. In a specific embodiment, the binding between the ephrin receptor JM domain and the cargo protein can be controlled by pH. In another specific embodiment, the binding between the ephrin receptor JM domain and the cargo protein can be controlled by ionic strength. In some embodiments, the binding between the ephrin receptor JM domain and the cargo protein can be controlled so that the cargo protein binds to the ephrin receptor JM domain in vitro but is released from the ephrin receptor JM domain in vivo. In other embodiments, the binding between the Ephrin receptor JM domain and a cargo protein can be controlled so that the cargo protein is released from the Ephrin receptor JM domain in a manner dependent on the intracellular compartment in which it is located. In certain embodiments, the Ephrin receptor JM domain contains phosphotyrosine, the cargo protein or SBD contains a domain capable of binding to phosphotyrosine, and the binding between the Ephrin receptor JM domain and the cargo protein is between the phosphotyrosine and the domain capable of binding to phosphotyrosine. In a specific embodiment, the domain capable of binding to phosphotyrosine is a PTB domain. In another specific embodiment, the domain capable of binding to phosphotyrosine is an SH2 domain.In specific embodiments, the ephrin receptor JM domain comprises: (i) an (X1)-Ptyr-(X2) motif (wherein Ptyr is phosphotyrosine, X1 is Y, P, V, I, T, or F, and X2 is I, V, L, or A); (ii) an (X3)-Ptyr-(X4) motif (wherein Ptyr is phosphotyrosine, X3 is T, A, or S, and X4 is E or G); or (iii) both (i) and (ii).

[0016] In various embodiments, the polypeptide can bind to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises an ephrin receptor KD at the C-terminus of the TM domain. In certain embodiments, the binding between the ephrin receptor KD and the cargo protein is a non-covalent bond. In certain embodiments, the binding between the ephrin receptor KD and the cargo protein is a reversible bond. In certain embodiments, the binding between the ephrin receptor KD and the cargo protein can be controlled. In a specific embodiment, the binding between the ephrin receptor KD and the cargo protein can be controlled by pH. In another specific embodiment, the binding between the ephrin receptor KD and the cargo protein can be controlled by ionic strength. In some embodiments, the binding between the ephrin receptor KD and the cargo protein can be controlled so that the cargo protein binds to the ephrin receptor KD in vitro but is released from the ephrin receptor KD in vivo. In other embodiments, the binding between the ephrin receptor KD and the cargo protein can be controlled so that the cargo protein is released from the ephrin receptor KD in a manner dependent on the intracellular compartment in which it is located. In certain embodiments, the ephrin receptor KD contains phosphotyrosine, the cargo protein or SBD contains a domain capable of binding to phosphotyrosine, and the binding between the ephrin receptor KD and the cargo protein is between the phosphotyrosine and the domain capable of binding to phosphotyrosine. In a specific embodiment, the domain capable of binding to phosphotyrosine is a PTB domain. In another specific embodiment, the domain capable of binding to phosphotyrosine is an SH2 domain. In a specific embodiment, the KD contains a (X7)-Ptyr-(X8) motif (wherein Ptyr is phosphotyrosine, X7 is T, V, or A, and X8 is E or T) in the activation loop.

[0017] In various embodiments, the polypeptide can bind to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises a SAM linker domain at the C-terminus of the TM domain. In certain embodiments, the bond between the SAM linker domain and the cargo protein is a non-covalent bond. In certain embodiments, the bond between the SAM linker domain and the cargo protein is a reversible bond. In certain embodiments, the bond between the SAM linker domain and the cargo protein can be controlled. In a specific embodiment, the bond between the SAM linker domain and the cargo protein can be controlled by pH. In another specific embodiment, the bond between the SAM linker domain and the cargo protein can be controlled by ionic strength. In some embodiments, the bond between the SAM linker domain and the cargo protein can be controlled so that the cargo protein binds to the SAM linker domain in vitro but is released from the SAM linker domain in vivo. In other embodiments, the bond between the SAM linker domain and the cargo protein can be controlled so that the cargo protein is released from the SAM linker domain in a manner dependent on the intracellular compartment in which it is located. In one embodiment, the SAM linker domain comprises a phosphorylated amino acid or a phosphomimetic amino acid, the cargo protein or SBD comprises a domain capable of binding to the phosphorylated amino acid or the phosphomimetic amino acid, and the bond between the SAM linker domain and the cargo protein is a bond between the phosphorylated amino acid or the phosphomimetic amino acid and the domain capable of binding to the phosphorylated amino acid or the phosphomimetic amino acid. In one embodiment, the SAM linker domain is an ephrin receptor SAM linker domain.

[0018] In various embodiments, the polypeptide can bind to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises a SAM domain at the C-terminus of the TM domain. In certain embodiments, the binding between the SAM domain and the cargo protein is a non-covalent bond. In certain embodiments, the binding between the SAM domain and the cargo protein is a reversible bond. In certain embodiments, the binding between the SAM domain and the cargo protein can be controlled. In a specific embodiment, the binding between the SAM domain and the cargo protein can be controlled by pH. In another specific embodiment, the binding between the SAM domain and the cargo protein can be controlled by ionic strength. In some embodiments, the binding between the SAM domain and the cargo protein can be controlled so that the cargo protein binds to the SAM domain in vitro but is released from the SAM domain in vivo. In other embodiments, the binding between the SAM domain and the cargo protein can be controlled so that the cargo protein is released from the SAM domain in a manner dependent on the intracellular compartment in which it is located. In some embodiments, the cargo protein or SBD comprises a second SAM domain, and the bond between the SAM domain and the cargo protein is the bond between the SAM domain and the second SAM domain. In some embodiments, the SAM domain comprises phosphotyrosine, the cargo protein or SBD comprises a domain capable of binding to phosphotyrosine, and the bond between the SAM domain and the cargo protein is the bond between the phosphotyrosine and the domain capable of binding to phosphotyrosine. In a specific embodiment, the domain capable of binding to phosphotyrosine is a PTB domain. In another specific embodiment, the domain capable of binding to phosphotyrosine is an SH2 domain. In a specific embodiment, the SAM domain comprises a phosphotyrosine in the α2 helix.In a specific embodiment, the phosphotyrosine in the α2 helix of the SAM domain is in a (X5)-Ptyr-(X6) motif, where Ptyr is phosphotyrosine, X5 is C, R, Q, or H, and X6 is Q, I, E, K, R, or T. In certain embodiments, the SAM domain is an ephrin receptor SAM domain.

[0019] In various embodiments, the polypeptide can bind to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises an ephrin receptor PDZ-binding motif (PBM) domain C-terminal to the TM domain. In certain embodiments, the binding between the ephrin receptor PBM domain and the cargo protein is a non-covalent bond. In certain embodiments, the binding between the ephrin receptor PBM domain and the cargo protein is a reversible bond. In certain embodiments, the binding between the ephrin receptor PBM domain and the cargo protein can be controlled. In a specific embodiment, the binding between the ephrin receptor PBM domain and the cargo protein can be controlled by pH. In another specific embodiment, the binding between the ephrin receptor PBM domain and the cargo protein can be controlled by ionic strength. In some embodiments, the binding between the ephrin receptor PBM domain and the cargo protein can be controlled so that the cargo protein binds to the ephrin receptor PBM domain in vitro but is released from the ephrin receptor PBM domain in vivo. In other embodiments, the binding between the Ephrin receptor PBM domain and the cargo protein can be controlled such that the cargo protein is released from the Ephrin receptor PBM domain in a manner that depends on the subcellular compartment they are located in. In certain embodiments, the cargo protein or SBD comprises a PDZ domain, and the binding between the Ephrin receptor PBM domain and the cargo protein is binding between the Ephrin receptor PBM domain and the PDZ domain.

[0020] In various embodiments, the cargo protein is a therapeutic protein. In a specific embodiment, the therapeutic protein is a therapeutic antibody or an antigen-binding fragment thereof. In another specific embodiment, the therapeutic protein is a gene editor or a transposase. In various embodiments, the cargo protein is a diagnostic protein. In a specific embodiment, the diagnostic protein is a fluorescent protein.

[0021] In various embodiments, the polypeptide lacks an ephrin receptor ligand binding domain (LBD). In various embodiments, the polypeptide comprises a mutated ephrin receptor LBD.

[0022] In various embodiments, a polypeptide comprises two distinct domains that allow the polypeptide to undergo heterodomain dimerization with another polypeptide identical to the polypeptide. In certain embodiments, a polypeptide comprises two distinct domains in a head-to-tail configuration that allow the polypeptide to undergo heterodomain dimerization with another polypeptide identical to the polypeptide.

[0023] In various embodiments, the TM domain is an ephrin receptor TM domain.

[0024] In various embodiments, any one or more of the ephrin receptor domains of the polypeptide are from or derived from EphAl, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphAlO, EphBl, EphB2, EphB3, EphB4, EphB6, or a combination thereof. In certain embodiments, any one or more of the ephrin receptor domains of the polypeptide are from or derived from EphA2, EphA4, EphB2, or a combination thereof.

[0025] In various embodiments, the polypeptide further comprises a modified Fc domain of an immunoglobulin. In certain embodiments, the modified Fc domain is at the N-terminus of the ephrin receptor CR domain. In specific embodiments, the modified Fc domain is fused to the remainder of the polypeptide via a linker sequence. In certain embodiments, the modified Fc domain a. is capable of specifically binding to the Fc-binding site of the neonatal Fc receptor (FcRn); and b. lacks the ability to form homodimers. In certain embodiments, the dissociation constant of the modified Fc domain bound to FcRn at a pH of 6.5 is at most 10 -4 In certain embodiments, the dissociation constant of the modified Fc domain bound to FcRn at a pH of 7.4 is at least 10 -4 In a specific embodiment, the modified Fc domain has the amino acid sequence [ka] (wherein X1, X2, X3, X4, X5, X6, X7, and X8 are each any amino acid). In a specific embodiment, the modified Fc domain is capable of specifically binding to an amino acid sequence between positions 135 and 158 of human FcRn (SEQ ID NO: 228) and / or mouse FcRn (SEQ ID NO: 227). In certain embodiments, the polypeptide does not substantially bind to C1q, FcγRI, FcγRII, or FcγRIII. In certain embodiments, the complement-dependent cytotoxicity (CDC) activity of the modified Fc domain, the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the modified Fc domain, the antibody-dependent cell-mediated phagocytosis (ADCP) activity of the modified Fc domain, and / or the antibody-dependent intracellular neutralization (ADIN) activity of the modified Fc domain is reduced by at least 10%, 20%, 30%, 40%, or 50% compared to the unmodified Fc domain. In certain embodiments, the complement-dependent cytotoxicity (CDC) activity of the modified Fc domain, the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the modified Fc domain, the antibody-dependent cell-mediated phagocytosis (ADCP) activity of the modified Fc domain, and / or the antibody-dependent intracellular neutralization (ADIN) activity of the modified Fc domain is reduced by at least 1.5, 2, 3, 4, or 5-fold compared to an unmodified Fc domain. In certain embodiments, the modified Fc domain comprises, from the N-terminus to the C-terminus: a. a modified CH2 domain that has been modified to have reduced effector function compared to an unmodified CH2 domain; and b. a modified CH3 domain that has been modified to lack the ability to form homodimers.

[0026] In various embodiments, the first Ephrin receptor FN III domain and the second Ephrin receptor FN III domain comprise different amino acid sequences.

[0027] In another aspect, provided herein is a method for delivering a therapeutic or diagnostic agent to a target cell or tissue, comprising providing the target cell or tissue with an extracellular vesicle or hybridosome described herein.

[0028] In another aspect, provided herein is a polypeptide comprising, from N-terminus to C-terminus: a. a targeting domain; b. an ephrin receptor CR domain; c. a first ephrin receptor FN III domain and a second ephrin receptor FN III domain; and d. a TM domain. In certain embodiments, the polypeptide lacks ephrin-binding activity. In certain embodiments, the targeting domain is selected from the group consisting of: scFv, (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Fd fragment, diabody, F(ab')3, disulfide-linked Fv, sdAb (VHH or nanobody), CDR, di-scFv, bi-scFv, tascFv (tandem scFv), triabody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, DARPin, centirin, affibody, affilin, affitin, anticalin, avimer, fynomer, Kunitz domain peptide, monobody (or adnectin), tribody, and nanophytin. In certain embodiments, the targeting domain specifically binds to a marker. In a specific embodiment, the marker is a tumor-associated antigen.In specific embodiments, the tumor-associated antigen is human epidermal growth factor receptor 2 (HER2), CD20, CD33, B-cell maturation antigen (BCMA), prostate-specific membrane antigen (PSMA), DLL3, ganglioside GD2 (GD2), CD123, anoctamin-1 (Ano1), mesothelin, carbonic ananhydrase IX (CAIX), tumor-associated calcium signaling receptor 2 (TROP2), carcinoembryonic antigen (CEA), claudin-18.2, receptor tyrosine kinase-like orphan receptor 1 (ROR1), trophoblast glycoprotein (5T4), glycoprotein non-metastatic melanoma protein B (GPNMB), folate receptor-alpha (FR-α), pregnancy-associated plasma protein A. (PAPP-A), CD37, epithelial cell adhesion molecule (EpCAM), CD2, CD19, CD30, CD38, CD40, CD52, CD70, CD79b, fms-like tyrosine kinase 3 (FLT3), glypican 3 (GPC3), B7 homolog 6 (B7H6), CC chemokine receptor type 4 (CCR4), C-X-C motif chemokine receptor 4 (CXCR4), receptor tyrosine kinase-like orphan receptor 2 (ROR2), CD133, HLA class I histocompatibility antigen, alpha chain E (HLA-E), epidermal growth factor receptor (EGFR / ERBB-1), insulin-like growth factor 1-receptor (IGF1R), and human epidermal growth factor receptor 3.

[0029] In another aspect, provided herein is a polypeptide comprising, from N-terminus to C-terminus: a. an ephrin receptor CR domain; b. a first ephrin receptor FN III domain and a second ephrin receptor FN III domain; c. a TM domain; and d. a cargo protein or cargo-binding domain. In certain embodiments, the polypeptide lacks ephrin-binding activity.

[0030] In another aspect, provided herein is a polypeptide comprising, from N-terminus to C-terminus: a. a targeting domain; b. an ephrin receptor CR domain; c. a first ephrin receptor FN III domain and a second ephrin receptor FN III domain; d. a TM domain; and e. a cargo protein or cargo-binding domain. In certain embodiments, the polypeptide lacks ephrin-binding activity. In certain embodiments, the targeting domain is selected from the group consisting of: scFv, (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Fd fragment, diabody, F(ab')3, disulfide-linked Fv, sdAb (VHH or nanobody), CDR, di-scFv, bi-scFv, tascFv (tandem scFv), triabody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, DARPin, centirin, affibody, affilin, affitin, anticalin, avimer, fynomer, Kunitz domain peptide, monobody (or adnectin), tribody, and nanophytin. In certain embodiments, the targeting domain specifically binds to a marker. In a specific embodiment, the marker is a tumor-associated antigen.In specific embodiments, the tumor-associated antigen is human epidermal growth factor receptor 2 (HER2), CD20, CD33, B-cell maturation antigen (BCMA), prostate-specific membrane antigen (PSMA), DLL3, ganglioside GD2 (GD2), CD123, anoctamin-1 (Ano1), mesothelin, carbonic ananhydrase IX (CAIX), tumor-associated calcium signaling receptor 2 (TROP2), carcinoembryonic antigen (CEA), claudin-18.2, receptor tyrosine kinase-like orphan receptor 1 (ROR1), trophoblast glycoprotein (5T4), glycoprotein non-metastatic melanoma protein B (GPNMB), folate receptor-alpha (FR-α), pregnancy-associated plasma protein A. (PAPP-A), CD37, epithelial cell adhesion molecule (EpCAM), CD2, CD19, CD30, CD38, CD40, CD52, CD70, CD79b, fms-like tyrosine kinase 3 (FLT3), glypican 3 (GPC3), B7 homolog 6 (B7H6), CC chemokine receptor type 4 (CCR4), C-X-C motif chemokine receptor 4 (CXCR4), receptor tyrosine kinase-like orphan receptor 2 (ROR2), CD133, HLA class I histocompatibility antigen, alpha chain E (HLA-E), epidermal growth factor receptor (EGFR / ERBB-1), insulin-like growth factor 1-receptor (IGF1R), and human epidermal growth factor receptor 3.

[0031] In various embodiments, the cargo protein or cargo-binding domain is fused to the remainder of the polypeptide via a linker. In certain embodiments, the cargo protein or cargo-binding domain is covalently fused to the remainder of the polypeptide via a linker. In specific embodiments, the linker is a peptide linker. In a specific embodiment, the peptide linker comprises the amino acid sequence of (GGGS)n (SEQ ID NO: 226), where n is an integer from 1 to 10. In another specific embodiment, the peptide linker comprises the amino acid sequence GGGS.

[0032] In various embodiments, the polypeptide comprises a cargo-binding domain that can bind to a cargo protein directly or indirectly via an SBD linked to the cargo protein. In specific embodiments, the bond between the cargo-binding domain and the cargo protein is a non-covalent bond. In specific embodiments, the bond between the cargo-binding domain and the cargo protein is a reversible bond. In specific embodiments, the bond between the cargo-binding domain and the cargo protein can be controlled. In specific embodiments, the bond between the cargo-binding domain and the cargo protein can be controlled by pH. In another specific embodiment, the bond between the cargo-binding domain and the cargo protein can be controlled by ionic strength. In some embodiments, the bond between the cargo-binding domain and the cargo protein can be controlled so that the cargo protein binds to the cargo-binding domain in vitro but is released from the cargo-binding domain in vivo. In other embodiments, the bond between the cargo-binding domain and the cargo protein can be controlled so that the cargo protein is released from the cargo-binding domain in a manner that depends on the intracellular compartment in which it is located. In a specific embodiment, the cargo-binding domain comprises a phosphotyrosine, the cargo protein or SBD comprises a domain capable of binding to phosphotyrosine, and the bond between the cargo-binding domain and the cargo protein is a bond between the phosphotyrosine and the domain capable of binding to phosphotyrosine. In a specific embodiment, the domain capable of binding to phosphotyrosine is a phosphotyrosine-binding (PTB) domain. In another specific embodiment, the domain capable of binding to phosphotyrosine is a Src homology 2 (SH2) domain. In a specific embodiment, the cargo-binding domain comprises a first sterile alpha-motif (SAM) domain, the cargo protein or SBD comprises a second SAM domain, and the bond between the cargo-binding domain and the cargo protein is a bond between the first SAM domain and the second SAM domain.In a specific embodiment, the cargo-binding domain comprises a PDZ-binding motif (PBM) domain, the cargo protein or the SBD comprises a PDZ domain, and the bond between the cargo-binding domain and the cargo protein is the bond between the PBM domain and the PDZ domain. In a specific embodiment, the cargo-binding domain comprises a PDZ domain, the cargo protein or the SBD comprises a PBM domain, and the bond between the cargo-binding domain and the cargo protein is the bond between the PDZ domain and the PBM domain.

[0033] In various embodiments, the polypeptide is a cargo protein.

[0034] In various embodiments, the polypeptide can bind to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises an ephrin receptor JM domain at the C-terminus of the TM domain. In certain embodiments, the binding between the ephrin receptor JM domain and the cargo protein is a non-covalent bond. In certain embodiments, the binding between the ephrin receptor JM domain and the cargo protein is a reversible bond. In certain embodiments, the binding between the ephrin receptor JM domain and the cargo protein can be controlled. In a specific embodiment, the binding between the ephrin receptor JM domain and the cargo protein can be controlled by pH. In another specific embodiment, the binding between the ephrin receptor JM domain and the cargo protein can be controlled by ionic strength. In some embodiments, the binding between the ephrin receptor JM domain and the cargo protein can be controlled so that the cargo protein binds to the ephrin receptor JM domain in vitro but is released from the ephrin receptor JM domain in vivo. In other embodiments, the binding between the Ephrin receptor JM domain and a cargo protein can be controlled so that the cargo protein is released from the Ephrin receptor JM domain in a manner dependent on the intracellular compartment in which it is located. In certain embodiments, the Ephrin receptor JM domain contains phosphotyrosine, the cargo protein or SBD contains a domain capable of binding to phosphotyrosine, and the binding between the Ephrin receptor JM domain and the cargo protein is between the phosphotyrosine and the domain capable of binding to phosphotyrosine. In a specific embodiment, the domain capable of binding to phosphotyrosine is a PTB domain. In another specific embodiment, the domain capable of binding to phosphotyrosine is an SH2 domain.In specific embodiments, the ephrin receptor JM domain comprises: (i) an (X1)-Ptyr-(X2) motif (wherein Ptyr is phosphotyrosine, X1 is Y, P, V, I, T, or F, and X2 is I, V, L, or A); (ii) an (X3)-Ptyr-(X4) motif (wherein Ptyr is phosphotyrosine, X3 is T, A, or S, and X4 is E or G); or (iii) both (i) and (ii).

[0035] In various embodiments, the polypeptide can bind to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises an ephrin receptor KD at the C-terminus of the TM domain. In certain embodiments, the binding between the ephrin receptor KD and the cargo protein is a non-covalent bond. In certain embodiments, the binding between the ephrin receptor KD and the cargo protein is a reversible bond. In certain embodiments, the binding between the ephrin receptor KD and the cargo protein can be controlled. In a specific embodiment, the binding between the ephrin receptor KD and the cargo protein can be controlled by pH. In another specific embodiment, the binding between the ephrin receptor KD and the cargo protein can be controlled by ionic strength. In some embodiments, the binding between the ephrin receptor KD and the cargo protein can be controlled so that the cargo protein binds to the ephrin receptor KD in vitro but is released from the ephrin receptor KD in vivo. In other embodiments, the binding between the ephrin receptor KD and the cargo protein can be controlled so that the cargo protein is released from the ephrin receptor KD in a manner dependent on the intracellular compartment in which it is located. In certain embodiments, the ephrin receptor KD contains phosphotyrosine, the cargo protein or SBD contains a domain capable of binding to phosphotyrosine, and the binding between the ephrin receptor KD and the cargo protein is between the phosphotyrosine and the domain capable of binding to phosphotyrosine. In a specific embodiment, the domain capable of binding to phosphotyrosine is a PTB domain. In another specific embodiment, the domain capable of binding to phosphotyrosine is an SH2 domain. In a specific embodiment, the KD contains a (X7)-Ptyr-(X8) motif (wherein Ptyr is phosphotyrosine, X7 is T, V, or A, and X8 is E or T) in the activation loop.

[0036] In various embodiments, the polypeptide can bind to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises a SAM linker domain at the C-terminus of the TM domain. In certain embodiments, the bond between the SAM linker domain and the cargo protein is a non-covalent bond. In certain embodiments, the bond between the SAM linker domain and the cargo protein is a reversible bond. In certain embodiments, the bond between the SAM linker domain and the cargo protein can be controlled. In a specific embodiment, the bond between the SAM linker domain and the cargo protein can be controlled by pH. In another specific embodiment, the bond between the SAM linker domain and the cargo protein can be controlled by ionic strength. In some embodiments, the bond between the SAM linker domain and the cargo protein can be controlled so that the cargo protein binds to the SAM linker domain in vitro but is released from the SAM linker domain in vivo. In other embodiments, the bond between the SAM linker domain and the cargo protein can be controlled so that the cargo protein is released from the SAM linker domain in a manner dependent on the intracellular compartment in which it is located. In one embodiment, the SAM linker domain comprises a phosphorylated amino acid or a phosphomimetic amino acid, the cargo protein or SBD comprises a domain capable of binding to the phosphorylated amino acid or the phosphomimetic amino acid, and the bond between the SAM linker domain and the cargo protein is a bond between the phosphorylated amino acid or the phosphomimetic amino acid and the domain capable of binding to the phosphorylated amino acid or the phosphomimetic amino acid. In one embodiment, the SAM linker domain is an ephrin receptor SAM linker domain.

[0037] In various embodiments, the polypeptide can bind to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises a SAM domain at the C-terminus of the TM domain. In certain embodiments, the binding between the SAM domain and the cargo protein is a non-covalent bond. In certain embodiments, the binding between the SAM domain and the cargo protein is a reversible bond. In certain embodiments, the binding between the SAM domain and the cargo protein can be controlled. In a specific embodiment, the binding between the SAM domain and the cargo protein can be controlled by pH. In another specific embodiment, the binding between the SAM domain and the cargo protein can be controlled by ionic strength. In some embodiments, the binding between the SAM domain and the cargo protein can be controlled so that the cargo protein binds to the SAM domain in vitro but is released from the SAM domain in vivo. In other embodiments, the binding between the SAM domain and the cargo protein can be controlled so that the cargo protein is released from the SAM domain in a manner dependent on the intracellular compartment in which it is located. In some embodiments, the cargo protein or SBD comprises a second SAM domain, and the bond between the SAM domain and the cargo protein is the bond between the SAM domain and the second SAM domain. In some embodiments, the SAM domain comprises phosphotyrosine, the cargo protein or SBD comprises a domain capable of binding to phosphotyrosine, and the bond between the SAM domain and the cargo protein is the bond between the phosphotyrosine and the domain capable of binding to phosphotyrosine. In a specific embodiment, the domain capable of binding to phosphotyrosine is a PTB domain. In another specific embodiment, the domain capable of binding to phosphotyrosine is an SH2 domain. In a specific embodiment, the SAM domain comprises a phosphotyrosine in the α2 helix.In a specific embodiment, the phosphotyrosine in the α2 helix of the SAM domain is in a (X5)-Ptyr-(X6) motif, where Ptyr is phosphotyrosine, X5 is C, R, Q, or H, and X6 is Q, I, E, K, R, or T. In certain embodiments, the SAM domain is an ephrin receptor SAM domain.

[0038] In various embodiments, the polypeptide can bind to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises an ephrin receptor PDZ-binding motif (PBM) domain C-terminal to the TM domain. In certain embodiments, the binding between the ephrin receptor PBM domain and the cargo protein is a non-covalent bond. In certain embodiments, the binding between the ephrin receptor PBM domain and the cargo protein is a reversible bond. In certain embodiments, the binding between the ephrin receptor PBM domain and the cargo protein can be controlled. In a specific embodiment, the binding between the ephrin receptor PBM domain and the cargo protein can be controlled by pH. In another specific embodiment, the binding between the ephrin receptor PBM domain and the cargo protein can be controlled by ionic strength. In some embodiments, the binding between the ephrin receptor PBM domain and the cargo protein can be controlled so that the cargo protein binds to the ephrin receptor PBM domain in vitro but is released from the ephrin receptor PBM domain in vivo. In other embodiments, the binding between the Ephrin receptor PBM domain and the cargo protein can be controlled such that the cargo protein is released from the Ephrin receptor PBM domain in a manner that depends on the subcellular compartment they are located in. In certain embodiments, the cargo protein or SBD comprises a PDZ domain, and the binding between the Ephrin receptor PBM domain and the cargo protein is binding between the Ephrin receptor PBM domain and the PDZ domain.

[0039] In various embodiments, the cargo protein is a therapeutic protein. In a specific embodiment, the therapeutic protein is a therapeutic antibody or an antigen-binding fragment thereof. In another specific embodiment, the therapeutic protein is a gene editor or a transposase. In various embodiments, the cargo protein is a diagnostic protein. In a specific embodiment, the diagnostic protein is a fluorescent protein.

[0040] In various embodiments, the polypeptide lacks an ephrin receptor ligand binding domain (LBD). In various embodiments, the polypeptide comprises a mutated ephrin receptor LBD.

[0041] In various embodiments, a polypeptide comprises two distinct domains that allow the polypeptide to undergo heterodomain dimerization with another polypeptide identical to the polypeptide. In certain embodiments, a polypeptide comprises two distinct domains in a head-to-tail configuration that allow the polypeptide to undergo heterodomain dimerization with another polypeptide identical to the polypeptide.

[0042] In various embodiments, the TM domain is an ephrin receptor TM domain.

[0043] In various embodiments, any one or more of the ephrin receptor domains of the polypeptide are from or derived from EphAl, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphAlO, EphBl, EphB2, EphB3, EphB4, EphB6, or a combination thereof. In certain embodiments, any one or more of the ephrin receptor domains of the polypeptide are from or derived from EphA2, EphA4, EphB2, or a combination thereof.

[0044] In various embodiments, the polypeptide further comprises a modified Fc domain of an immunoglobulin. In certain embodiments, the modified Fc domain is at the N-terminus of the ephrin receptor CR domain. In specific embodiments, the modified Fc domain is fused to the remainder of the polypeptide via a linker sequence. In certain embodiments, the modified Fc domain a. is capable of specifically binding to the Fc-binding site of the neonatal Fc receptor (FcRn); and b. lacks the ability to form homodimers. In certain embodiments, the dissociation constant of the modified Fc domain bound to FcRn at a pH of 6.5 is at most 10 -4 In certain embodiments, the dissociation constant of the modified Fc domain bound to FcRn at a pH of 7.4 is at least 10 -4 In a specific embodiment, the modified Fc domain has the amino acid sequence [ka] (wherein X1, X2, X3, X4, X5, X6, X7, and X8 are each any amino acid). In a specific embodiment, the modified Fc domain is capable of specifically binding to an amino acid sequence between positions 135 and 158 of human FcRn (SEQ ID NO: 228) and / or mouse FcRn (SEQ ID NO: 227). In certain embodiments, the polypeptide does not substantially bind to C1q, FcγRI, FcγRII, or FcγRIII. In certain embodiments, the complement-dependent cytotoxicity (CDC) activity of the modified Fc domain, the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the modified Fc domain, the antibody-dependent cell-mediated phagocytosis (ADCP) activity of the modified Fc domain, and / or the antibody-dependent intracellular neutralization (ADIN) activity of the modified Fc domain is reduced by at least 10%, 20%, 30%, 40%, or 50% compared to the unmodified Fc domain. In certain embodiments, the complement-dependent cytotoxicity (CDC) activity of the modified Fc domain, the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the modified Fc domain, the antibody-dependent cell-mediated phagocytosis (ADCP) activity of the modified Fc domain, and / or the antibody-dependent intracellular neutralization (ADIN) activity of the modified Fc domain is reduced by at least 1.5, 2, 3, 4, or 5-fold compared to an unmodified Fc domain. In certain embodiments, the modified Fc domain comprises, from the N-terminus to the C-terminus: a. a modified CH2 domain that has been modified to have reduced effector function compared to an unmodified CH2 domain; and b. a modified CH3 domain that has been modified to lack the ability to form homodimers.

[0045] In various embodiments, the first Ephrin receptor FN III domain and the second Ephrin receptor FN III domain comprise different amino acid sequences.

[0046] In another aspect, provided herein is a nucleic acid encoding a polypeptide described herein.

[0047] In another aspect, provided herein is an expression vector comprising a nucleic acid described herein.

[0048] In another aspect, provided herein is a cell comprising a nucleic acid or expression vector described herein.

[0049] In another aspect, provided herein is a method of producing EVs, the method comprising: a. transfecting cells with a nucleic acid or expression vector described herein; b. culturing the cells under conditions suitable for the production of the EVs; and c. collecting EVs secreted by the cells.

[0050] In another aspect, provided herein is a method of producing a hybridosome, the method comprising contacting a first EV with a second EV, thereby combining the first EV with the second EV to produce the hybridosome, wherein the first EV is produced in vitro and the first EV comprises (i) a membrane and (ii) a fusogenic, ionizable cationic lipid, and wherein the second EV is produced by the method described above.

[0051] In another aspect, provided herein is a method for purifying EVs or hybridosomes, the method comprising: a. providing the EVs or hybridosomes, wherein the EVs or hybridosomes comprise a first binding partner, wherein the first binding partner is capable of binding to the Fc-binding site of FcRn in a pH-dependent manner; and b. contacting the EVs or hybridosomes comprising the first binding partner with a second binding partner at a first pH, wherein the second binding partner comprises the Fc-binding site of FcRn and is associated with a solid matrix; and c. eluting the EVs or hybridosomes comprising the first binding partner from the solid matrix at a second pH. In certain embodiments, the method further comprises a washing step at the first pH. In certain embodiments, the first pH is less than 6.5. In certain embodiments, the second pH is greater than 7.4. In certain embodiments, the Fc-binding site of FcRn comprises the amino acid sequence of SEQ ID NO: 230.

[0052] In another aspect, provided herein is a method for purifying EVs or hybridosomes, the method comprising: a. providing the EVs or hybridosomes, wherein the EVs or hybridosomes comprise a first binding partner, wherein the first binding partner is capable of binding to the Fc-binding site of FcRn in a pH-dependent manner and comprises or consists of a polypeptide described herein; and b. contacting the EVs or hybridosomes comprising the first binding partner with a second binding partner at a first pH, wherein the second binding partner comprises the Fc-binding site of FcRn and is associated with a solid matrix; and c. eluting the EVs or hybridosomes comprising the first binding partner from the solid matrix at a second pH. In certain embodiments, the method further comprises a wash step at the first pH. In certain embodiments, the first pH is less than 6.5. In certain embodiments, the second pH is greater than 7.4. In certain embodiments, the Fc-binding site of FcRn comprises the amino acid sequence of SEQ ID NO: 230.

[0053] In some embodiments, the polypeptides provided herein are signal-neutral in that they have reduced forward (i.e., luminal) signaling capabilities due to the lack of an endodomain or portion thereof (e.g., a sterile alpha motif (SAM) domain and / or a PDZ domain) or because the kinase activity of the parent Eph receptor has been inactivated, e.g., by mutation and / or deletion. Furthermore, the scaffold is preferably extracellularly inactive, preferably because the ligand-binding domain has been modified or deleted to reduce or abolish binding to ephrins, the natural ligands of the Eph receptor.

[0054] Importantly, such modified Eph receptor variants of the present disclosure have both accessible and free N- and C-termini to which molecules of interest (e.g., cargo, targeting domains, or purification domains) can be fused. For example, a polypeptide can be fused in-frame with one or more targeting domains, allowing nanovesicles (e.g., EVs and hybridosomes) containing such polypeptides to be targeted to specific cell types upon administration to a subject. For the fusion moiety to be functional, it is beneficial to maintain a certain distance between the fusion moiety and the surface of the nanovesicle (e.g., EV or hybridosome). Fusing a targeting moiety to the N-terminus of the ligand-binding domain (LBD) of an Eph receptor-derived polypeptide results in a structure that is flexible enough to bend and / or reconfigure, yet stable. Furthermore, because the ectodomain of an Eph receptor protrudes from the membrane, it provides a long protrusion for reach.

[0055] In certain embodiments, the polypeptides disclosed herein and nanovesicles (e.g., EVs and hybridosomes) comprising these polypeptides are suitable for therapeutic applications.

[0056] In one aspect, the present disclosure provides a polypeptide derived from an Eph receptor, comprising: i. contains an ephrin ligand binding domain that exhibits reduced or no ephrin binding when compared to the parent Eph receptor; and ii. comprises a transmembrane domain; The polypeptide is provided.

[0057] Preferably, the polypeptide is fused to one or more molecules of interest, preferably proteins.

[0058] In another aspect, nucleic acids encoding the polypeptides described herein are provided.

[0059] In another aspect, an expression vector is provided that includes a nucleic acid described herein.

[0060] In yet another aspect, a cell containing such a nucleic acid or expression vector is provided. The exogenous nucleic acid or expression vector can be transiently or stably introduced into the cell. In a preferred embodiment, such a cell is a source cell capable of producing nanovesicles (e.g., EVs and hybridosomes) under suitable conditions.

[0061] In another aspect, the present disclosure relates to nanovesicles (e.g., EVs or hybridosomes) comprising the polypeptides disclosed herein. In one embodiment, the nanovesicles are derived from source cells (i.e., extracellular vesicles or "EVs"). In one embodiment, the nanovesicles are natural / synthetic hybrids (e.g., hybridosomes).

[0062] In another embodiment, there is provided a method for producing nanovesicles (e.g., EVs or hybridosomes) whose surfaces are decorated with one or more heterologous polypeptides (e.g., targeting domains), comprising: (i) providing a nucleic acid or expression vector encoding a polypeptide disclosed herein fused to one or more proteins of interest; (ii) transfecting the cells with a nucleic acid or expression vector described herein; (iii) culturing the cells under suitable conditions so that nanovesicles are produced; and (iv) purifying the nanovesicles so produced from the cell culture. A method is provided, comprising:

[0063] Also provided are compositions comprising the nanovesicles described herein, the nucleic acids described herein, the expression vectors described herein, and / or the cells described herein. Such compositions can be used in the treatment of a disease or disorder.

[0064] Also provided is a method of treating a disease or disorder, comprising administering to a subject a therapeutically effective amount of a composition described herein. 3.1 Exemplary Implementations 1. An extracellular vesicle (EV) comprising a polypeptide, the polypeptide comprising, in the N-terminal to C-terminal direction: a. Ephrin receptor cysteine-rich (CR) domain; b. a first ephrin receptor fibronectin type III (FN III) domain and a second ephrin receptor FN III domain; and c. transmembrane (TM) domain; Including, wherein the polypeptide lacks (i) ephrin-binding activity, (ii) ephrin receptor kinase activity, or (iii) both ephrin-binding activity and ephrin receptor kinase activity. The extracellular vesicles (EVs). 2. A hybridosome comprising a polypeptide, wherein the polypeptide is, from N-terminus to C-terminus: a. Ephrin receptor CR domain; b. a first ephrin receptor FN III domain and a second ephrin receptor FN III domain; and c. TM domain; Including, wherein the polypeptide lacks (i) ephrin-binding activity, (ii) ephrin receptor kinase activity, or (iii) both ephrin-binding activity and ephrin receptor kinase activity. The hybridosome. 3. An EV according to paragraph 1 or a hybridosome according to paragraph 2, wherein the polypeptide lacks ephrin-binding activity. 4. An EV or hybridosome according to any one of paragraphs 1 to 3, wherein the polypeptide further comprises a targeting domain at the N-terminus of the ephrin receptor CR domain. 5. The EV or hybridosome of paragraph 4, wherein the targeting domain is selected from the group consisting of: scFv, (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Fd fragment, diabody, F(ab')3, disulfide-linked Fv, sdAb (VHH or nanobody), CDR, di-scFv, bi-scFv, tascFv (tandem scFv), triabody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, DARPin, centilin, affibody, affilin, affitin, anticalin, avimer, fynomer, Kunitz domain peptide, monobody (or adnectin), tribody, and nanophytin. 6. The EV or hybridosome according to paragraph 4 or 5, wherein the targeting domain specifically binds to a marker. 7. The EV or hybridosome according to paragraph 6, wherein the marker is a tumor-associated antigen. 8. The tumor-associated antigen is human epidermal growth factor receptor 2 (HER2), CD20, CD33, B-cell maturation antigen (BCMA), prostate-specific membrane antigen (PSMA), DLL3, ganglioside GD2 (GD2), CD123, anoctamin-1 (Ano1), mesothelin, carbonic anhydrase IX (CAIX), tumor-associated calcium signaling protein 2 (TROP2), carcinoembryonic antigen (CEA), claudin-18.2, receptor tyrosine kinase-like orphan receptor 1 (ROR1), trophoblast glycoprotein (5T4), glycoprotein non-metastatic melanoma protein B (GPNMB), folate receptor-alpha (FR-α), pregnancy-associated plasma protein A (PAPP-A), CD37, 8. The EV or hybridosome of paragraph 7, wherein the EV or hybridosome is selected from the group consisting of epithelial cell adhesion molecule (EpCAM), CD2, CD19, CD30, CD38, CD40, CD52, CD70, CD79b, fms-like tyrosine kinase 3 (FLT3), glypican 3 (GPC3), B7 homolog 6 (B7H6), CC chemokine receptor type 4 (CCR4), C-X-C motif chemokine receptor 4 (CXCR4), receptor tyrosine kinase-like orphan receptor 2 (ROR2), CD133, HLA class I histocompatibility antigen, alpha chain E (HLA-E), epidermal growth factor receptor (EGFR / ERBB-1), insulin-like growth factor 1 receptor (IGF1R), and human epidermal growth factor receptor 3. 9. The EV or hybridosome of any one of paragraphs 1 to 8, wherein the polypeptide further comprises a cargo protein or cargo-binding domain at the C-terminus of the TM domain. 10. The EV or hybridosome according to paragraph 9, wherein the cargo protein or cargo-binding domain is fused to the remainder of the polypeptide via a linker. 11. The EV or hybridosome of paragraph 10, wherein the cargo protein or cargo-binding domain is covalently fused to the remainder of the polypeptide via a linker. 12. EVs or hybridosomes according to paragraph 10 or 11, wherein the linker is a peptide linker. 13. The EV or hybridosome according to paragraph 12, wherein the peptide linker comprises the amino acid sequence (GGGS)n (SEQ ID NO: 226), where n is an integer between 1 and 10. 14. The EV or hybridosome of paragraph 12, wherein the peptide linker comprises the amino acid sequence GGGS. 15. The EV or hybridosome of any one of paragraphs 9 to 14, wherein the polypeptide comprises a cargo-binding domain capable of binding to a cargo protein directly or indirectly via a scaffold-binding domain (SBD) linked to the cargo protein. 16. The EV or hybridosome according to paragraph 15, wherein the bond between the cargo-binding domain and the cargo protein is a non-covalent bond. 17. The EV or hybridosome according to paragraph 15 or 16, wherein the bond between the cargo-binding domain and the cargo protein is a reversible bond. 18. The EV or hybridosome according to any one of paragraphs 15 to 17, wherein the binding between the cargo-binding domain and the cargo protein can be controlled. 19. The EV or hybridosome according to paragraph 18, wherein the binding between the cargo-binding domain and the cargo protein can be controlled by pH. 20. The EV or hybridosome according to paragraph 18, wherein the binding between the cargo-binding domain and the cargo protein can be controlled by ionic strength. 21. The EV or hybridosome of any one of paragraphs 15 to 20, wherein the binding between the cargo-binding domain and the cargo protein can be controlled such that the cargo protein binds to the cargo-binding domain in vitro but is released from the cargo-binding domain in vivo. 22. The EV or hybridosome according to any one of paragraphs 15 to 20, wherein the binding between the cargo-binding domain and the cargo protein can be controlled such that the cargo proteins are released from the cargo-binding domain in a manner that depends on the intracellular compartment in which they are located. 23. The EV or hybridosome according to any one of paragraphs 15 to 22, wherein the cargo-binding domain comprises a phosphotyrosine, the cargo protein or the SBD comprises a domain capable of binding to a phosphotyrosine, and the bond between the cargo-binding domain and the cargo protein is a bond between the phosphotyrosine and the domain capable of binding to the phosphotyrosine. 24. The EV or hybridosome according to paragraph 23, wherein the domain capable of binding to phosphotyrosine is a phosphotyrosine-binding (PTB) domain. 25. The EV or hybridosome according to paragraph 23, wherein the domain capable of binding to phosphotyrosine is an Src homology 2 (SH2) domain. 26. The EV or hybridosome of any one of paragraphs 15 to 22, wherein the cargo-binding domain comprises a first sterile alpha-motif (SAM) domain, the cargo protein or the SBD comprises a second SAM domain, and the bond between the cargo-binding domain and the cargo protein is the bond between the first SAM domain and the second SAM domain. 27. The EV or hybridosome of any one of paragraphs 15 to 22, wherein the cargo-binding domain comprises a PDZ-binding motif (PBM) domain, the cargo protein or the SBD comprises a PDZ domain, and the bond between the cargo-binding domain and the cargo protein is a bond between the PBM domain and the PDZ domain. 28. The EV or hybridosome of any one of paragraphs 15 to 22, wherein the cargo-binding domain comprises a PDZ domain, the cargo protein or the SBD comprises a PBM domain, and the bond between the cargo-binding domain and the cargo protein is a bond between the PDZ domain and the PBM domain. 29. The EV or hybridosome according to any one of paragraphs 9 to 14, wherein the polypeptide is a cargo protein. 30. An EV or hybridosome described in any one of paragraphs 1 to 8, wherein the polypeptide is capable of binding to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises an ephrin receptor JM domain C-terminal to the TM domain. 31. The EV or hybridosome according to paragraph 30, wherein the bond between the ephrin receptor JM domain and the cargo protein is a non-covalent bond. 32. The EV or hybridosome according to paragraph 30 or 31, wherein the binding between the ephrin receptor JM domain and the cargo protein is a reversible binding. 33. An EV or hybridosome according to any one of paragraphs 30 to 32, wherein the binding between the ephrin receptor JM domain and the cargo protein can be controlled. 34. The EV or hybridosome according to paragraph 33, wherein the binding between the ephrin receptor JM domain and the cargo protein can be controlled by pH. 35. The EV or hybridosome according to paragraph 33, wherein the binding between the ephrin receptor JM domain and the cargo protein can be controlled by ionic strength. 36. An EV or hybridosome according to any one of paragraphs 30 to 35, wherein the binding between the ephrin receptor JM domain and the cargo protein can be controlled so that the cargo protein binds to the ephrin receptor JM domain in vitro but is released from the ephrin receptor JM domain in vivo. 37. An EV or hybridosome according to any one of paragraphs 30 to 35, wherein the binding between the ephrin receptor JM domain and the cargo protein can be controlled such that the cargo proteins are released from the ephrin receptor JM domain in a manner that depends on the intracellular compartment in which they are located. 38. An EV or hybridosome according to any one of paragraphs 30 to 37, wherein the ephrin receptor JM domain comprises a phosphotyrosine, the cargo protein or the SBD comprises a domain capable of binding to phosphotyrosine, and the bond between the ephrin receptor JM domain and the cargo protein is a bond between the phosphotyrosine and the domain capable of binding to phosphotyrosine. 39. The EV or hybridosome according to paragraph 38, wherein the domain capable of binding to phosphotyrosine is a PTB domain. 40. The EV or hybridosome according to paragraph 38, wherein the domain capable of binding to phosphotyrosine is an SH2 domain. 41. The ephrin receptor JM domain is (i) the (X1)-Ptyr-(X2) motif (where Ptyr is phosphotyrosine, X1 is Y, P, V, I, T, or F, and X2 is I, V, L, or A); (ii) a (X3)-Ptyr-(X4) motif, where Ptyr is phosphotyrosine, X3 is T, A, or S, and X4 is E or G; or (iii) Both (i) and (ii) 41. The EV or hybridosome according to any one of paragraphs 38 to 40, comprising: 42. An EV or hybridosome described in any one of paragraphs 1 to 8, wherein the polypeptide is capable of binding to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises an ephrin receptor KD at the C-terminus of the TM domain. 43. The EV or hybridosome according to paragraph 42, wherein the bond between the ephrin receptor KD and the cargo protein is a non-covalent bond. 44. An EV or hybridosome according to paragraph 42 or 43, wherein the binding between the ephrin receptor KD and the cargo protein is a reversible binding. 45. An EV or hybridosome according to any one of paragraphs 42 to 44, wherein the binding between the ephrin receptor KD and the cargo protein can be controlled. 46. ​​An EV or hybridosome according to paragraph 45, wherein the binding between the ephrin receptor KD and the cargo protein can be controlled by pH. 47. An EV or hybridosome according to paragraph 45, wherein the binding between the ephrin receptor KD and the cargo protein can be controlled by ionic strength. 48. An EV or hybridosome described in any one of paragraphs 42 to 47, wherein the binding between the ephrin receptor KD and the cargo protein can be controlled so that the cargo protein binds to the ephrin receptor KD in vitro but is released from the ephrin receptor KD in vivo. 49. An EV or hybridosome according to any one of paragraphs 42 to 47, wherein the binding between the ephrin receptor KD and the cargo protein can be controlled so that the cargo protein is released from the ephrin receptor KD in a manner that depends on the intracellular compartment in which it is located. 50. An EV or hybridosome described in any one of paragraphs 42 to 49, wherein the ephrin receptor KD contains a phosphotyrosine, the cargo protein or the SBD contains a domain capable of binding to phosphotyrosine, and the bond between the ephrin receptor KD and the cargo protein is a bond between the phosphotyrosine and the domain capable of binding to phosphotyrosine. 51. An EV or hybridosome according to paragraph 50, wherein the domain capable of binding to phosphotyrosine is a PTB domain. 52. An EV or hybridosome according to paragraph 50, wherein the domain capable of binding to phosphotyrosine is an SH2 domain. 53. The EV or hybridosome of any one of paragraphs 50 to 52, wherein the KD comprises a (X7)-Ptyr-(X8) motif in the activation loop (wherein Ptyr is phosphotyrosine, X7 is T, V, or A, and X8 is E or T). 54. An EV or hybridosome described in any one of paragraphs 1 to 8, wherein the polypeptide is capable of binding to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises a SAM linker domain at the C-terminus of the TM domain. 55. The EV or hybridosome according to paragraph 54, wherein the bond between the SAM linker domain and the cargo protein is a non-covalent bond. 56. The EV or hybridosome according to paragraph 54 or 55, wherein the bond between the SAM linker domain and the cargo protein is a reversible bond. 57. An EV or hybridosome according to any one of paragraphs 54 to 56, wherein the binding between the SAM linker domain and the cargo protein can be controlled. 58. The EV or hybridosome according to paragraph 57, wherein the binding between the SAM linker domain and the cargo protein can be controlled by pH. 59. The EV or hybridosome according to paragraph 57, wherein the binding between the SAM linker domain and the cargo protein can be controlled by ionic strength. 60. An EV or hybridosome according to any one of paragraphs 54 to 59, wherein the binding between the SAM linker domain and the cargo protein can be controlled so that the cargo protein binds to the SAM linker domain in vitro but is released from the SAM linker domain in vivo. 61. The EV or hybridosome according to any one of paragraphs 54 to 59, wherein the binding between the SAM linker domain and the cargo protein can be controlled so that the cargo proteins are released from the SAM linker domain in a manner that depends on the intracellular compartment in which they are located. 62. An EV or hybridosome according to any one of paragraphs 54 to 61, wherein the SAM linker domain comprises a phosphorylated amino acid or a phospho-mimetic amino acid, the cargo protein or the SBD comprises a domain capable of binding to the phosphorylated amino acid or phospho-mimetic amino acid, and the bond between the SAM linker domain and the cargo protein is a bond between the phosphorylated amino acid or phospho-mimetic amino acid and the domain capable of binding to the phosphorylated amino acid or phospho-mimetic amino acid. 63. An EV or hybridosome according to any one of paragraphs 54 to 62, wherein the SAM linker domain is an ephrin receptor SAM linker domain. 64. An EV or hybridosome described in any one of paragraphs 1 to 8, wherein the polypeptide is capable of binding to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises a SAM domain C-terminal to the TM domain. 65. The EV or hybridosome according to paragraph 64, wherein the bond between the SAM domain and the cargo protein is a non-covalent bond. 66. The EV or hybridosome according to paragraph 64 or 65, wherein the bond between the SAM domain and the cargo protein is a reversible bond. 67. An EV or hybridosome according to any one of paragraphs 64 to 66, wherein the binding between the SAM domain and the cargo protein can be controlled. 68. The EV or hybridosome according to paragraph 67, wherein the binding between the SAM domain and the cargo protein can be controlled by pH. 69. The EV or hybridosome according to paragraph 67, wherein the binding between the SAM domain and the cargo protein can be controlled by ionic strength. 70. An EV or hybridosome according to any one of paragraphs 64 to 69, wherein the binding between the SAM domain and the cargo protein can be controlled so that the cargo protein binds to the SAM domain in vitro but is released from the SAM domain in vivo. 71. The EV or hybridosome of any one of paragraphs 64 to 69, wherein the binding between the SAM domain and the cargo protein can be controlled so that the cargo proteins are released from the SAM domain in a manner that depends on the intracellular compartment in which they are located. 72. The EV or hybridosome of any one of paragraphs 64 to 71, wherein the cargo protein or the SBD comprises a second SAM domain, and the bond between the SAM domain and the cargo protein is a bond between the SAM domain and the second SAM domain. 73. The EV or hybridosome according to any one of paragraphs 64 to 71, wherein the SAM domain comprises a phosphotyrosine, the cargo protein or the SBD comprises a domain capable of binding to a phosphotyrosine, and the bond between the SAM domain and the cargo protein is a bond between the phosphotyrosine and the domain capable of binding to the phosphotyrosine. 74. The EV or hybridosome according to paragraph 73, wherein the domain capable of binding to phosphotyrosine is a PTB domain. 75. The EV or hybridosome according to paragraph 73, wherein the domain capable of binding to phosphotyrosine is an SH2 domain. 76. The EV or hybridosome of any one of paragraphs 73 to 75, wherein the SAM domain comprises a phosphotyrosine in the α2 helix. 77. The EV or hybridosome of paragraph 76, wherein the phosphotyrosine in the α2 helix of the SAM domain is in a (X5)-Ptyr-(X6) motif (wherein Ptyr is phosphotyrosine, X5 is C, R, Q, or H, and X6 is Q, I, E, K, R, or T). 78. An EV or hybridosome according to any one of paragraphs 64 to 77, wherein the SAM domain is an ephrin receptor SAM domain. 79. An EV or hybridosome according to any one of paragraphs 1 to 8, wherein the polypeptide is capable of binding to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises an ephrin receptor PDZ binding motif (PBM) domain C-terminal to the TM domain. 80. The EV or hybridosome of paragraph 79, wherein the bond between the ephrin receptor PBM domain and the cargo protein is a non-covalent bond. 81. The EV or hybridosome according to paragraph 79 or 80, wherein the binding between the ephrin receptor PBM domain and the cargo protein is a reversible binding. 82. An EV or hybridosome according to any one of paragraphs 79 to 81, wherein the binding between the ephrin receptor PBM domain and the cargo protein can be controlled. 83. The EV or hybridosome according to paragraph 82, wherein the binding between the ephrin receptor PBM domain and the cargo protein can be controlled by pH. 84. The EV or hybridosome according to paragraph 82, wherein the binding between the ephrin receptor PBM domain and the cargo protein can be controlled by ionic strength. 85. An EV or hybridosome according to any one of paragraphs 79 to 84, wherein the binding between the ephrin receptor PBM domain and the cargo protein can be controlled such that the cargo protein binds to the ephrin receptor PBM domain in vitro but is released from the ephrin receptor PBM domain in vivo. 86. The EV or hybridosome of any one of paragraphs 79 to 84, wherein the binding between the ephrin receptor PBM domain and the cargo protein can be controlled such that the cargo protein is released from the ephrin receptor PBM domain in a manner that depends on the intracellular compartment in which it is located. 87. An EV or hybridosome according to any one of paragraphs 79 to 86, wherein the cargo protein or the SBD comprises a PDZ domain, and the binding between the ephrin receptor PBM domain and the cargo protein is the binding between the ephrin receptor PBM domain and the PDZ domain. 88. The EV or hybridosome according to any one of paragraphs 9 to 87, wherein the cargo protein is a therapeutic protein. 89. The EV or hybridosome according to paragraph 88, wherein the therapeutic protein is a therapeutic antibody or an antigen-binding fragment thereof. 90. The EV or hybridosome according to paragraph 88, wherein the therapeutic protein is a gene editor or a transposase. 91. The EV or hybridosome according to any one of paragraphs 9 to 87, wherein the cargo protein is a diagnostic protein. 92. The EV or hybridosome according to paragraph 91, wherein the diagnostic protein is a fluorescent protein. 93. The EV or hybridosome of any one of paragraphs 1 to 92, wherein the polypeptide lacks an ephrin receptor ligand binding domain (LBD). 94. The EV or hybridosome of any one of paragraphs 1 to 92, wherein the polypeptide comprises a mutated ephrin receptor LBD. 95. The EV or hybridosome of any one of paragraphs 1 to 94, wherein the polypeptide comprises two distinct domains that enable the polypeptide to undergo heterodomain dimerization with another polypeptide identical to the polypeptide. 96. An EV or hybridosome described in any one of paragraphs 1 to 94, wherein the polypeptide comprises two different domains in a head-to-tail arrangement that enable the polypeptide to undergo heterodomain dimerization with another polypeptide identical to the polypeptide. 97. An EV or hybridosome according to any one of paragraphs 1 to 96, wherein the TM domain is an ephrin receptor TM domain. 98. The EV or hybridosome of any one of paragraphs 1 to 97, wherein any one or more of the ephrin receptor domains of the polypeptide are or are derived from EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, or a combination thereof. 99. The EV or hybridosome of any one of paragraphs 1 to 97, wherein any one or more of the ephrin receptor domains of the polypeptide are or are derived from EphA2, EphA4, EphB2, or a combination thereof. 100. The EV or hybridosome of any one of paragraphs 1 to 99, wherein the polypeptide further comprises a modified Fc domain of an immunoglobulin. 101. The EV or hybridosome of paragraph 100, wherein the modified Fc domain is N-terminal to the ephrin receptor CR domain. 102. The EV or hybridosome of paragraph 101, wherein the modified Fc domain is fused to the remainder of the polypeptide by a linker sequence. 103. The modified Fc domain is a. capable of specifically binding to the Fc binding site of the neonatal Fc receptor (FcRn); and B. lacks the ability to form homodimers; EVs or hybridosomes according to any one of paragraphs 100 to 102. 104. The dissociation constant of the modified Fc domain bound to FcRn at a pH of 6.5 is at most 10 -4 EV or hybridosome according to any one of paragraphs 100 to 103, having a value of M. 105. The dissociation constant of the modified Fc domain bound to FcRn at a pH of 7.4 is at least 10 -4 EV or hybridosome according to any one of paragraphs 100 to 104, having a value of M. 106. The modified Fc domain has the amino acid sequence [ka] (wherein X1, X2, X3, X4, X5, X6, X7, and X8 are each any amino acid) 107. The EV or hybridosome according to any one of paragraphs 100 to 106, wherein the modified Fc domain is capable of specifically binding to an amino acid sequence between positions 135 and 158 of human FcRn (SEQ ID NO: 228) and / or mouse FcRn (SEQ ID NO: 227). 108. The EV or hybridosome of any one of paragraphs 100 to 107, wherein the polypeptide does not substantially bind to C1q, FcγRI, FcγRII, or FcγRIII. 109. a. the complement-dependent cytotoxicity (CDC) activity of the modified Fc domain; b. antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the modified Fc domain; c. antibody-dependent cell-mediated phagocytosis (ADCP) activity of the modified Fc domain; and / or d. Antibody-dependent intracellular neutralization (ADIN) activity of the modified Fc domain is reduced by at least 10%, 20%, 30%, 40%, or 50% compared to the unmodified Fc domain : EVs or hybridosomes according to any one of paragraphs 100 to 108. 110. a. the complement-dependent cytotoxicity (CDC) activity of the modified Fc domain; b. antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the modified Fc domain; c. antibody-dependent cell-mediated phagocytosis (ADCP) activity of the modified Fc domain; and / or d. Antibody-dependent intracellular neutralization (ADIN) activity of the modified Fc domain is reduced by at least 1.5, 2, 3, 4, or 5 fold compared to the unmodified Fc domain : EVs or hybridosomes according to any one of paragraphs 100 to 109. 111. The modified Fc domain comprises, from the N-terminus to the C-terminus: a. a modified CH2 domain that has been modified to decrease effector function relative to the unmodified CH2 domain; and b. A modified CH3 domain that has been modified so as to lack the ability to form homodimers EVs or hybridosomes according to any one of paragraphs 100 to 110, comprising: 112. The EV or hybridosome of any one of paragraphs 1 to 111, wherein the first ephrin receptor FN III domain and the second ephrin receptor FN III domain comprise different amino acid sequences. 113. A method for delivering a therapeutic or diagnostic agent to a target cell or tissue, the method comprising providing the target cell or tissue with an extracellular vesicle or hybridosome described in any one of paragraphs 1 to 112. 114. From the N-terminus to the C-terminus, a. Targeting domain; b. Ephrin receptor CR domain; c. a first ephrin receptor FN III domain and a second ephrin receptor FN III domain; and d. TM Domain A polypeptide comprising: 115. The polypeptide of paragraph 114, wherein said polypeptide lacks ephrin-binding activity. 116. The polypeptide of paragraph 114 or 115, wherein the targeting domain is selected from the group consisting of: scFv, (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Fd fragment, diabody, F(ab')3, disulfide-linked Fv, sdAb (VHH or nanobody), CDR, di-scFv, bi-scFv, tascFv (tandem scFv), triabody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, DARPin, centirin, affibody, affilin, affitin, anticalin, avimer, fynomer, Kunitz domain peptide, monobody (or adnectin), tribody, and nanophytin. 117. The polypeptide of any one of paragraphs 114-116, wherein the targeting domain specifically binds to a marker. 118. The polypeptide of paragraph 117, wherein the marker is a tumor-associated antigen. 119. The tumor-associated antigen is selected from the group consisting of human epidermal growth factor receptor 2 (HER2), CD20, CD33, B-cell maturation antigen (BCMA), prostate-specific membrane antigen (PSMA), DLL3, ganglioside GD2 (GD2), CD123, anoctamin-1 (Ano1), mesothelin, carbonic anhydrase IX (CAIX), tumor-associated calcium signaling protein 2 (TROP2), carcinoembryonic antigen (CEA), claudin-18.2, receptor tyrosine kinase-like orphan receptor 1 (ROR1), trophoblast glycoprotein (5T4), glycoprotein non-metastatic melanoma protein B (GPNMB), folate receptor-α (FR-α), pregnancy-associated plasma protein A (PAPP-A), CD 37, the polypeptide of paragraph 118, being selected from the group consisting of epithelial cell adhesion molecule (EpCAM), CD2, CD19, CD30, CD38, CD40, CD52, CD70, CD79b, fms-like tyrosine kinase 3 (FLT3), glypican 3 (GPC3), B7 homolog 6 (B7H6), CC chemokine receptor type 4 (CCR4), C-X-C motif chemokine receptor 4 (CXCR4), receptor tyrosine kinase-like orphan receptor 2 (ROR2), CD133, HLA class I histocompatibility antigen, alpha chain E (HLA-E), epidermal growth factor receptor (EGFR / ERBB-1), insulin-like growth factor 1-receptor (IGF1R), and human epidermal growth factor receptor 3. 120. From the N-terminus to the C-terminus, a. Ephrin receptor CR domain; b. a first ephrin receptor FN III domain and a second ephrin receptor FN III domain; c. TM domains; and d. cargo protein or cargo-binding domain A polypeptide comprising: 121. The polypeptide of paragraph 120, wherein the polypeptide lacks ephrin-binding activity. 122. From the N-terminus to the C-terminus, a. Targeting domain; b. Ephrin receptor CR domain; c. a first ephrin receptor FN III domain and a second ephrin receptor FN III domain; d. TM domains; and e. cargo protein or cargo-binding domain A polypeptide comprising: 123. The polypeptide of paragraph 122, wherein the polypeptide lacks ephrin-binding activity. 124. The polypeptide of paragraph 122 or 123, wherein the targeting domain is selected from the group consisting of: scFv, (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Fd fragment, diabody, F(ab')3, disulfide-linked Fv, sdAb (VHH or nanobody), CDR, di-scFv, bi-scFv, tascFv (tandem scFv), triabody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, DARPin, centirin, affibody, affilin, affitin, anticalin, avimer, fynomer, Kunitz domain peptide, monobody (or adnectin), tribody, and nanophytin. 125. The polypeptide of any one of paragraphs 122-124, wherein the targeting domain specifically binds to a marker. 126. The polypeptide of paragraph 125, wherein the marker is a tumor-associated antigen. 127. The tumor-associated antigen is human epidermal growth factor receptor 2 (HER2), CD20, CD33, B-cell maturation antigen (BCMA), prostate-specific membrane antigen (PSMA), DLL3, ganglioside GD2 (GD2), CD123, anoctamin-1 (Ano1), mesothelin, carbonic anhydrase IX (CAIX), tumor-associated calcium signaling pathway 2 (TROP2), carcinoembryonic antigen (CEA), claudin-18.2, receptor tyrosine kinase-like orphan receptor 1 (ROR1), trophoblast glycoprotein (5T4), glycoprotein non-metastatic melanoma protein B (GPNMB), folate receptor-α (FR-α), pregnancy-associated plasma protein A (PAPP-A), CD 37, the polypeptide of paragraph 126, being selected from the group consisting of epithelial cell adhesion molecule (EpCAM), CD2, CD19, CD30, CD38, CD40, CD52, CD70, CD79b, fms-like tyrosine kinase 3 (FLT3), glypican 3 (GPC3), B7 homolog 6 (B7H6), CC chemokine receptor type 4 (CCR4), C-X-C motif chemokine receptor 4 (CXCR4), receptor tyrosine kinase-like orphan receptor 2 (ROR2), CD133, HLA class I histocompatibility antigen, alpha chain E (HLA-E), epidermal growth factor receptor (EGFR / ERBB-1), insulin-like growth factor 1-receptor (IGF1R), and human epidermal growth factor receptor 3. 128. The polypeptide of any one of paragraphs 120 to 127, wherein the cargo protein or cargo-binding domain is fused to the remainder of the polypeptide via a linker. 129. The polypeptide of paragraph 128, wherein the cargo protein or cargo-binding domain is covalently fused to the remainder of the polypeptide via a linker. 130. The polypeptide of paragraph 128 or 129, wherein the linker is a peptide linker. 131. The polypeptide of paragraph 130, wherein the peptide linker comprises the amino acid sequence (GGGS)n (SEQ ID NO: 226), where n is an integer from 1 to 10. 132. The polypeptide of paragraph 130, wherein the peptide linker comprises the amino acid sequence GGGS. 133. The polypeptide of any one of paragraphs 120 to 132, wherein the polypeptide comprises a cargo-binding domain capable of binding to a cargo protein directly or indirectly via an SBD linked to the cargo protein. 134. The polypeptide of paragraph 133, wherein the bond between the cargo-binding domain and the cargo protein is a non-covalent bond. 135. The polypeptide of paragraph 133 or 134, wherein the bond between the cargo-binding domain and the cargo protein is a reversible bond. 136. The polypeptide of any one of paragraphs 133 to 135, wherein the binding between the cargo-binding domain and the cargo protein can be controlled. 137. The polypeptide of paragraph 136, wherein the binding between the cargo-binding domain and the cargo protein can be controlled by pH. 138. The polypeptide according to paragraph 136, wherein the binding between the cargo-binding domain and the cargo protein can be controlled by ionic strength. 139. The polypeptide of any one of paragraphs 133 to 138, wherein the binding between the cargo-binding domain and the cargo protein can be controlled such that the cargo protein binds to the cargo-binding domain in vitro but is released from the cargo-binding domain in vivo. 140. The polypeptide of any one of paragraphs 133 to 138, wherein the binding between the cargo-binding domain and the cargo protein can be controlled such that the cargo proteins are released from the cargo-binding domain in a manner that depends on the intracellular compartment in which they are located. 141. The polypeptide of any one of paragraphs 133 to 140, wherein the cargo-binding domain comprises a phosphotyrosine, the cargo protein or the SBD comprises a domain capable of binding to a phosphotyrosine, and the bond between the cargo-binding domain and the cargo protein is a bond between the phosphotyrosine and the domain capable of binding to the phosphotyrosine. 142. The polypeptide according to paragraph 141, wherein the domain capable of binding to phosphotyrosine is a PTB domain. 143. The polypeptide according to paragraph 141, wherein the domain capable of binding to phosphotyrosine is an SH2 domain. 144. The polypeptide of any one of paragraphs 133 to 140, wherein the cargo-binding domain comprises a first SAM domain, the cargo protein or the SBD comprises a second SAM domain, and the bond between the cargo-binding domain and the cargo protein is the bond between the first SAM domain and the second SAM domain. 145. The polypeptide of any one of paragraphs 133 to 140, wherein the cargo-binding domain comprises a PBM domain, the cargo protein or the SBD comprises a PDZ domain, and the bond between the cargo-binding domain and the cargo protein is the bond between the PBM domain and the PDZ domain. 146. The polypeptide of any one of paragraphs 133 to 140, wherein the cargo-binding domain comprises a PDZ domain, the cargo protein or the SBD comprises a PBM domain, and the bond between the cargo-binding domain and the cargo protein is the bond between the PDZ domain and the PBM domain. 147. The polypeptide of any one of paragraphs 120 to 132, wherein the polypeptide is a cargo protein. 148. The polypeptide of any one of paragraphs 114 to 119, wherein the cargo-binding domain comprises a PBM domain, the cargo protein or the SBD comprises a PDZ domain, and the bond between the cargo-binding domain and the cargo protein is the bond between the PBM domain and the PDZ domain. 149. The polypeptide of paragraph 148, wherein the bond between the ephrin receptor JM domain and the cargo protein is a non-covalent bond. 150. The polypeptide of paragraph 148 or 149, wherein the binding between the ephrin receptor JM domain and the cargo protein is a reversible binding. 151. The polypeptide of any one of paragraphs 148 to 150, wherein the binding between the ephrin receptor JM domain and the cargo protein can be regulated. 152. The polypeptide of paragraph 151, wherein the binding between the ephrin receptor JM domain and the cargo protein can be controlled by pH. 153. The polypeptide of paragraph 151, wherein the binding between the ephrin receptor JM domain and the cargo protein can be controlled by ionic strength. 154. The polypeptide of any one of paragraphs 148 to 153, wherein the binding between the ephrin receptor JM domain and the cargo protein can be controlled so that the cargo protein binds to the ephrin receptor JM domain in vitro but is released from the ephrin receptor JM domain in vivo. 155. The polypeptide of any one of paragraphs 148 to 153, wherein the binding between the ephrin receptor JM domain and the cargo protein can be controlled such that the cargo protein is released from the ephrin receptor JM domain in a manner that depends on the intracellular compartment in which it is located. 156. The polypeptide of any one of paragraphs 148 to 155, wherein the Ephrin receptor JM domain comprises a phosphotyrosine, the cargo protein or the SBD comprises a domain capable of binding to phosphotyrosine, and the bond between the Ephrin receptor JM domain and the cargo protein is a bond between the phosphotyrosine and the domain capable of binding to phosphotyrosine. 157. The polypeptide according to paragraph 156, wherein the domain capable of binding to phosphotyrosine is a PTB domain. 158. The polypeptide according to paragraph 156, wherein the domain capable of binding to phosphotyrosine is an SH2 domain. 159. The ephrin receptor JM domain is (i) the (X1)-Ptyr-(X2) motif (where Ptyr is phosphotyrosine, X1 is Y, P, V, I, T, or F, and X2 is I, V, L, or A); (ii) a (X3)-Ptyr-(X4) motif, where Ptyr is phosphotyrosine, X3 is T, A, or S, and X4 is E or G; or (iii) Both (i) and (ii) 159. The polypeptide of any one of paragraphs 156 to 158, comprising: 160. The polypeptide of any one of paragraphs 114 to 119, wherein the polypeptide is capable of binding to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises an ephrin receptor KD at the C-terminus of the TM domain. 161. The polypeptide according to paragraph 160, wherein the bond between the ephrin receptor KD and the cargo protein is a non-covalent bond. 162. The polypeptide according to paragraph 160 or 161, wherein the binding between the ephrin receptor KD and the cargo protein is a reversible binding. 163. The polypeptide according to any one of paragraphs 160 to 162, wherein the binding between the ephrin receptor KD and the cargo protein can be regulated. 164. The polypeptide according to paragraph 163, wherein the binding between the ephrin receptor KD and the cargo protein can be controlled by pH. 165. The polypeptide according to paragraph 163, wherein the binding between the ephrin receptor KD and the cargo protein can be controlled by ionic strength. 166. A polypeptide described in any one of paragraphs 160 to 165, wherein the binding between the ephrin receptor KD and the cargo protein can be controlled so that the cargo protein binds to the ephrin receptor KD in vitro but is released from the ephrin receptor KD in vivo. 167. A polypeptide described in any one of paragraphs 160 to 165, wherein the binding between the ephrin receptor KD and the cargo protein can be controlled so that the cargo protein is released from the ephrin receptor KD in a manner that depends on the intracellular compartment in which it is located. 168. A polypeptide described in any one of paragraphs 160 to 167, wherein the ephrin receptor KD comprises a phosphotyrosine, the cargo protein or the SBD comprises a domain capable of binding to phosphotyrosine, and the bond between the ephrin receptor KD and the cargo protein is a bond between the phosphotyrosine and the domain capable of binding to phosphotyrosine. 169. The polypeptide according to paragraph 168, wherein the domain capable of binding to phosphotyrosine is a PTB domain. 170. The polypeptide according to paragraph 168, wherein the domain capable of binding to phosphotyrosine is an SH2 domain. 171. The polypeptide of any one of paragraphs 168 to 170, wherein the KD comprises a (X7)-Ptyr-(X8) motif in the activation loop, where Ptyr is phosphotyrosine, X7 is T, V, or A, and X8 is E or T. 172. The polypeptide of any one of paragraphs 114 to 119, wherein the polypeptide is capable of binding to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises a SAM linker domain C-terminal to the TM domain. 173. The polypeptide of paragraph 172, wherein the bond between the SAM linker domain and the cargo protein is a non-covalent bond. 174. The polypeptide of paragraph 172 or 173, wherein the bond between the SAM linker domain and the cargo protein is a reversible bond. 175. The polypeptide of any one of paragraphs 172 to 174, wherein the binding between the SAM linker domain and the cargo protein can be controlled. 176. The polypeptide of paragraph 175, wherein the binding between the SAM linker domain and the cargo protein can be controlled by pH. 177. The polypeptide of paragraph 175, wherein the binding between the SAM linker domain and the cargo protein can be controlled by ionic strength. 178. The polypeptide of any one of paragraphs 172 to 177, wherein the bond between the SAM linker domain and the cargo protein can be controlled such that the cargo protein binds to the SAM linker domain in vitro but is released from the SAM linker domain in vivo. 179. The polypeptide of any one of paragraphs 172 to 177, wherein the binding between the SAM linker domain and the cargo protein can be controlled such that the cargo proteins are released from the SAM linker domain in a manner that depends on the intracellular compartment in which they are located. 180. The polypeptide of any one of paragraphs 172 to 179, wherein the SAM linker domain comprises a phosphorylated amino acid or a phosphomimetic amino acid, the cargo protein or the SBD comprises a domain capable of binding to the phosphorylated amino acid or phosphomimetic amino acid, and the bond between the SAM linker domain and the cargo protein is a bond between the phosphorylated amino acid or phosphomimetic amino acid and the domain capable of binding to the phosphorylated amino acid or phosphomimetic amino acid. 181. The polypeptide of any one of paragraphs 172 to 180, wherein the SAM linker domain is an ephrin receptor SAM linker domain. 182. The polypeptide of any one of paragraphs 114 to 119, wherein the polypeptide is capable of binding to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises a SAM domain C-terminal to the TM domain. 183. The polypeptide of paragraph 182, wherein the bond between the SAM domain and the cargo protein is a non-covalent bond. 184. The polypeptide of paragraph 182 or 183, wherein the bond between the SAM domain and the cargo protein is a reversible bond. 185. The polypeptide of any one of paragraphs 182 to 184, wherein the binding between the SAM domain and the cargo protein can be controlled. 186. The polypeptide of paragraph 185, wherein the binding between the SAM domain and the cargo protein can be controlled by pH. 187. The polypeptide of paragraph 185, wherein the binding between the SAM domain and the cargo protein can be controlled by ionic strength. 188. The polypeptide of any one of paragraphs 182 to 187, wherein the binding between the SAM domain and the cargo protein can be controlled such that the cargo protein binds to the SAM domain in vitro but is released from the SAM domain in vivo. 189. The polypeptide of any one of paragraphs 182 to 187, wherein the binding between the SAM domain and the cargo protein can be controlled such that the cargo proteins are released from the SAM domain in a manner that depends on the intracellular compartment in which they are located. 190. The polypeptide of any one of paragraphs 182 to 189, wherein the cargo protein or the SBD comprises a second SAM domain, and the bond between the SAM domain and the cargo protein is the bond between the SAM domain and the second SAM domain. 191. The polypeptide of any one of paragraphs 182 to 189, wherein the SAM domain comprises a phosphotyrosine, the cargo protein or the SBD comprises a domain capable of binding to a phosphotyrosine, and the bond between the SAM domain and the cargo protein is a bond between the phosphotyrosine and the domain capable of binding to the phosphotyrosine. 192. The polypeptide according to paragraph 191, wherein the domain capable of binding to phosphotyrosine is a PTB domain. 193. The polypeptide according to paragraph 191, wherein the domain capable of binding to phosphotyrosine is an SH2 domain. 194. The polypeptide of any one of paragraphs 191-193, wherein the SAM domain comprises a phosphotyrosine in the α2 helix. 195. The polypeptide of paragraph 194, wherein the phosphotyrosine in the α2 helix of the SAM domain is in a (X5)-Ptyr-(X6) motif (wherein Ptyr is phosphotyrosine, X5 is C, R, Q, or H, and X6 is Q, I, E, K, R, or T). 196. The polypeptide of any one of paragraphs 182 to 195, wherein the SAM domain is an ephrin receptor SAM domain. 197. The polypeptide of any one of paragraphs 114 to 119, wherein the polypeptide is capable of binding to a cargo protein directly or indirectly via an SBD linked to the cargo protein, and further comprises an ephrin receptor PDZ binding motif (PBM) domain C-terminal to the TM domain. 198. The polypeptide of paragraph 197, wherein the bond between the ephrin receptor PBM domain and the cargo protein is a non-covalent bond. 199. The polypeptide of paragraph 197 or 198, wherein the binding between the ephrin receptor PBM domain and the cargo protein is a reversible binding. 200. The polypeptide of any one of paragraphs 197 to 199, wherein the binding between the ephrin receptor PBM domain and the cargo protein can be controlled. 201. The polypeptide of paragraph 200, wherein the binding between the ephrin receptor PBM domain and the cargo protein can be controlled by pH. 202. The polypeptide of paragraph 200, wherein the binding between the ephrin receptor PBM domain and the cargo protein can be controlled by ionic strength. 203. The polypeptide of any one of paragraphs 197 to 202, wherein the binding between the Ephrin receptor PBM domain and the cargo protein can be controlled such that the cargo protein binds to the Ephrin receptor PBM domain in vitro but is released from the Ephrin receptor PBM domain in vivo. 204. The polypeptide of any one of paragraphs 197 to 202, wherein the binding between the ephrin receptor PBM domain and the cargo protein can be controlled such that the cargo protein is released from the ephrin receptor PBM domain in a manner that depends on the intracellular compartment in which it is located. 205. The polypeptide of any one of paragraphs 197 to 204, wherein the cargo protein or the SBD comprises a PDZ domain, and the binding between the ephrin receptor PBM domain and the cargo protein is the binding between the ephrin receptor PBM domain and the PDZ domain. 206. The polypeptide of any one of paragraphs 120 to 205, wherein the cargo protein is a therapeutic protein. 207. The polypeptide of paragraph 206, wherein said therapeutic protein is a therapeutic antibody or antigen-binding fragment thereof. 208. The EV or hybridosome according to paragraph 206, wherein the therapeutic protein is a gene editor or a transposase. 209. The polypeptide of any one of paragraphs 120 to 205, wherein the cargo protein is a diagnostic protein. 210. The polypeptide of paragraph 209, wherein the diagnostic protein is a fluorescent protein. 211. The polypeptide of any one of paragraphs 114 to 210, wherein said polypeptide lacks an ephrin receptor ligand binding domain (LBD). 212. The polypeptide of any one of paragraphs 114 to 210, wherein said polypeptide comprises a mutated ephrin receptor LBD. 213. The polypeptide of any one of paragraphs 114 to 210, wherein the polypeptide comprises two distinct domains that enable the polypeptide to undergo heterodomain dimerization with another polypeptide identical to the polypeptide. 214. The polypeptide of any one of paragraphs 114 to 210, wherein the polypeptide comprises two different domains in a head-to-tail arrangement that enable the polypeptide to undergo heterodomain dimerization with another polypeptide identical to the polypeptide. 215. The polypeptide of any one of paragraphs 114 to 214, wherein the TM domain is an ephrin receptor TM domain. 216. The polypeptide of any one of paragraphs 114 to 215, wherein any one or more of the ephrin receptor domains of said polypeptide are or are derived from EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, or a combination thereof. 217. The polypeptide of any one of paragraphs 114 to 215, wherein any one or more of the ephrin receptor domains of said polypeptide are or are derived from EphA2, EphA4, EphB2, or a combination thereof. 218. The polypeptide of any one of paragraphs 114 to 217, wherein the polypeptide further comprises a modified Fc domain of an immunoglobulin. 219. The polypeptide of paragraph 218, wherein the modified Fc domain is N-terminal to the ephrin receptor CR domain. 220. The polypeptide of paragraph 219, wherein the modified Fc domain is fused to the remainder of the polypeptide by a linker sequence. 221. The modified Fc domain is a. capable of specifically binding to the Fc binding site of the neonatal Fc receptor (FcRn); and B. lacks the ability to form homodimers; A polypeptide described in any one of paragraphs 218 to 220. 222. The dissociation constant of the modified Fc domain bound to FcRn at a pH of 6.5 is at most 10 -4 222. The polypeptide of any one of paragraphs 218 to 221, having a value of M. 223. The dissociation constant of the modified Fc domain bound to FcRn at a pH of 7.4 is at least 10 -4 223. The polypeptide of any one of paragraphs 218 to 222, having a value of M. 224. The modified Fc domain has the amino acid sequence [ka] (wherein X1, X2, X3, X4, X5, X6, X7, and X8 are each any amino acid). 225. The polypeptide of any one of paragraphs 218 to 224, wherein the modified Fc domain is capable of specifically binding to an amino acid sequence between positions 135 and 158 of human FcRn (SEQ ID NO: 228) and / or mouse FcRn (SEQ ID NO: 227). 226. The polypeptide of any one of paragraphs 218-225, wherein the polypeptide does not substantially bind to C1q, FcγRI, FcγRII, or FcγRIII. 227. a. the complement-dependent cytotoxicity (CDC) activity of the modified Fc domain; b. antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the modified Fc domain; c. antibody-dependent cell-mediated phagocytosis (ADCP) activity of the modified Fc domain; and / or d. Antibody-dependent intracellular neutralization (ADIN) activity of the modified Fc domain is reduced by at least 10%, 20%, 30%, 40%, or 50% compared to the unmodified Fc domain : A polypeptide described in any one of paragraphs 218 to 226. 228. a. the complement-dependent cytotoxicity (CDC) activity of the modified Fc domain; b. antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the modified Fc domain; c. antibody-dependent cell-mediated phagocytosis (ADCP) activity of the modified Fc domain; and / or d. Antibody-dependent intracellular neutralization (ADIN) activity of the modified Fc domain is reduced by at least 1.5, 2, 3, 4, or 5 fold compared to the unmodified Fc domain, : A polypeptide described in any one of paragraphs 218 to 227. 229. The modified Fc domain comprises, from the N-terminus to the C-terminus: a. a modified CH2 domain that has been modified to decrease effector function relative to the unmodified CH2 domain; and b. A modified CH3 domain that has been modified so as to lack the ability to form homodimers 229. The polypeptide of any one of paragraphs 218 to 228, comprising: 230. The polypeptide of any of paragraphs 114 to 229, wherein the first Ephrin receptor FN III domain and the second Ephrin receptor FN III domain comprise different amino acid sequences. 231. A nucleic acid encoding a polypeptide according to any one of paragraphs 114 to 230. 232. An expression vector comprising the nucleic acid of paragraph 231. 233. A cell comprising a nucleic acid according to paragraph 231 or an expression vector according to paragraph 232. 234. A method for producing EVs, comprising: a. transfecting a cell with the nucleic acid of Paragraph 231 or the expression vector of Paragraph 232; b. culturing the cells under conditions suitable for the production of the EVs; and c. Recovering EVs secreted by the cells. The method comprising: 235. A method for producing hybridosomes, comprising contacting a first EV with a second EV, thereby combining the first EV with the second EV and producing the hybridosome; wherein the first EV is produced in vitro and comprises (i) a membrane, and (ii) a fusogenic, ionizable cationic lipid; and wherein said second EVs are produced by the method of paragraph 234. 236. A method for purifying EVs or hybridosomes, comprising: a. providing the EV or hybridosome, wherein the EV or hybridosome comprises a first binding partner, wherein the first binding partner is capable of binding to the Fc-binding site of FcRn in a pH-dependent manner; and b. contacting the EVs or hybridosomes containing the first binding partner with a second binding partner at a first pH, wherein the second binding partner comprises the Fc binding site of the FcRn and is associated with a solid matrix; and c. eluting the EVs or hybridosomes containing the first binding partner from the solid matrix at a second pH. 237. The method of paragraph 236, wherein the method further comprises a washing step at the first pH. 238. The method of paragraph 236 or 237, wherein the first pH is less than 6.5. 239. The method of any one of claims 236-238, wherein the second pH is greater than 7.4. 240. The method of any one of claims 236-239, wherein the Fc binding site of FcRn comprises the amino acid sequence of SEQ ID NO: 230. 241. A method for purifying EVs or hybridosomes, comprising: a. providing the EV or hybridosome, wherein the EV or hybridosome comprises a first binding partner, wherein the first binding partner is capable of binding to the Fc-binding site of FcRn in a pH-dependent manner and comprises or consists of a polypeptide according to any one of paragraphs 111 to 224; and b. contacting the EVs or hybridosomes containing the first binding partner with a second binding partner at a first pH, wherein the second binding partner comprises the Fc binding site of the FcRn and is associated with a solid matrix; and c. eluting the EVs or hybridosomes containing the first binding partner from the solid matrix at a second pH. 242. The method of paragraph 241, wherein the method further comprises a washing step at the first pH. 243. The method of paragraph 241 or 242, wherein the first pH is less than 6.5. 244. The method of any one of claims 241-243, wherein the second pH is greater than 7.4. 245. The method of any one of claims 241-244, wherein the Fc binding site of FcRn comprises the amino acid sequence of SEQ ID NO: 230. [Brief explanation of the drawings]

[0065] (4. Brief description of the drawings) [Figure 1]Figure 1A is a diagram of the structure of an Eph receptor. Figure 1B is a diagram of exemplary versions of scaffold proteins containing the following domains: (i) CRD-FNIII-FNIII-TM, (ii) CRD-FNIII-FNIII-TM-JM, (iii) CRD-FNIII-FNIII-TM-JM-KD, (iv) CRD-FNIII-FNIII-TM-JM-SAM-PBM, (v) LBD*-CRD-FNIII-FNIII-TM, (vi) LBD*-CRD-FNIII-FNIII-TM-JM, (vii) LBD*-CRD-FNIII-FNIII-TM-JM-KD, or (viii) LBD*-CRD-FNIII-FNIII-TM-JM-SAM-PBM. LBD* denotes a mutated LBD that has reduced ephrin binding compared to the wild-type LBD.

[0066] [Figure 2] Figure 2A shows a homodomain dimerization interface and a dimer of polypeptides, and Figure 2B shows a heterodomain dimerization interface and an oligomer of polypeptides in a head-to-tail configuration.

[0067] [Figure 3] Figure 3A shows the interaction of Ephrin2 receptor-binding domain residue E129 with EphA4 LBD residue R106. Figure 3B shows the interaction of EphA4 FNIII residues N504 and N07 with EphA4 LBD residue R106 in the head-to-tail cluster. Mutagenesis of EphA4 LBD residue R106 to E impairs EphA4-ephrin2 interaction. R106 interacts (H-bonds) with N504 and T507. To preserve the head-to-tail configuration with impaired ephrin binding, a mutation, T507N, can be introduced, creating a new H-bond with R106E, while N504 can be left intact and maintain its interaction with R106E.

[0068] [Figure 4]Figure 4. Diagram of the FNIII-LBD (head-to-tail) interaction between two EphA4 scaffolds and the N-terminus linked to the fusion moiety via a linker.

[0069] [Figure 5] Figure 5. Diagram of ephrin-independent LBD-LBD interactions between two EphA4 LBDs and the N-terminus linked to the fusion moiety via a linker distal to the homodomain dimerization interface.

[0070] [Figure 6] Figure 6. Diagram of the interactions between two EphA4 scaffolds via the CRD and LBD homodomain interfaces and the N-terminus linked to the fusion moiety via a linker.

[0071] [Figure 7] Figure 7. Diagram of the interaction between two LBD truncated EphA4 scaffolds via the CRD homodomain interface and the N-terminus linked to the fusion moiety via a linker.

[0072] [Figure 8] Figure 8. Exemplary structures of new scaffolds derived from Eph receptors with mutated (including truncated) or no ligand binding domain (LBD) linked to a targeting domain and / or modified Fc domain.

[0073] [Figure 9] Figure 9. Exemplary schematic of cargo protein loading by covalent attachment to scaffold polypeptides during EV biogenesis.

[0074] [Figure 10] Figure 10. Exemplary schematic of phosphotyrosine-based reversible binding of cargo proteins to scaffold polypeptides via scaffold-binding domains (SBDs) during EV biogenesis.

[0075] [Figure 11] Figure 11. Alignment of LBD sequences of human Eph receptors showing β-strands D-M and corresponding loops (SEQ ID NO: 243 from EphAl, SEQ ID NO: 244 from EphA2, SEQ ID NO: 245 from EphA3, SEQ ID NO: 246 from EphA4, SEQ ID NO: 247 from EphA5, SEQ ID NO: 248 from EphA6, SEQ ID NO: 249 from EphA7, SEQ ID NO: 250 from EphA8, SEQ ID NO: 251 from EphAlO, SEQ ID NO: 252 from EphBl, SEQ ID NO: 253 from EphB2, SEQ ID NO: 254 from EphB3, and SEQ ID NO: 255 from EphB4).

[0076] [Figure 12] Figure 12. Illustrative schematic of several scaffold proteins interacting with adaptor proteins.

[0077] [Figure 13] Figure 13. Western blot showing EVs purified from conditioned medium contained full-length scaffold proteins with intraluminal turboluc.

[0078] [Figure 14] Figures 14A and 14B show binding curves from FcRn binding immunoassays using EVs expressing modified Fc domains (Figure 14A), native EVs (Figure 14A), human IgG1 (Figure 14B), and mouse IgG1 (Figure 14B).

[0079] [Figure 15] Figure 15. Anti-EphA4 Western blot showing detection of EphA4 fusion protein expressed from constructs in concentrated conditioned medium loaded onto an scFcRn column. Lane 1 is the load, lane 2 is a flow-through sample, and lane 3 is a sample of the elution fraction.

[0080] [Figure 16]Figure 16. Percentage of cells that were RFP+ as determined by flow cytometry of color-switched HEK293T cells expressing EphA2 and treated with various doses of Cre mRNA-loaded hybridosomes derived from EVs containing scaffold proteins and LNPs targeting EphA2 or mouse CD64 as a non-targeting control.

[0081] [Figure 17] Figure 17A. Schematic of lentiviral polycistronic construct for non-covalent loading of cargo into the lumen of EVs. Figure 17B. Anti-turboluc Western blot showing the presence of turboluc-SH2-SBX100 protein in harvested EV samples. Figure 17C. Luminescence of trypsin-treated and untreated harvested turboluc-SH2-SBX100 EVs.

[0082] [Figure 18] Figure 18. DNA vector copy numbers per ul of mouse plasma at days 3, 6, 21, and 24 after IV administration of EV and LNP formulations containing scaffold proteins presenting modified Fc domains.

[0083] [Figure 19]Figure 19. Crystal structures of the following Eph receptor ectodomain chains obtained from the Protein Data Bank (PDB) and superimposed according to Xu Q, Dunbrack RL Jr., Bioinformatics. 2012;28(21):2763-2772: EphB2_MOUSE_lbd (PDB:1kgy), EphB4_HUMAN_lbd (PDB:2bba), EphB2_HUMAN_lbd (PDB:2qbx), EphA4_HUMAN_lbd (PDB:2wo1), EphA2_HUMAN_lbd_fn3_fn3 (PDB:3fl7), EphA2_HUMAN_lbd (PDB:3mbw), EphA2_HUMAN_lbd_fn3(PDB:3mx0), EphA7_HUMAN_lbd(PDB:3nru), EphB3_HUMAN_lbd(PDB:3p1i), Schematic diagram of EphA5_HUMAN_lbd (PDB:4et7), EphA3_HUMAN_lbd (PDB:4l0p), and EphA4_HUMAN_lbd (PDB:4w50). DETAILED DESCRIPTION OF THE INVENTION

[0084] (5. Detailed Description) The present disclosure is based, at least in part, on the discovery that ephrin (Eph) receptors can be sorted into nanovesicles. Thus, ephrin receptors enable the transport, trafficking, or shuttling of cargo (e.g., cargo proteins) into nanovesicles (e.g., extracellular vesicles (EVs) or hybridosomes). Therefore, ephrin receptors, particularly ephrin receptor mutants modified to exhibit reduced or no ephrin binding and therefore reduced or no reverse signaling, can be used as neutral protein scaffolds suitable for loading cargo (e.g., cargo proteins) onto or within nanovesicles. This makes the polypeptides described herein containing ephrin receptor domains (also referred to herein as ephrin receptor-derived polypeptides) attractive protein scaffolds for displaying cargo (e.g., cargo proteins) on or within nanovesicles (e.g., EVs and hybridosomes).

[0085] Provided herein are polypeptides that include a transmembrane domain and further include an ectodomain and / or an endodomain that can be used to load a cargo (e.g., a cargo protein) onto or into the surface of nanovesicles (e.g., EVs and hybridosomes). The cargo (e.g., cargo protein) can be a portion of a polypeptide described herein. That is, the cargo (e.g., cargo protein) can be fused to the remainder of the polypeptide (e.g., via a linker). Alternatively, the cargo (e.g., cargo protein) can be bound (preferably reversibly bound) to the polypeptide via a domain capable of binding to the cargo (e.g., cargo protein), i.e., a cargo-binding domain. The cargo-binding domain can bind to the cargo (e.g., cargo protein) directly or indirectly via a scaffold-binding domain (SBD) linked to the cargo (e.g., cargo protein). As used herein, the singular forms "a," "an," and "the" include plural referents. Thus, the polypeptides described herein can be used to deliver one or more (e.g., 1, 2, 3, 4, 5, or more) cargoes, and the polypeptides described herein can comprise one or more (e.g., 1, 2, 3, 4, 5, or more) cargoes or one or more (e.g., 1, 2, 3, 4, 5, or more) cargo-binding domains.

[0086] The polypeptides described herein can further comprise one or more functional moieties, such as a targeting domain capable of targeting the nanovesicle (e.g., EV or hybridosome) to a particular organ, tissue, or cell type, and / or a purification domain capable of facilitating the purification of the nanovesicle (e.g., EV or hybridosome).

[0087] Preferably, the polypeptides described herein comprise one or more domains from or derived from one or more Eph receptors. Such polypeptides are also referred to herein as Eph receptor-derived polypeptides or polypeptides derived from Eph receptors. Ephrin receptor-derived polypeptides may or may not comprise one or more domains (e.g., transmembrane domains) from or derived from non-Ephrin receptor proteins. In various embodiments, the polypeptides described herein comprise an ectodomain or fragment thereof (e.g., flexible domain) from or derived from one or more Ephrin receptors, a transmembrane domain from or derived from an Ephrin receptor or non-Ephrin receptor transmembrane protein, and, optionally, an endodomain or fragment thereof from or derived from one or more Ephrin receptors.

[0088] In particular, provided herein are polypeptides comprising at least an ephrin receptor cysteine-rich (CR) domain, two ephrin receptor fibronectin type III (FN III) domains (i.e., a first ephrin receptor FN III domain (ephrin receptor FN1 domain) and a second ephrin receptor FN III domain (ephrin receptor FN2 domain)), and a transmembrane (TM) domain (e.g., an ephrin receptor TM domain), and optionally, a cargo-binding domain, an ephrin receptor juxtamembrane (JM) domain, an ephrin receptor kinase domain (KD), a sterile alpha-motif (SAM) linker domain (e.g., an ephrin receptor linker-SAM domain), a SAM domain (e.g., an ephrin receptor SAM domain), an ephrin receptor PDZ-binding motif (PBM) domain, a targeting domain, a purification domain, a modified Fc domain, and / or a ligand-binding domain (LBD). Various aspects and embodiments of the polypeptides are described in Section 5.2. The ephrin receptor LBD, ephrin receptor CR domain (CRD), ephrin receptor FN III domain, and TM domain (e.g., ephrin receptor TM domain) are further described in Section 5.2.1. The ephrin receptor JM domain, ephrin receptor KD, SAM linker domain (e.g., ephrin receptor SAM linker domain), SAM domain (e.g., ephrin receptor SAM domain), and ephrin receptor PBM domain are further described in Section 5.2.2. Cargo binding domains are further described in Section 5.2.3. Targeting domains and purification domains are further described in Section 5.2.4. Modified Fc domains are further described in Section 5.2.5.

[0089] The polypeptides described herein can be used to deliver cargo (e.g., cargo proteins), for example, via extracellular vesicles (EVs) or hybridosomes, for example, for therapeutic or diagnostic applications. The cargo (e.g., cargo proteins) can be a portion of the polypeptide. That is, the cargo (e.g., cargo proteins) can be fused to the remainder of the polypeptide (e.g., via a linker). Alternatively, the cargo (e.g., cargo proteins) can be linked (preferably reversibly linked) to the polypeptide via a cargo-binding domain. The cargo-binding domain can be directly linked to the cargo (e.g., cargo proteins) or indirectly linked via an SBD linked to the cargo (e.g., cargo proteins). The cargo-binding domain can be either an ephrin receptor domain (e.g., an ephrin receptor JM domain, an ephrin receptor KD, an ephrin receptor SAM linker domain, an ephrin receptor SAM domain, or an ephrin receptor PBM domain), or a domain that can bind to cargo but is not an ephrin receptor domain. The reversible bond between the cargo (e.g., cargo protein) and the cargo-binding domain can be, but is not limited to, a phosphotyrosine-based bond (e.g., a bond between a phosphotyrosine and a phosphotyrosine-binding (PTB) domain, or a bond between a phosphotyrosine and a Src homology 2 (SH2) domain), a SAM domain-based bond, a PDZ domain-based bond, or a DH-PH motif-based bond. Cargo (e.g., cargo proteins) and cargo-binding domains are further described in Section 5.2.3. Ephrin receptor domains are further described in Sections 5.2.1 and 5.2.2.

[0090] Any one or more of the ephrin receptor domains described herein can be from or derived from the same or different ephrin receptors. In some embodiments, the polypeptides described herein comprise ephrin receptor domains from or derived from the same ephrin receptor. In other embodiments, the polypeptides described herein comprise ephrin receptor domains from or derived from two, three, or more than three ephrin receptors. In specific embodiments, any one or more of the ephrin receptor domains of the polypeptides described herein are from or derived from EphA1, EphA2, EphHA3, EphA4, EphA5, EphHA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, or a combination thereof. In specific embodiments, any one or more of the ephrin receptor domains of the polypeptides described herein are or are derived from EphA2, EphA4, EphB2, or a combination thereof.

[0091] Any one or more of the ephrin receptor domains described herein can be wild-type or mutant ephrin receptor domains. In certain embodiments, the ephrin receptor domains described herein have at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the corresponding domain of a wild-type ephrin receptor (e.g., an ephrin receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 212-225). In certain embodiments, the ephrin receptor domains described herein comprise the amino acid sequence of the corresponding domain of a wild-type ephrin receptor (e.g., an ephrin receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 212-225), except for one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, seven amino acid mutations, or more than seven amino acid mutations. The mutations can be substitutions, insertions, deletions, or any combination thereof.

[0092] In various embodiments, adaptor proteins can be used to bring the polypeptides described herein into close proximity (e.g., clustered) with one another on nanovesicles (e.g., EVs or hybridosomes). Adaptor proteins are further described in Section 5.2.3(c).

[0093] Also provided are nucleic acids encoding the polypeptides described herein, expression vectors comprising the nucleic acids described herein, and cells comprising the nucleic acids or expression vectors described herein, all of which are further described in Section 5.3.

[0094] Further provided are nanovesicles (e.g., EVs and hybridosomes) comprising the polypeptides described herein. Nanovesicles (e.g., EVs and hybridosomes) are further described in Section 5.4.

[0095] Methods for producing or purifying nanovesicles (e.g., EVs or hybridosomes) are also provided and are further described in Section 5.4.

[0096] Compositions and kits comprising the polypeptides, nanovesicles (e.g., EVs or hybridosomes), nucleic acids, expression vectors, or cells described herein are provided and are further described in Section 5.5.

[0097] Therapeutic and diagnostic uses of the polypeptides, nanovesicles (e.g., EVs or hybridosomes), compositions, or kits described herein are provided and further described in Section 5.6.

[0098] (5.1 Definition) As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "polypeptide" can include two or more such molecules, and the like.

[0099] As used herein, the terms "about" and "approximately," when used to modify a quantity specified by a numerical value or range, indicate not only the numerical value but also reasonable deviations from values ​​known to those of ordinary skill in the art, e.g., ±20%, ±10%, or ±5%, within the intended meaning of the recited value. It is further understood that slight variations above and below the stated range can be used to achieve substantially the same results as values ​​within the range. Also, unless otherwise indicated, disclosure of a range is intended as a continuous range, including every value between the minimum and maximum values.

[0100] Terms such as "comprising," "having," "including," "containing," and the like are to be read expansively or open-endedly and without limitation.

[0101] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. The terms "at least one" and "at least one of" include, for example, 1, 2, 3, 4, or 5 or more elements.

[0102] The term "nanovesicles" refers to lipid nanovesicles derived from source cells (i.e., extracellular vesicles), as well as synthetic lipid nanoparticles and natural / synthetic hybrids (e.g., hybridosomes). Nanovesicles typically comprise lipids or fatty acids and polypeptides, and may further comprise a payload, targeting moiety, or other molecule. Furthermore, when teachings herein refer to a singular nanovesicle, it should be understood that all such teachings are equally relevant and applicable to a plurality of nanovesicles and populations of nanovesicles. When describing medical and scientific uses and applications of nanovesicles, it will be apparent to those skilled in the art that the present disclosure generally relates to a plurality of nanovesicles, i.e., populations of nanovesicles that may include thousands, millions, billions, or even trillions of nanovesicles. As can be seen from the experimental section below, nanovesicles can be as dense as 10 nanovesicles per unit volume (e.g., per ml). 5 , 10 8 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 18 , 10 25 , 10 30 The nanovesicles may be present in a concentration of particles, or any other number greater than, less than, or anywhere in between. Individual nanovesicles, when present in plurality, constitute a nanovesicle population. It should be understood, therefore, that the present disclosure relates to both individual nanovesicles and populations comprising nanovesicles.

[0103] The terms "extracellular vesicles," "EVs," or "exosomes" are used interchangeably herein and are understood to refer to any type of vesicle obtained from any cell, including, for example, microvesicles (e.g., any vesicles shed from the plasma membrane of a cell), exosomes (e.g., any vesicles derived from the endosomal, lysosomal, and / or endo-lysosomal pathways), apoptotic bodies, ARMMs (arrestin domain-containing protein 1 [ARRDC1]-mediated microvesicles), fusosomes, microparticles, and cell-derived vesicular structures. Extracellular vesicles typically have hydrodynamic diameters ranging from 20 nm to 1000 nm and can contain a variety of macromolecular cargoes (or "payloads") within the interior space, displayed on the outer surface of the extracellular vesicle, and / or spanning the membrane. The cargo can include nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example, and not limitation, extracellular vesicles include apoptotic bodies, cell fragments, vesicles obtained from cells by direct or indirect manipulation (e.g., by continuous extrusion, sonication, or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). Extracellular vesicles can be obtained from living or dead organisms, explanted tissues or organs, and / or cultured cells.

[0104] As used herein, the term "hybridosome" refers to a hybrid biocompatible carrier comprising at least one extracellular vesicle (EV)-derived structural and bioactive element (e.g., lipid, carbohydrate, fatty acid, polynucleotide, or polypeptide) and at least one artificial drug encapsulation module (EDEM) with a tunable fusogenic moiety. The fusogenic moiety may be a fusogenic lipid or any other fusogenic component that enhances or enables membrane disruption or lipid mixing between the membrane and the lipid bilayer. Hybridosomes are generated by combining one EV with one EDEM, one EV with several EDEMs, several EVs with one EDEM, or several EVs with several EDEMs. The combining event can be controlled by the size of the EV and EDEM, their respective charges, and the conditions applied during the combining reaction, such as the EV / EDEM ratio, pH, temperature, and reaction time. Hybridosomes and methods for producing them are described in detail in WO2015110957, the entire contents of which are incorporated herein by reference.

[0105] The term "Eph receptor" or "ephrin receptor" refers to a subfamily of receptor tyrosine kinases (RTKs) that bind to a group of cell membrane-associated ligands known as ephrins. Through ligand-induced activation of their kinase domains, Eph receptors transduce signals from the extracellular to the intracellular space. Thus, Eph receptors mediate contact-associated intercellular communication by interacting with ephrins on neighboring cells. Binding of an Eph receptor to an ephrin results in activation of the Eph receptor's kinase domain. Eph receptor-ephrin binding events can lead to endocytosis of the receptor-ligand complex, and the activated receptor continues to transduce signals from the intracellular compartment until it is inactivated by dephosphorylation and degradation or transported back to the cell surface. In humans, the Eph receptor family has a highly conserved overall structure, with the EphA and EphB receptor classes sharing the same structural features and domains. Eph receptor domains are cataloged in the National Center for Biotechnology Information (NCBI) Conserved Domain Database, including a list of sequence / structure / function relationships. The class consists of 10 EphA and 5 EphB members, classified according to sequence homology. The ectodomain of the Eph receptor domain contains a conserved N-terminal ligand-binding domain (LBD, SMART accession number SM00615), which binds the receptor to its ephrin ligands. The LBD of Eph receptors consists of a β-strand DM and corresponding loops, as shown in Figure 11. Complex formation between Eph receptors and ephrins revolves around the insertion of the ephrin GH loop into the Eph receptor hydrophobic channel, formed by a convex sheet of four β-strands combined with the DE, JK, and GH loops. The major sequence differences between EphA and EphB receptors reside in the region of the ligand-binding domain, which determines ephrin subclass binding specificity.Adjacent to the LBD is a cysteine-rich region containing the sushi domain and epidermal growth factor (EGF)-like domain, followed by two fibronectin type III domains (FN1 and FN2). Transitioning from the transmembrane domain, the cytoplasmic Eph receptor region contains a kinase domain, a sterile alpha motif (SAM) domain, and a PDZ-binding motif (PBM). The LBD is unique to this RTK family and shares no significant amino acid sequence homology with other known proteins. See Figure 1A for a schematic representation of a wild-type Eph receptor and Figure 1B for a schematic representation of an exemplary scaffold protein containing domains derived from or derived from an Eph receptor.

[0106] As used herein, the term "domain" refers to a unit (e.g., segment) of a polypeptide that can fold independently into a stable tertiary structure. Domains are typically responsible for distinct functional properties of a protein and can often be added, removed, or transferred to other proteins without loss of function of the remainder of the protein and / or the domain. Several different domains can be combined in different combinations to form multi-domain polypeptides. Traditionally, the length of a polypeptide spanning a domain has been elucidated using atomic coordinates from the experimentally determined three-dimensional structure of the protein. More recently, proteins lacking an experimentally determined three-dimensional (3D) structure have been assigned to domains using computational methods based on sequence homology. Because many proteins have unsolved structures, sequence-based approaches have attracted more attention. As reviewed in Wang, Yan et al., Computational and structural biotechnology journal, vol. 19, pp. 1145-1153, February 2, 2021, sequence-based approaches include template-based methods, homology modeling-based methods, and machine learning-based methods, depending on whether the prediction method uses 3D structures or homologous sequences. Some computationally predicted domains have been cataloged in publicly available databases (e.g., Pfam: The protein families database in 2021; J. Mistry, S. et al., Nucleic Acids Research (2020)) or the NCBI Conserved Domain Database (CDD) https: / / www.ncbi.nlm.nih.gov / Structure / cdd / cdd.shtml).

[0107] The term "interdomain linker" refers to a polypeptide segment that connects two adjacent domains. Interdomain linkers provide flexibility for facilitating domain movement and adjusting interdomain shape, as described in Bhaskara RM et al., J Biomol Struct Dyn. 2013 Dec; 31(12):1467-80. Interdomain linkers regulate the interactions of adjacent domains through their length, conformation, intermolecular interactions, and local structure, thereby affecting the overall interdomain shape. The above-mentioned databases based on predicted structural domains (Pfam database or NCBI Conserved Domain Database) provide generalizations of domains and can only provide estimates of domain boundaries (e.g., to distinguish between residues within a domain and residues that are interdomain linkers). Therefore, the domain sequences described herein (e.g., the sequences in Tables 2-20) may include polypeptide sequences containing the corresponding domains and interdomain linkers. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues at the N-terminus or C-terminus of a cataloged domain sequence can be an interdomain linker. One skilled in the art can determine the segments of the polypeptide chain that correspond to the domains and interdomain linkers, and where the transition from the domain to the interdomain linker occurs (i.e., at the domain boundary).

[0108] The term "ectodomain" of an ephrin receptor is well known in the art and refers to the extracellular portion of an Eph receptor, ie, the portion of the RTK that is outside the plasma membrane and lacks the signal peptide.

[0109] A "ligand binding domain" or "LBD" is a peptide region that specifically binds to one or more particular receptor ligands. When multiple ligands are present, the ligands share sufficient binding determinants to detectably bind to the binding domain. Optionally, the binding domain is a contiguous sequence of amino acids.

[0110] As used herein, the term "surface-decorated" refers to nanoparticles comprising a scaffold protein to which a molecule of interest (e.g., a protein) is attached. The scaffold protein can be altered by chemical, physical, or biological methods, or by being produced from cells that have been modified by chemical, physical, or biological methods. Specifically, the scaffold protein can be altered by genetic engineering such that cells previously modified by genetic engineering produce such modified scaffold protein.

[0111] As used herein, the term "biologically active molecule" refers to an agent that has activity in a biological system (e.g., a cell or a human subject), including but not limited to, a protein, polypeptide, or peptide (including but not limited to, a structural protein, an enzyme, a cytokine (e.g., interferon and / or interleukin), an antibiotic, a polyclonal or monoclonal antibody, or an effective portion thereof, e.g., an Fv fragment (the antibody or portion thereof can be natural, synthetic, or humanized), a peptide hormone, a receptor, a signaling molecule, or a protein); a nucleic acid, as defined below (including but not limited to, an oligonucleotide or modified oligonucleotide); Examples of biologically active molecules include, but are not limited to, nucleotides, antisense oligonucleotides or modified antisense oligonucleotides, cDNA, genomic DNA, artificial or natural chromosomes (e.g., yeast artificial chromosomes) or portions thereof, RNA (including mRNA, tRNA, rRNA, or ribozymes), or peptide nucleic acids (PNAs); viruses or virus-like particles; nucleotides or ribonucleotides or synthetic analogs thereof (which may be modified or unmodified); amino acids or analogs thereof (which may be modified or unmodified); non-peptide (e.g., steroid) hormones; proteoglycans; lipids; or carbohydrates. In some embodiments, the biologically active molecule comprises a therapeutic molecule (e.g., an antigen), a targeting moiety (e.g., an antibody or antigen-binding fragment thereof), an adjuvant, an immunomodulator, or any combination thereof. In some embodiments, the biologically active molecule comprises a macromolecule (e.g., a protein, an antibody, an enzyme, a peptide, DNA, RNA, or any combination thereof). In some embodiments, the biologically active molecule comprises a small molecule (e.g., an antisense oligomer (ASO), a phosphorodiamidate morpholino oligomer (PMO), a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), an siRNA, STING, a drug, or any combination thereof). In some embodiments, the biologically active molecule is exogenous to the EV, i.e., not naturally found in the EV.

[0112] As used herein, the term "scaffold protein" refers to a polypeptide that can be used to anchor a payload or any other compound of interest (e.g., a cargo protein) to a nanovesicle. In some embodiments, the scaffold protein is a polypeptide that does not naturally occur in EVs. In some embodiments, the scaffold protein comprises a synthetic polypeptide. In some embodiments, the scaffold protein comprises a modified protein, where the corresponding unmodified protein is naturally present in EVs, e.g., exosomes. In some embodiments, the scaffold protein comprises a protein that naturally occurs in EVs, or a fragment thereof, e.g., a fragment of an EV protein, where the protein is expressed at a level higher than the naturally occurring level. In some embodiments, the scaffold protein comprises a fusion protein comprising (i) a naturally occurring EV protein or a fragment thereof and (ii) a heterologous peptide (e.g., an antigen-binding domain, a cargo protein, a modified Fc, or any combination thereof). As used herein, the term "scaffold protein" of the present disclosure or grammatical variations thereof can be:

[0113] (i) ephrin receptors (naturally expressed, chemically or enzymatically synthesized, or recombinantly produced) spanning the membrane of nanovesicles, e.g., exosomes;

[0114] (ii) any functional fragment of (i);

[0115] (iii) any functional variant of (i) or (ii);

[0116] (iv) a derivative of any of (i) to (iii);

[0117] (v) any peptide corresponding to a domain or combination thereof derived from a protein of (i) that can span the membrane of a nanovesicle, e.g., an exosome, or a molecule containing such a peptide;

[0118] (vi) a polypeptide derived from an ephrin receptor described herein

[0119] (vii) any of molecules (i)-(vi) comprising at least one unnatural amino acid; or

[0120] (viii) any combination of (i) to (vii);

[0121] This makes it suitable for use as a scaffold for targeting (attaching) a payload, e.g., a biologically active molecule, to the surface (e.g., if the biologically active molecule comprises a targeting domain) and / or lumen (e.g., if the biologically active molecule comprises a cargo protein) of a nanovesicle, e.g., an exosome.

[0122] The term "fragment" with respect to a polypeptide refers to any amino acid sequence present in the polypeptide that is shorter than the parent polypeptide due to an N-terminal and / or C-terminal deletion compared to the parent protein, but that is still capable of performing the intended function of the parent polypeptide.

[0123] The terms "source cells" or "EV source cells" or "cell source" or "EV-producing cells" or "producer cells" or any other similar terminology may be understood to relate to any type of cell that is capable of producing EVs under suitable conditions, e.g. in suspension culture or in adherent culture or in any other type of culture system.

[0124] The term "specifically binds" refers to a molecule (e.g., an antigen-binding molecule) that binds to an epitope or target with greater affinity, greater avidity, and / or longer duration than it binds to another epitope or non-target compound (e.g., a structurally different antigen) in a sample. In some embodiments, a molecule (e.g., an antigen-binding molecule) that specifically binds to an epitope or target binds to the epitope or target with at least 5-fold greater affinity than other epitopes or non-target compounds, e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 100-fold, 1000-fold, 10,000-fold, or more. As used herein, the terms "specific binding of," "specifically binds to," or "is specific for" a particular epitope or target refer to, for example, a specific binding affinity for the epitope or target to which it binds, e.g., 10-fold. -4 M or less, e.g., 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 Equilibrium dissociation constant K below M d It will be appreciated by those skilled in the art that a molecule (e.g., an antigen-binding molecule) that specifically binds to a target from one species may also specifically bind to an ortholog of that target.

[0125] The term "isolated" indicates that a substance, such as a polypeptide, nucleic acid, or cell, has been removed from its normal physiological environment, e.g., a natural source, or that the polypeptide or nucleic acid has been synthesized. Use of the term "isolated" indicates that a naturally occurring sequence has been removed from its normal cellular (e.g., chromosomal) environment. Thus, the sequence may be in a cell-free solution or may be located in a different cellular environment. "Isolated," with respect to a polypeptide or nucleic acid molecule, refers to a polymer of two or more amino acids or nucleotides linked together, including a polypeptide or nucleic acid molecule that has been isolated from a natural source or that has been synthesized. The term "isolated" does not imply that the sequence is the only amino acid or nucleotide chain present, but rather means that it is essentially free, e.g., from non-amino acid and / or non-nucleic acid materials, respectively, that naturally accompany it. An "isolated cell" refers to a cell that has been separated from molecules and / or cellular components that naturally accompany the cell.

[0126] In the context of nanovesicles, the terms "isolate," "isolated," and "isolating," or "purify," "purified," and "purifying," as well as "extracted" and "extracting," are used interchangeably and refer to the state of a desired nanovesicle preparation (e.g., a plurality of known or unknown quantities and / or concentrations) following one or more processes of purification, e.g., selection or enrichment of a desired nanovesicle preparation. In some embodiments, isolation or purification, as used herein, is a process of removing, or partially removing (e.g., fractionating), nanovesicles from a sample containing source cells.

[0127] The terms "polynucleotide" and "nucleic acid" refer interchangeably to a chain of nucleotides of any length and include DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding polypeptides. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a strand by DNA or RNA polymerase. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and their analogs. Examples of polynucleotides contemplated herein include single- and double-stranded DNA, single- and double-stranded RNA, and hybrid molecules comprising a mixture of single- and double-stranded DNA and single- and double-stranded RNA.

[0128] The term "amino acid sequence" is used interchangeably with the terms "polypeptide," "protein," and "peptide." The conventional one-letter or three-letter code for amino acid residues is used, and amino acid sequences are presented in the standard amino- to carboxy-terminal orientation (i.e., N→C).

[0129] As used herein, "kinase-dead domain" refers to an Eph receptor that is defective in intracellular signaling. The kinase domain of the corresponding wild-type Eph receptor can either be absent (partially or entirely) or rendered non-functional by one or more mutations.

[0130] The term "parent" or "reference" with respect to a polypeptide or polynucleotide sequence means a polypeptide or polynucleotide sequence that serves as a template sequence used to generate modified (or mutant) forms of the polypeptide or polynucleotide.

[0131] The terms "wild-type," "native," and "naturally occurring" with respect to Eph receptors are used herein to refer to domains having a sequence that occurs in nature. A wild-type polypeptide is understood to include the mature form of the polypeptide. A "mature" polypeptide (or a variant thereof) is one in which the signal sequence is not present, e.g., cleaved from the immature form of the polypeptide during or after expression of the polypeptide.

[0132] As used herein, the term "mutant" with respect to a mutant polypeptide or mutant polynucleotide is used interchangeably with "variant." Variants with respect to a given wild-type Eph receptor reference sequence can include naturally occurring allelic variants. A "non-naturally" occurring Eph receptor domain refers to a variant or mutant domain that does not occur in a natural cell and is produced by genetic modification of a parent Eph receptor polynucleotide, e.g., using genetic engineering or mutagenesis techniques, to introduce appropriate modifications into the nucleic acid sequence encoding the polypeptide, or by protein / peptide synthesis. A "variant" includes any sequence that contains at least one amino acid mutation relative to the wild-type. Mutations can include one or more amino acid substitutions, insertions, and deletions (e.g., truncations), as well as frameshifts or rearrangements in another protein. Similarly, the term "variant" with respect to a polynucleotide refers to a polynucleotide whose nucleotide sequence differs from a particular parent polynucleotide. The identity of the parent polypeptide or polynucleotide will be clear from the context. A variant can contain one or more specified substitutions, insertions, and / or deletions, as well as percent sequence identity with the parent sequence.

[0133] The term "amino acid substitution" refers to the replacement of at least one existing amino acid residue with another different amino acid residue (substitute amino acid residue). The substitute amino acid residue may be a "naturally occurring amino acid residue" and may be selected from the group consisting of alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gin, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0134] The term "amino acid insertion" refers to the incorporation of at least one amino acid residue at a predetermined position in the amino acid sequence. In one embodiment, the insertion is of one or two amino acid residues. The inserted amino acid residue can be any naturally occurring or non-naturally occurring amino acid residue. The term "amino acid deletion" refers to the removal of at least one amino acid residue at a predetermined position in the amino acid sequence.

[0135] The term "non-naturally occurring amino acid residue" refers to an amino acid residue other than the naturally occurring amino acid residues listed above, which can be covalently linked to an adjacent amino acid residue in a polypeptide chain. Examples of non-naturally occurring amino acid residues are norleucine, ornithine, norvaline, and homoserine. Further examples are described in Ellman et al., Meth. Enzym. 202 (1991) 301-336. Exemplary methods for the synthesis of non-naturally occurring amino acid residues are reported, for example, in Noren et al., Science 244 (1989) 182 and Ellman et al. (supra).

[0136] "Percent (%) sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. The same principle applies to nucleic acid sequences. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways within the skill of those in the art, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. Two molecules with the same primary amino acid or nucleic acid sequence are identical, regardless of any chemical and / or biological modifications. A sequence that is longer than any of the sequences provided herein, for example, because it contains additional domains, is still considered identical to the reference sequence disclosed herein when sequence identity is shown over a comparison window, such as a comparison window covering the entire claimed sequence.

[0137] The terms "heterologous" or "exogenous" refer to a molecule that is not normally found in a given context, e.g., within a cell or polypeptide. For example, an exogenous or heterologous molecule can be introduced into a cell and only be present after manipulating the cell, e.g., by transfection or other forms of genetic engineering. As another example, a heterologous amino acid sequence can be present in a protein where it is not found in nature.

[0138] The term "endogenous" with respect to a polynucleotide or polypeptide refers to a polynucleotide or polypeptide that occurs naturally within a host cell.

[0139] A "fused" polypeptide sequence is joined via a peptide bond between two polypeptide sequences of interest.

[0140] The terms "associated with," "bound to," "linked to," "conjugated to," and grammatical variations thereof, are used interchangeably herein to refer to direct or indirect interactions between two or more elements. Two elements can be associated with each other through covalent or non-covalent bonds and / or interactions. In some embodiments, a first element, e.g., a targeting domain, is associated with a second element, e.g., a scaffold protein, through a peptide bond. In some embodiments, a first element (e.g., a cargo protein linked to a scaffold-binding domain) is associated with a second element, e.g., a scaffold protein, through a non-covalent interaction, e.g., a phosphotyrosine-based bond (e.g., a bond between a phosphotyrosine and a phosphotyrosine-binding (PTB) domain, a bond between a phosphotyrosine and a Src homology 2 (SH2) domain), an electrostatic interaction, a hydrogen bond, a van der Waals interaction, a hydrophobic interaction, an ion-induced dipole, a dipole-induced dipole, an ionic bond, a coordinate bond, chelation, or a combination thereof. The first and second elements can be directly associated, e.g., where the scaffold protein is linked to the cargo protein by a peptide bond; or the first element can be associated with the second element through an indirect association, e.g., where the cargo protein is associated with the scaffold protein through the interaction of the scaffold protein with an intermediate scaffold-binding domain, where the scaffold protein binds to a scaffold-binding domain that is covalently linked to the cargo protein.

[0141] The phrase "binding partner" refers to a molecule that is a member of a specific binding pair, which is one of two different molecules that specifically binds to, and is thereby defined as complementary to, the other molecule of the pair. For example, one member of a specific binding pair can have an area on its surface or within a cavity that specifically binds to the other member of the specific binding pair in a particular spatial and polar configuration.

[0142] As used herein, the term "dimerizing agent" or "dimerization agent" refers to one member of at least two elements that interact with each other to form a multimer (e.g., a dimer). In some embodiments, the dimerizing agent is a first binding partner that interacts with a second binding partner. In some embodiments, the dimerizing agent is a first binding partner that interacts with a second binding partner and / or a third binding partner. Any dimerizing agent can be used in the compositions and methods disclosed herein. In some embodiments, the dimerizing agent can be a polypeptide, a polynucleotide, a fatty acid, a small molecule, or any combination thereof.

[0143] As used herein, the term "adapter protein" refers to a polypeptide dimerizer, which can comprise two or more scaffold-binding domains that simultaneously interact with two scaffold proteins and / or a third scaffold protein. The adaptor protein can simultaneously interact with multiple scaffold proteins and, through its interactions with the scaffold proteins, spatially confine the scaffold proteins to the membrane of a cell or nanovesicle, thus functioning as a dimerization or oligomerization agent.

[0144] As used herein, the term "chemically-induced dimerizer" refers to a dimerizer (e.g., a first binding partner and / or a second binding partner) that forms a dimer in the presence of a small molecule. In some embodiments, the chemically-induced dimerizer is selected from the first and second binding partners of a chemically-induced dimer selected from the group consisting of: (i) FKBP and FKBP (FK1012); (ii) FKBP and calcineurin A (CNA) (FK506); (iii) FKBP and CyP-Fas (FKCsA); (iv) FKBP and FRB (rapamycin); (v) GyrB and GyrB (coumermycin); (vi) GAI and GID1 (gibberellin); (vii) Snap-tag and HaloTag (HaXS); (viii) eDHFR and HaloTag (TMP-HTag); and (ix) BCL-xL and Fab(AZ1) (ABT-737).

[0145] As used herein, the term "scaffold-binding domain" refers to a first member of at least two binding partners that interact with each other to form a multimer (e.g., a dimer), where at least a second member is a scaffold protein, a cargo-binding domain present on a scaffold protein, or a cargo-binding domain covalently attached to a scaffold protein.

[0146] The term "pharmaceutically acceptable" refers to active compounds, materials, compositions, carriers, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0147] In a medical / physiological context, ie, in the context of a physiological condition, the term "prevent" refers to reducing the probability that an organism will contract or develop an abnormal disease.

[0148] The terms "subject" and "individual" are used interchangeably herein and refer to a human or non-human animal, typically a mammal. A subject may be a mammalian species such as a rabbit, mouse, rat, guinea pig, dog, cat, pig, cow, horse, monkey, or human. Thus, the methods, uses, and compositions described herein are applicable to both human and veterinary use. When a subject is a human receiving medical treatment for a disease or disorder, the subject is also referred to as a "patient."

[0149] Whenever an aspect or embodiment is described herein with the word "comprising," it is understood that other similar aspects described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0150] The scope and meaning of any use of a term will be apparent from the specific context in which the term is used. Certain further definitions of selected terms used throughout this document are provided in the appropriate context of the detailed description, where applicable. Furthermore, depending on the particular implementation, selected definitions, implementations, or ranges may not apply.

[0151] Any embodiment specifically and explicitly recited herein may form the basis of a disclaimer, either alone or in combination with one or more additional embodiments.

[0152] 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 the disclosure set forth herein belongs. All publications and patents mentioned herein are incorporated by reference in their entirety for the purpose of describing and disclosing, for example, the constructs and methodologies described in the publications that may be used in connection with the presently described disclosure.

[0153] Various aspects of the disclosure are described in further detail in the following subsections, it being understood that the various embodiments, preferences, and ranges may be freely combined.

[0154] 5.2 Polypeptides of the Present Disclosure One aspect of the present invention relates to the identification, use, and modification of transmembrane polypeptides suitable for use as scaffolds for targeting (tethering) payloads, e.g., biologically active molecules (e.g., cargo proteins), to the surface and / or lumen of nanovesicles (e.g., EVs and hybridosomes). Another aspect of the present invention relates to the production and use of nanovesicles comprising scaffold polypeptides. One or more transmembrane polypeptides identified herein can be selectively used depending on the producer cell, production conditions, purification method, or intended use of the nanovesicles (e.g., EVs and hybridosomes).

[0155] Provided herein are polypeptides that include a transmembrane domain and further include an ectodomain and / or an endodomain that can be used to load a cargo (e.g., a cargo protein) onto or into a nanovesicle (e.g., EVs and hybridosomes). The cargo (e.g., cargo protein) can be a portion of a polypeptide described herein. That is, the cargo (e.g., cargo protein) can be fused to the remainder of the polypeptide (e.g., via a linker). Alternatively, the cargo (e.g., cargo protein) can be bound (preferably reversibly bound) to the polypeptide via a cargo-binding domain. The cargo-binding domain can be bound directly to the cargo (e.g., cargo protein) or indirectly via a scaffold-binding domain (SBD) linked to the cargo (e.g., cargo protein). As used herein, the singular forms "a," "an," and "the" include plural referents. Thus, the polypeptides described herein can be used to deliver one or more (e.g., one, two, three, four, five, or more) cargoes, and the polypeptides described herein can comprise one or more (e.g., one, two, three, four, five, or more) cargo-binding domains.

[0156] The polypeptides described herein can further comprise one or more functional moieties, such as a targeting domain capable of targeting the nanovesicle (e.g., EV or hybridosome) to a particular organ, tissue, or cell type, and / or a purification domain capable of facilitating the purification of the nanovesicle (e.g., EV or hybridosome).

[0157] In certain aspects, the polypeptides described herein comprise one or more domains from or derived from one or more ephrin receptors and are located in the membrane of a nanovesicle. Such polypeptides are also referred to herein as ephrin receptor-derived polypeptides or polypeptides derived from ephrin receptors. Ephrin receptor-derived polypeptides may or may not comprise one or more domains (e.g., transmembrane domains) from or derived from non-ephrin receptor proteins. In various embodiments, the polypeptides described herein comprise an ectodomain or fragment thereof (e.g., flexible domain) from or derived from one or more ephrin receptors, a transmembrane domain from or derived from an ephrin receptor or a non-ephrin receptor transmembrane protein, and, optionally, an endodomain or fragment thereof from or derived from one or more ephrin receptors.

[0158] Wild-type Eph receptors typically consist of an ectodomain, a transmembrane domain, and an endodomain. The ectodomain contains, in N→C order, a ligand-binding domain (LBD), a cysteine-rich region containing the sushi domain, and an epidermal growth factor (EGF)-like domain, followed by two fibronectin type III domains (FN1 and FN2 domains). The ectodomain is further described in Section 5.2.1. The cysteine-rich region and the two fibronectin type III domains are hereafter referred to as "flexible domains." The endodomain contains a juxtamembrane domain, a kinase domain, a sterile alpha motif (SAM) domain, and a PDZ-binding motif (the endodomain is further described in Section 5.2.2).

[0159] In particular, provided herein are certain scaffold polypeptides comprising at least an ephrin receptor cysteine-rich (CR) domain, two ephrin receptor fibronectin type III (FN III) domains (i.e., a first ephrin receptor FN III domain (ephrin receptor FN1 domain) and a second ephrin receptor FN III domain (ephrin receptor FN2 domain)), and a transmembrane (TM) domain (e.g., an ephrin receptor TM domain), and optionally a cargo-binding domain, an ephrin receptor juxtamembrane (JM) domain, an ephrin receptor kinase domain (KD), a sterile α-motif (SAM) linker domain (e.g., an ephrin receptor SAM linker domain), a SAM domain (e.g., an ephrin receptor SAM domain), an ephrin receptor PDZ-binding motif (PBM) domain, a targeting domain, a purification domain, a modified Fc domain, and / or a ligand-binding domain (LBD).

[0160] In one aspect, provided herein is a polypeptide comprising, from N-terminus to C-terminus: a. an ephrin receptor CR domain; b. a first ephrin receptor FN III domain; and a second ephrin receptor FN III domain; and c. a TM domain (e.g., an ephrin receptor TM domain). In some embodiments, the polypeptide is fused to a cargo (e.g., a cargo protein). In some embodiments, the polypeptide associates with (i.e., binds to) a cargo (e.g., a cargo protein).

[0161] In one aspect, provided herein is a polypeptide comprising, from N-terminus to C-terminus: a. a targeting domain; b. an ephrin receptor CR domain; c. a first ephrin receptor FN III domain and a second ephrin receptor FN III domain; and d. a TM domain (e.g., an ephrin receptor TM domain). In some embodiments, the polypeptide is fused to a cargo (e.g., a cargo protein). In some embodiments, the polypeptide associates with (i.e., binds to) a cargo (e.g., a cargo protein).

[0162] In one aspect, provided herein is a polypeptide comprising, from N-terminus to C-terminus: a. an ephrin receptor CR domain; b. a first ephrin receptor FN III domain and a second ephrin receptor FN III domain; c. a TM domain (e.g., an ephrin receptor TM domain); and d. a cargo protein or cargo-binding domain.

[0163] In one aspect, provided herein is a polypeptide comprising, from N-terminus to C-terminus: a. a targeting domain; b. an ephrin receptor CR domain; c. a first ephrin receptor FN III domain and a second ephrin receptor FN III domain; d. a TM domain (e.g., an ephrin receptor TM domain); and e. a cargo protein or cargo-binding domain.

[0164] In a specific embodiment, the polypeptide lacks ephrin-binding activity. In a specific embodiment, the polypeptide lacks ephrin receptor LBD. In a specific embodiment, the polypeptide is an inactivated ephrin receptor LBD, for example, a modified (e.g., mutated) ephrin receptor LBD that lacks ephrin-binding activity. In a specific embodiment, the ephrin receptor LBD can become inactivated due to one or more mutations in one or more outer domains of the ephrin receptor LBD.

[0165] In a specific embodiment, the polypeptide lacks ephrin receptor kinase activity. In a specific embodiment, the polypeptide lacks ephrin receptor KD. In a specific embodiment, the polypeptide comprises an inactivated ephrin receptor KD, for example, a modified (e.g., mutated) ephrin receptor KD that lacks ephrin receptor kinase activity. In a specific embodiment, the ephrin receptor KD can become inactivated due to one or more mutations in one or more outer domains of the ephrin receptor KD.

[0166] In a specific embodiment, the polypeptide lacks both ephrin-binding activity and ephrin receptor kinase activity. In a specific embodiment, the polypeptide lacks both the ephrin receptor LBD and the ephrin receptor KD. In a specific embodiment, the polypeptide comprises an inactivated ephrin receptor LBD, e.g., a modified (e.g., mutated) ephrin receptor LBD that lacks ephrin-binding activity and lacks the ephrin receptor KD. In a specific embodiment, the polypeptide lacks the ephrin receptor LBD and comprises an inactivated ephrin receptor KD, e.g., a modified (e.g., mutated) ephrin receptor KD that lacks ephrin receptor kinase activity. In a specific embodiment, the polypeptide comprises an inactivated ephrin receptor LBD, e.g., a modified (e.g., mutated) ephrin receptor LBD that lacks ephrin-binding activity, and comprises an inactivated ephrin receptor KD, e.g., a modified (e.g., mutated) ephrin receptor KD that lacks ephrin receptor kinase activity. In a specific embodiment, the Ephrin Receptor LBD can become inactivated due to one or more mutations in one or more domains outside the Ephrin Receptor LBD. In a specific embodiment, the Ephrin Receptor KD can become inactivated due to one or more mutations in one or more domains outside the Ephrin Receptor KD.

[0167] In certain aspects, provided herein are Eph receptor-derived polypeptides that can function as signal-neutral protein scaffolds in nanovesicles (e.g., extracellular vesicles (EVs) and hybridosomes) for attachment of molecules of interest. In certain embodiments, the polypeptides are membrane-bound and (i) have reduced or no ability to activate cytoplasmic kinases within cells, and (ii) have reduced or no ability to bind ligands to ephrins expressed on other cells.

[0168] The polypeptides provided herein have several advantages over previously described protein scaffolds. They are endocytic recycling proteins and therefore can be sorted by source cells into nanovesicles (e.g., EVs and hybridosomes). Ectodomains (particularly Eph receptor ectodomains) or fragments thereof can be fused to molecules of interest, thereby enabling the engineering of nanovesicles (e.g., EVs and hybridosomes) for additional functionality, such as cell-type-specific targeting, receptor decoys, or purification. The protein scaffolds protrude from the membrane, thereby allowing access to the fused moiety. Furthermore, the long protrusions allow the polypeptides described herein the flexibility to bend and / or reconfigure while maintaining stability. Stable membrane anchoring can streamline the construction of the resulting fusion proteins, in that the molecule of interest can be directed to the lumen of the nanovesicle (e.g., EV or hybridosome) or the outer surface or interior of the cell. Both the N-terminus and C-terminus of the polypeptide are accessible and free for attachment of biologically active molecules (eg, fusion moieties).

[0169] Furthermore, the polypeptides described herein have superior characteristics to protein scaffolds previously described in the art because they contain homodimer interaction interfaces that confer a propensity for oligomerization, e.g., clustering, thereby enabling high-density surface decoration on nanovesicles (e.g., EVs and hybridosomes) (see, e.g., Figures 2A, 5, 6, and 7). The key residues involved in clustering at the Eph LBD-LBD interface (e.g., D104, K116, E117, and T144 of EphA2) and homodomain dimerization at the CRD-CRD interface (e.g., the CRD homodomain dimerization motif: The amino acid sequence GX1WX2VX3X4G (where X1=E or K, X2=L or M, X3=P or A, and X4=V, I, or L) (SEQ ID NO: 239) is highly conserved among EphA and EphB receptors. In certain embodiments, the polypeptides described herein comprise domains capable of undergoing heterodomain dimerization, which can result in oligomerization (e.g., heterodomain dimerization between LBD-FN1) (see, e.g., Figures 2B and 4). In certain embodiments, the polypeptides undergo heterodomain dimerization in a head-to-tail configuration (e.g., dimerization between the LBD-FN1 of EphA4 and EphA2 or dimerization between the LBD-FN2 of EphB6).

[0170] In certain embodiments, the clustering tendency can be further enhanced or disrupted by modifications of the amino sequences of the domains. Further examples of clustering modifications are described in the following sections.

[0171] Several assays for detecting clustering of the disclosed polypeptides are available, including microscopy techniques for visualizing polypeptide clustering in membranes, including, but not limited to, confocal microscopy and lateral membrane diffusion with fluorescence correlation spectroscopy. For example, the polypeptides and / or polypeptide-specific antibodies can be labeled, and these labels can be detected to visualize the clustering of polypeptide elements. In one example of this type of assay, cells containing the polypeptide of interest are contacted with a polypeptide-specific antibody and a fluorescently labeled secondary antibody that binds to the polypeptide-specific antibody. Examples of such techniques include Mely, Y. et al., 2013. Fluorescent methods to study biological membranes. Berlin: Springer Berlin Heidelberg. He L, Hristova K (2014). Quantifying the effects of mutations on receptor tyrosine kinase (RTK) activation in mammalian cells. Quantification of the effects of mutations on receptor tyrosine kinase (RTK) activation in mammalian cells. Receptor Tyrosine Kinases: Methods and Protocols. 81-87; and Christopher King et al., Fully quantified spectral imaging reveals in vivo membrane protein interactions, Integrative Biology, Vol. 8, 2nd edition, February 2016, pp. 216-229.

[0172] In one embodiment, a polypeptide derived from an Eph receptor is provided, wherein the polypeptide comprises: i. contains an ephrin ligand binding domain that exhibits reduced or no binding to ephrin compared to the parent Eph receptor; and ii. Contains a transmembrane domain.

[0173] Although the parent Eph receptor can be derived from any mammalian species, including human, mouse, or rat, the parent Eph receptor is preferably a human Eph receptor (hEph). In some embodiments, the parent Eph receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 212-225 and fragments thereof. However, in some embodiments, the sources of the individual domains of the polypeptide may be mixed. For example, a polypeptide may comprise the LBD of one receptor (e.g., a mutated LBD of EphA2, see SEQ ID NO: 16), a flexible domain of another receptor (e.g., a flexible domain of EphA4, see SEQ ID NO: 63), and a transmembrane domain of a third receptor (e.g., the TM domain of EphA1, see SEQ ID NO: 74).

[0174] Any combination of deletions, substitutions, additions, modifications, and insertions into Eph receptor-derived polypeptides can be made, provided that the resulting variants retain the desired characteristics, which can be screened for using appropriate methods. Of particular interest are substitutions, preferably conservative substitutions. The polypeptides described herein can contain one or more such substitutions, e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more.

[0175] 5.2.1 Ectodomain and Transmembrane Domain As explained above, the ephrin receptor ectodomain contains, in N-to-C order, a ligand-binding domain (LBD), a cysteine-rich region (CR domain) containing the sushi domain, and an epidermal growth factor (EGF)-like domain, followed by two fibronectin type III domains (FN III domains—FN1 and FN2 domains). The transmembrane domain is C-terminal to the two FN III domains.

[0176] In certain embodiments, the polypeptides described herein comprise an ephrin receptor ectodomain and an ephrin receptor transmembrane domain (TM domain) from or derived from the same ephrin receptor. In certain embodiments, the polypeptides described herein comprise an ephrin receptor ectodomain and an ephrin receptor TM domain from or derived from two different ephrin receptors. In certain embodiments, the polypeptides described herein comprise a wild-type ephrin receptor ectodomain and a wild-type ephrin receptor TM domain. In certain embodiments, the polypeptides described herein comprise a mutant ephrin receptor ectodomain and a mutant ephrin receptor TM domain. In certain embodiments, the polypeptides described herein comprise a wild-type ephrin receptor ectodomain and a mutant ephrin receptor TM domain. In certain embodiments, the polypeptides described herein comprise a mutant ephrin receptor ectodomain and a wild-type ephrin receptor TM domain. In various embodiments, the mutant Ephrin receptor ectodomain has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the wild-type Ephrin receptor ectodomain, hi various embodiments, the mutant Ephrin receptor TM domain has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the wild-type Ephrin receptor TM domain.

[0177] In certain embodiments, the polypeptides described herein comprise an ephrin receptor CR domain (wild-type or mutant ephrin receptor CR domain), a first ephrin receptor FN III domain (wild-type or mutant first ephrin receptor FN III domain), a second ephrin receptor FN III domain (wild-type or mutant second ephrin receptor FN III domain), and an ephrin receptor TM domain (wild-type or mutant ephrin receptor TM domain), wherein all four ephrin receptor domains are from or are derived from the same ephrin receptor. In certain embodiments, the polypeptides described herein comprise an Ephrin receptor CR domain (wild-type or mutant Ephrin receptor CR domain), a first Ephrin receptor FN III domain (wild-type or mutant first Ephrin receptor FN III domain), a second Ephrin receptor FN III domain (wild-type or mutant second Ephrin receptor FN III domain), and an Ephrin receptor TM domain (wild-type or mutant Ephrin receptor TM domain), wherein these four Ephrin receptor domains are from or are derived from two, three, or four Ephrin receptors. In various embodiments, the mutant Ephrin receptor domain has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the wild-type Ephrin receptor domain.

[0178] Both the ectodomain and endodomain of a native Eph receptor can contain protease cleavage sites. In some embodiments, it may be beneficial to remove one or more protease cleavage sites so that the polypeptide remains intact in a cellular environment. In some embodiments, one or more cleavage sites are specific for a metalloprotease, such as ADAM (a disintegrin and metalloprotein, a member of the zinc protease superfamily). In some embodiments, one or more cleavage sites are specific for γ-secretase.

[0179] In some embodiments, one cleavage site may be located between the FN2 domain and the transmembrane domain. For example, one or more modifications in the amino acid stretch 533-547 of the EphA4 fragment (SEQ ID NO: 226) can be made to remove a protease cleavage site. In specific embodiments, such modifications are mutations. Thus, the polypeptide can comprise a sequence in which, compared to the parent Eph receptor, one or more mutations are present at amino acid positions 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, and / or 547 of SEQ ID NO: 226. A further example of removing a cleavage site between the FN2 domain and the transmembrane domain is one or more modifications, e.g., an S536E modification, at amino acid position 536 of the EphB2 ectodomain (SEQ ID NO: 208).

[0180] In further embodiments, Eph receptors can be made more resistant to protease cleavage by one or more modifications of amino acids in the FN1 domain. For example, EphB2 can be made resistant to protease cleavage by one or more modifications in FN1 at amino acids L356 and I395 of SEQ ID NO: 208 (e.g., L356A, I395A).

[0181] In further embodiments, Eph receptors can be made more resistant to protease cleavage by one or more modifications of amino acids in the transmembrane domain, for example, EphB2 can be made resistant to protease cleavage by one or more modifications in the transmembrane domain at amino acid A562 of SEQ ID NO: 208 (e.g., A562S).

[0182] Thus, the polypeptide can comprise a sequence in which one or more mutations are present in the FN1, FN2, and / or transmembrane domains when compared to the parent Eph receptor. In some embodiments, the polypeptide is more resistant to cleavage and has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations in the ectodomain and / or transmembrane domains when compared to the parent Eph receptor.

[0183] In certain embodiments, the polypeptides of the present disclosure do not contain a protease cleavage site. In some embodiments, the polypeptides contain one, two, or three protease cleavage sites.

[0184] (a) Ligand-binding domain (LBD) In various embodiments, the polypeptides described herein exhibit reduced or no ephrin-binding activity. In certain embodiments, the polypeptides described herein lack the ephrin receptor ligand-binding domain (LBD). See FIG. 1B for a schematic diagram of exemplary Eph receptor-derived polypeptides with mutant (including truncated) ligand-binding domains (LBDs) or no LBD. Reduced or absent ephrin ligand binding can be useful in that the Eph receptor-derived polypeptides of the present disclosure do not induce reverse signaling in cells with which they come into close contact. This can be beneficial when the polypeptides described herein are present on nanovesicles (e.g., EVs and hybridosomes), in that the respective signaling pathways of the target cells are not triggered upon contact of the nanovesicles with the target cells.

[0185] In certain embodiments, the Eph receptor-derived polypeptides described herein contain an ephrin ligand-binding domain that is modified to exhibit reduced or no binding to ephrin. The parent Eph receptor can serve as a reference for determining the affinity of ephrin binding. In some embodiments, the ephrin ligand-binding domain is modified by mutation (e.g., substitution, insertion, and / or deletion), preferably by substitution of one or more amino acids. Receptor-ligand binding activity can be measured using methods known in the art. See, e.g., Elliott S. et al. (1997) Blood, 89:493-502.

[0186] In some embodiments, ephrin ligand binding is reduced by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to the parent Eph receptor.

[0187] In some embodiments, the binding affinity of a polypeptide of the disclosure to an ephrin is at least 2-fold less, at least 3-fold less, at least 4-fold less, at least 5-fold less, at least 6-fold less, at least 7-fold less, at least 8-fold less, at least 9-fold less, at least 10-fold less, at least 15-fold less, at least 20-fold less, at least 25-fold less, at least 30-fold less, at least 35-fold less, at least 40-fold less, at least 45-fold less, at least 50-fold less, at least 100-fold less, at least 150-fold less, or 10-50-fold less, 50-100-fold less, 100-150-fold less, 150-200-fold less, or more than 200-fold less than the binding affinity of the parent Eph receptor.

[0188] In various embodiments, the Eph receptor-derived polypeptides of the present disclosure are those in which the polypeptide has a substantially reduced or eliminated affinity or activity, e.g., binding affinity (e.g., K D) and / or activation activity (e.g., when the ligand is an agonist for an ephrin receptor, e.g., K A and / or EC 50 ) and / or inhibitory activity (e.g., when the ligand is an antagonist for an ephrin receptor, e.g., K I and / or IC 50 In such embodiments, the polypeptide comprises one or more mutations that cause it to have an affinity for an ephrin (measurable as a function of affinity) of about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% to 20%, about 20% to 40%, about 50%, about 40% to 60%, about 60% to 80%, or about 80% to 100% of that of the parent Eph receptor. In some embodiments, the binding affinity is at least 2-fold lower, at least 3-fold lower, at least 4-fold lower, at least 5-fold lower, at least 6-fold lower, at least 7-fold lower, at least 8-fold lower, at least 9-fold lower, at least 10-fold lower, at least 15-fold lower, at least 20-fold lower, at least 25-fold lower, at least 30-fold lower, at least 35-fold lower, at least 40-fold lower, at least 45-fold lower, at least 50-fold lower, at least 100-fold lower, at least 150-fold lower, or 10-50-fold lower, 50-100-fold lower, 100-150-fold lower, 150-200-fold lower, or more than 200-fold lower, compared to the parent Eph receptor.

[0189] Methods for analyzing the binding affinity and binding kinetics between ephrins and LBDs are known in the art. These methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore™ (GE Healthcare, Piscataway, NJ)), kinetic exclusion assays (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), bio-layer interferometry (e.g., Octet® (ForteBio, Menlo Park, CA)), and Western blot analysis. In some embodiments, ELISA is used to determine binding affinity. In some embodiments, surface plasmon resonance (SPR) is used to determine binding affinity and / or binding kinetics. In some embodiments, kinetic exclusion assays are used to determine binding affinity and / or binding kinetics. In some embodiments, bio-layer interferometry assays are used to determine binding affinity and / or binding kinetics.

[0190] Specific positions within the LBD constitute ephrin-binding sites and interact with ephrins. A non-exhaustive list of LBD amino acid positions that interact with ephrins and constitute ephrin-binding sites is cataloged in the NCBI Conserved Domain Database (CDD) (see, e.g., positions in Table 1). Thus, in certain embodiments, the parent Eph receptor comprises an LBD in which arginine (R) is substituted for glutamic acid (E) in the loop between the G and H β-strands (e.g., R104 for EphA1 / 3, R103 for EphA2, R106 for EphA4 / 8, R107 for EphA7, R109 for EphA6, R110 for EphA10, R135 for EphA5, R94 for EphB1, R95 for EphB2, R115 for EphB3, or R112 for EphB6). In some embodiments, leucine L95 of EphB4 can be substituted with arginine (R). Additionally, or alternatively, the amino acid at position 154 of the parent EphA4 LBD is substituted with alanine (A) (see SEQ ID NO: 15). One of skill in the art will be well able to identify additional positions that reduce or eliminate ephrin binding. In some embodiments, the LBD of the polypeptide has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the LBD of a wild-type ephrin receptor (e.g., an ephrin receptor LBD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14).

[0191] In some embodiments, the LBD of the polypeptide is the LBD of a wild-type ephrin receptor (eg, an ephrin receptor LBD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14).

[0192] Table 1. Location of the LBD ephrin binding site (CDD: cl02704) [Table 1]

[0193] In one aspect, the polypeptides described herein can comprise an LBD, wherein the LBD exhibits a three-dimensional structure that is superimposable with the LBD structure of a wild-type ephrin receptor. In certain embodiments, the polypeptides described herein comprise an LBD, wherein the LBD exhibits a three-dimensional structure that is superimposable with the wild-type Eph receptor LBD at portions between their equivalent Cα positions with a root mean square deviation (RMSD) of at most 1, 2, 4, 4, 5, 6, 7, 8, 9, or 10 Å. For example, the structures of the unbound EphA2 LBD and EphB2 LBD can be superimposed with an RMSD of 1.7 Å between corresponding Cα positions, as described in Himanen, JP et al. (2009), EMBO Reports, 10: 722-728. As a further example, the structures of unbound EphB4 LBD and EphB2 LBD can be superimposed with an overall RMSD of 1.08 Å between equivalent Cα positions, as described in Chrencik et al., Structure, 14, 2, (2006), 321-330. Methods for comparing two biological structures by calculating the RMSD of superimposed structures are well known in the art (Xu, Y., Xu, D., and Liang, J., 2007. Computational methods for protein structure prediction and modeling. Springer). Two identical structures will exhibit an RMSD of zero, whereas two different structures will exhibit a value proportional to their dissimilarity. Further examples of superimposed structures are shown in Figure 19.

[0194] In some embodiments, the scaffold polypeptides described herein comprise an LBD domain, wherein the LBD has the reduced ephrin-binding activity described above and has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the LBD domain of a wild-type ephrin receptor (e.g., an ephrin receptor LBD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14 shown in Table 2).

[0195] Table 2. LBD (CDD Superfamily: cl02704, ProRule: PRU00883) [Table 2] TIFF2024514157000008.tif244170TIFF2024514157000009.tif89170

[0196] In some embodiments, the ligand-binding domain comprises the amino acid sequence of a wild-type ephrin receptor LBD (e.g., an ephrin receptor LBD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14), excluding one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, seven amino acid mutations, or more than seven amino acid mutations. The mutations can be substitutions, insertions, deletions, or any combination thereof.

[0197] In some embodiments, the ligand-binding domain comprises an amino acid sequence shown in Table 2.

[0198] In some embodiments, the ligand-binding domain comprises an amino acid sequence shown in Table 2.

[0199] Table 3. Exemplary LBDs with ephrin-binding site mutations [Table 3]

[0200] In some embodiments, the ligand-binding domain comprises the amino acid sequence of a wild-type ephrin receptor (e.g., an ephrin receptor LBD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 14), and its length is 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more at the N-terminus and / or C-terminus of the amino acid sequence of the ligand-binding domain of a wild-type ephrin receptor (e.g., SEQ ID NOs: 1 to 14).

[0201] In some embodiments, the LBD comprises a chimera of amino acid sequences encoding beta strands and connecting loops from different Eph receptors (e.g., the EphA2 parent sequence with the HJ loop and J beta strand and JK loop derived from the EphA4 LBD, as described in Li Peng et al., Journal of Molecular Biology, 2011).

[0202] In some embodiments, a fragment of the ephrin receptor binding domain is inserted into the BC loop of the LBD via a linker, thereby mimicking the ligand-bound state of the LBD and promoting clustering at the LBD clustering interface. For example, the GH loop of the receptor binding domain of ephrin A2 can be inserted into the BC loop of the EphA4 LBD set forth in SEQ ID NO: 17. In some embodiments, the ligand-binding domain comprises the amino acid sequence set forth in Table 2.

[0203] Table 3

[0204] In some embodiments, the modified LBD can have the property of inducing head-to-tail heterodomain dimerization, i.e., the modified LBD can bind to the FN2 domain of another Eph receptor-derived polypeptide, thereby increasing oligomerization of the polypeptide on the cell surface or nanovesicles (e.g., EVs and hybridosomes). In one embodiment, the Eph receptor-derived polypeptide comprises an FN2 domain modified to enhance oligomerization. In certain embodiments, the LBD is modified to decrease ephrin binding, and the FN2 is modified to improve head-to-tail binding, for example, in EphA4, by substituting glutamic acid for arginine at position 106 and asparagine for threonine at position 507 in FN2 domain 2 (R106E and T507N, respectively). The vicinity of these residues is shown in Figure 3B. The interaction of Ephrin2 receptor-binding domain residue E129 with EphA4 LBD residue R106 is shown in Figure 3A.

[0205] (b) Cysteine-rich (CR) domain and fibronectin type III (FN III) domain The cysteine-rich region and the two fibronectin type III domains (i.e., FN1 and FN2) are referred to as "flexible domains." In some embodiments, the flexible domains of the polypeptides described herein are selected from the sequences in Table 7.

[0206] Polypeptides of the present disclosure may or may not comprise a flexible domain of a parent Eph receptor. In some embodiments, the flexible domain is partially or entirely absent. For example, the flexible domain can be partially or entirely replaced by another polypeptide, such as a linker or functional protein. Additionally or alternatively, a protein sequence of interest (e.g., a targeting domain and / or a purification domain) can be inserted or attached to a flexible domain of the polypeptide (e.g., a CR domain, an FN1 domain, or an FN2 domain), thereby adding additional functionality. In certain embodiments, a polypeptide of the present disclosure comprises a flexible domain of an Eph receptor.

[0207] In some embodiments, the polypeptide comprises a cysteine-rich region, an FN1 domain, and / or an FN2 domain of a parent Eph receptor, wherein the cysteine-rich region, the FN1 domain, and / or the FN2 domain comprises one or more modifications to increase interaction between one or more polypeptides. In certain embodiments, the one or more modifications are mutations, which can be substitutions, insertions, deletions, or any combination thereof.

[0208] In a specific embodiment, the parent Eph receptor comprises SEQ ID NO: 202, wherein the amino acid at position 504 is substituted with aspartic acid (D) and / or the amino acid at position 507 is substituted with aspartic acid (D). In a further specific embodiment, amino acid position 154 of the parent LBD is substituted with alanine (A). It is within routine experimentation to identify additional mutations within the flexible domain of this or other parent Eph receptors to increase oligomerization of the resulting polypeptides.

[0209] In some embodiments, the polypeptide comprises a CRD homodomain dimerization motif that increases the interaction between two or more polypeptides. In one embodiment, the CRD homodomain dimerization motif is GX1WX2VX3X4G (wherein X1 = E or K, X2 = L or M, X3 = P or A, and X4 = V, I, or L) (SEQ ID NO: 239).

[0210] In some embodiments, the CR domain of the polypeptide has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a CR domain of a wild-type ephrin receptor (e.g., an ephrin receptor CR domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-31). In some embodiments, the CR domain of the polypeptide is a CR domain of a wild-type ephrin receptor (e.g., an ephrin receptor CR domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-31).

[0211] In one aspect, the polypeptides described herein can comprise a CRD, wherein the CRD exhibits a three-dimensional structure that is superimposable with the CRD structure of a wild-type Ephrin receptor. In certain embodiments, the polypeptides described herein can comprise a CRD, wherein the CRD exhibits a three-dimensional structure that is superimposable with the CRD of a wild-type Eph receptor with a root mean square deviation (RMSD) of at most 1, 2, 4, 4, 5, 6, 7, 8, 9, or 10 Å between its equivalent Cα positions.

[0212] Table 4. CRD sequences [Table 4] TIFF2024514157000012.tif187170

[0213] In some embodiments, the CR domain comprises the amino acid sequence of a wild-type ephrin receptor CR domain (e.g., an ephrin receptor CR domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-31), excluding one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, seven amino acid mutations, or more than seven amino acid mutations. The mutations can be substitutions, insertions, deletions, or any combination thereof.

[0214] In some embodiments, the FN1 domain of the polypeptide has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the FN1 domain of a wild-type ephrin receptor (e.g., an ephrin receptor FN1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-45). In some embodiments, the FN1 domain of the polypeptide is the FN1 domain of a wild-type ephrin receptor (e.g., an ephrin receptor FN1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-45).

[0215] In one aspect, the polypeptides described herein can comprise FN1, which exhibits a three-dimensional structure that is superimposable with the FN1 structure of a wild-type Ephrin receptor. In certain embodiments, the polypeptides described herein can comprise FN1, which exhibits a three-dimensional structure that is superimposable with the wild-type Eph receptor FN1 at a root mean square deviation (RMSD) of at most 1, 2, 4, 4, 5, 6, 7, 8, 9, or 10 Å between its equivalent Cα positions.

[0216] Table 5. FNIII-1(CDD:365830 ProRule: PRU00316) [Table 5] TIFF2024514157000014.tif154170

[0217] In some embodiments, the FN1 domain comprises the amino acid sequence of a wild-type ephrin receptor FN1 domain (e.g., an ephrin receptor FN1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-45), excluding one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, seven amino acid mutations, or more than seven amino acid mutations. The mutations can be substitutions, insertions, deletions, or any combination thereof.

[0218] In some embodiments, the FN2 domain of the polypeptide has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the FN2 domain of a wild-type ephrin receptor (e.g., an ephrin receptor FN2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-59). In some embodiments, the FN2 domain of the polypeptide is an FN2 domain of a wild-type ephrin receptor (e.g., an ephrin receptor FN2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-59).

[0219] In one aspect, the polypeptides described herein can comprise FN2, which exhibits a three-dimensional structure that is superimposable with the FN2 structure of a wild-type Ephrin receptor. In certain embodiments, the polypeptides described herein can comprise FN2, which exhibits a three-dimensional structure that is superimposable with the wild-type Eph receptor FN2 at a root mean square deviation (RMSD) of at most 1, 2, 4, 4, 5, 6, 7, 8, 9, or 10 Å between its equivalent Cα positions.

[0220] Table 6. FNIII-2(CDD:365830 ProRule: PRU00316) [Table 6] TIFF2024514157000016.tif115170

[0221] In some embodiments, the FN2 domain comprises the amino acid sequence of a wild-type ephrin receptor FN2 domain (e.g., an ephrin receptor FN2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-59), excluding one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, seven amino acid mutations, or more than seven amino acid mutations. The mutations can be substitutions, insertions, deletions, or any combination thereof.

[0222] In some embodiments, the first Ephrin receptor FN III domain (i.e., FN1) and the second Ephrin receptor FN III domain (i.e., FN2) comprise different amino acid sequences. In some embodiments, the first Ephrin receptor FN III domain (i.e., FN1) and the second Ephrin receptor FN III domain (i.e., FN2) comprise the same amino acid sequence.

[0223] In one aspect, the polypeptides described herein can comprise a flexible domain, which exhibits a three-dimensional structure that is superimposable with the flexible domain structure of a wild-type Ephrin receptor. In certain embodiments, the polypeptides described herein can comprise a flexible domain, which exhibits a three-dimensional structure that is superimposable with the flexible domain of a wild-type Eph receptor with a root mean square deviation (RMSD) of at most 1, 2, 4, 4, 5, 6, 7, 8, 9, or 10 Å between its equivalent Cα positions.

[0224] In some embodiments, the flexible domain of the polypeptide has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a flexible domain of a wild-type ephrin receptor (e.g., a flexible domain of an ephrin receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-73). In some embodiments, the flexible domain of the polypeptide is a flexible domain of a wild-type ephrin receptor (e.g., a flexible domain of an ephrin receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-73).

[0225] Table 7. Exemplary flexible domains (CRD+FN1+FN2) [Table 7] TIFF2024514157000018.tif249170TIFF2024514157000019.tif104170

[0226] (c) Transmembrane (TM) domain A transmembrane domain of the present disclosure is a polypeptide domain of a membrane-associated or transmembrane protein that is embedded in and traverses the cell membrane at least once and includes one or more transmembrane regions. Such transmembrane regions or functional fragments thereof can be used as membrane anchors for polypeptides of the present disclosure (particularly Eph receptor-derived polypeptides). Transmembrane domains useful in such polypeptides of the present disclosure can be derived from transmembrane proteins associated with any of the various membranes of a cell, including, but not limited to, the plasma membrane, the endoplasmic reticulum membrane, the Golgi complex membrane, the lysosomal membrane, the nuclear membrane, and the mitochondrial membrane. In certain embodiments, the transmembrane domain is derived from a mammalian protein, preferably a human protein.

[0227] A transmembrane domain in a polypeptide of the present disclosure (particularly a polypeptide derived from an Eph receptor) includes all or a portion of the transmembrane region of a transmembrane protein that normally traverses the membrane of a cell with which the transmembrane protein is normally associated. A transmembrane domain can include not only the transmembrane region of a transmembrane protein, but also additional amino acids of a transmembrane protein located in adjacent regions either upstream (N-terminal) and / or downstream (C-terminal) to the transmembrane region or the membrane-embedded region of the transmembrane protein. For example, in certain embodiments, the entire transmembrane region of a transmembrane protein is used. In further embodiments, the entire transmembrane region and all or a portion of any upstream or downstream region of the membrane-embedded portion of the transmembrane protein can be used. Additional amino acids located either upstream (N-terminus) and / or downstream (C-terminus) from the membrane-embedded portion of a transmembrane protein that may be part of a transmembrane domain of a polypeptide of the disclosure (particularly an Eph receptor-derived polypeptide) may have a variety of sizes, including, but not limited to, 1 to 10 amino acids, 1 to 20 amino acids, 1 to 30 amino acids, 1 to 40 amino acids, 1 to 50 amino acids, 1 to 60 amino acids, 1 to 70 amino acids, 1 to 80 amino acids, 1 to 90 amino acids, 1 to 100 amino acids, 1 to 200 amino acids, 1 to 300 amino acids, 1 to 400 amino acids, 1 to 500 amino acids, 1 to 600 amino acids, 1 to 700 amino acids, 1 to 800 amino acids, and 1 to 900 amino acids. In some embodiments, the transmembrane domain lacks at least 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 800 amino acids from the N-terminus of the native transmembrane protein. In some embodiments, the transmembrane domain lacks at least 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 800 amino acids from the C-terminus of the native transmembrane protein. In some embodiments, the transmembrane domain lacks at least 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 800 amino acids from both the N-terminus and the C-terminus of the native transmembrane protein. In some embodiments, the transmembrane domain lacks one or more functional or structural domains of the native transmembrane protein.

[0228] The transmembrane domain, in some embodiments, is the transmembrane domain of a parent Eph receptor (e.g., an Eph receptor TM domain, such as an Eph receptor TM domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-87). The transmembrane domain of the polypeptides described herein can include the entire cytoplasmic region attached to the transmembrane region of a transmembrane protein, or a truncation of the cytoplasmic region by one or more amino acids, e.g., to eliminate undesired signaling functions of the cytoplasmic tail. As noted above, if all or a portion of the membrane-embedded (transmembrane) region and adjacent cytoplasmic C-terminal region of a kinase transmembrane protein is to be used as the transmembrane domain of a fusion protein of the present disclosure, any known functional kinase signal should be eliminated or disrupted so that a fusion protein comprising the transmembrane region and any adjacent cytoplasmic region does not activate host cells.

[0229] In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a transmembrane domain of a wild-type ephrin receptor (e.g., an ephrin receptor TM domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-87). In some embodiments, the transmembrane domain of the polypeptide is a transmembrane domain of a wild-type ephrin receptor (e.g., an ephrin receptor TM domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-87).

[0230] Table 8. TM Domains [Table 8]

[0231] In some embodiments, the transmembrane domain comprises the amino acid sequence of a wild-type ephrin receptor transmembrane domain (e.g., an ephrin receptor TM domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-87), excluding one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, seven amino acid mutations, or more than seven amino acid mutations. The mutations can be substitutions, insertions, deletions, or any combination thereof.

[0232] In some embodiments, the polypeptide comprises a transmembrane domain homo-domain dimerization motif that increases the interaction between two or more polypeptides in the transmembrane domain. In some embodiments, the transmembrane domain homo-domain dimerization motif is a transmembrane leucine zipper motif. In some embodiments, the transmembrane domain homo-dimerization motif is a transmembrane glycine zipper motif. Methods for modifying and assaying transmembrane domain dimerization are known in the art. See, for example, Bocharov et al., J Biol Chem. 2008 Oct 24;283(43):29385-95.

[0233] In some embodiments, the transmembrane domain comprises the amino acid sequence of a wild-type ephrin receptor transmembrane domain (e.g., an ephrin receptor TM domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 74 to 87), and its length is 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more at the N-terminus and / or C-terminus of the amino acid sequence of the wild-type ephrin receptor transmembrane domain (e.g., SEQ ID NOs: 74 to 87).

[0234] Knowing that a transmembrane region is derived from a particular type of transmembrane protein suggests a preferred orientation and location of the transmembrane domain for the polypeptides of the present disclosure (especially Eph receptor-derived polypeptides). This is particularly important for type I and type II transmembrane proteins, which have fixed orientations and locations of their N- and C-termini relative to the cytoplasm and nanovesicle lumen on either side of the transmembrane region. For example, when the transmembrane region of a type I transmembrane protein is used as the transmembrane domain of a polypeptide of the present disclosure, the polypeptide will be oriented in a position distal to the membrane. Thus, the most common configuration of a polypeptide of the present disclosure having a transmembrane domain derived from a type I transmembrane protein comprises a linear structure from N- to C-terminus shown as follows: (1) (ectodomain)-L-(transmembrane domain) (where L in the formula represents a direct peptide bond connecting the two domains or a linker of one or more amino acid residues).

[0235] In a specific embodiment, the polypeptides described herein that comprise a type I-derived transmembrane domain preferably comprise an N-terminal signal sequence that can direct the N-terminus of the polypeptide through the ER membrane and into the ER lumen.

[0236] As a non-limiting example, the Eph receptor-derived polypeptides described herein are constructed by inserting an ectodomain after the N-terminal signal peptide of a type I-derived transmembrane domain, such as that of EphA4 (SEQ ID NO: 77), or a transmembrane domain that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the transmembrane domain of EphA4 (SEQ ID NO: 77).

[0237] 5.2.2 Endodomains (Juxtamembrane (JM) Domain, Kinase Domain (KD), Sterile α-Motif (SAM) Linker Domain, SAM Domain, and PDZ-Binding Motif (PBM) Domain) In wild-type ephrin receptors, the ectodomain is connected to the transmembrane domain, which extends into the cell to the juxtamembrane (JM) domain, which connects the kinase domain, which is part of the endodomain.

[0238] As outlined above, the endodomain of wild-type ephrin receptors contains a juxtamembrane (JM) domain, a kinase domain (KD), a sterile alpha motif (SAM) linker domain, a SAM domain, and a PDZ-binding motif (PBM) domain. Among Eph receptors, EphB6 and EphA10 have altered essential motifs that contribute to their catalytic tyrosine kinase activity, rendering them catalytically deficient. In some cases, Eph receptors can be naturally expressed splicing isoforms that do not contain the KD, SAM linker domain, SAM domain, and / or PBM domain, such as mouse EphA4 and EphA7 isoforms.

[0239] The polypeptides of the present disclosure may optionally comprise a JM domain, a KD, a SAM linker domain, a SAM domain, and / or a PBM domain, or fragments thereof. In certain embodiments, the polypeptides described herein comprise, from N- to C-terminal, an Ephrin receptor CR domain, two Ephrin receptor FN III domains, a TM domain (e.g., an Ephrin receptor TM domain), and further comprise one, two, three, four, or all of the following domains C-terminal to the TM domain (e.g., an Ephrin receptor TM domain): an Ephrin receptor JM domain, an Ephrin receptor KD, a SAM linker domain (e.g., an Ephrin receptor SAM linker domain), a SAM domain (e.g., an Ephrin receptor SAM domain), and an Ephrin receptor PBM domain.

[0240] In specific embodiments, the polypeptides described herein comprise, from N-terminal to C-terminal, an ephrin receptor CR domain, two ephrin receptor FN III domains, a TM domain (e.g., an ephrin receptor TM domain), and further comprise an ephrin receptor JM domain C-terminal to the TM domain (e.g., the ephrin receptor TM domain) (e.g., fused to the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (e.g., with or without a linker, such as a peptide linker described herein)).

[0241] In some embodiments, the polypeptides of the present disclosure (particularly Eph receptor-derived polypeptides) lack forward signaling capability. Thus, in some embodiments, the polypeptide lacks ephrin receptor kinase activity. In some embodiments, the polypeptide completely lacks the endodomain of the parent Eph receptor. In some embodiments, the polypeptide lacks a portion of the endodomain of the parent Eph receptor. In some embodiments, the polypeptide lacks the kinase domain of the parent Eph receptor or a fragment thereof. In some embodiments, the polypeptide lacks a tyrosine amino acid in the kinase domain of the parent Eph receptor. In some embodiments, the polypeptide lacks the SAM linker domain of the parent Eph receptor. In some embodiments, the polypeptide lacks the SAM domain of the parent Eph receptor. In some embodiments, the polypeptide lacks the PBM domain of the parent Eph receptor.

[0242] Therefore, in a specific embodiment, the polypeptide provided herein is signal-neutral with respect to the forward (i.e., luminal) signal transduction ability of the cell that expresses the receptor.The ability of anterograde signal transduction can be tested by methods known in the art.See, for example, Germano, S., 2015, Receptor tyrosine kinases. Totowa, NJ: Humana Press.

[0243] In certain embodiments, the cellular localization, ubiquitination, and trafficking of a polypeptide can be redirected by modifying key residues in the KD and JM domains. Methods for modifying these processes are known in the art, as described, for example, in Sabet, O. et al., "Ubiquitination switches EphA2 vesicular traffic from a continuous safeguard to a finite signaling mode." Nat. Commun. 6:804.

[0244] In certain embodiments, polypeptides of the present disclosure (particularly Eph receptor-derived polypeptides) lack both ephrin receptor kinase activity and ephrin-binding activity. In a specific embodiment, polypeptides of the present disclosure (particularly Eph receptor-derived polypeptides) lack both an ephrin receptor kinase domain and an ephrin receptor ligand-binding domain. In a specific embodiment, polypeptides of the present disclosure (particularly Eph receptor-derived polypeptides) lack an ephrin receptor kinase domain and comprise a modified ephrin receptor ligand-binding domain described in Section 5.2.1. In a specific embodiment, polypeptides of the present disclosure (particularly Eph receptor-derived polypeptides) comprise a modified ephrin receptor kinase domain described herein and lack an ephrin receptor ligand-binding domain. In a specific embodiment, polypeptides of the present disclosure (particularly Eph receptor-derived polypeptides) comprise a modified ephrin receptor kinase domain described herein and comprise a modified ephrin receptor ligand-binding domain described in Section 5.2.1.

[0245] (a) Juxtamembrane domain In specific embodiments, the polypeptides described herein comprise, from N-terminal to C-terminal, an ephrin receptor CR domain, two ephrin receptor FN III domains, a TM domain (e.g., an ephrin receptor TM domain), and further comprise an ephrin receptor JM domain C-terminal to the TM domain (e.g., the ephrin receptor TM domain) (e.g., fused to the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (e.g., with or without a linker, such as a peptide linker described herein)).

[0246] In some embodiments, the JM domain of the polypeptide has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the JM domain of a wild-type ephrin receptor (e.g., an ephrin receptor JM domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 88-101 in Table 9). In some embodiments, the JM domain of the polypeptide is a JM domain of a wild-type ephrin receptor (e.g., an ephrin receptor JM domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 88-101).

[0247] Table 9. JM (CDD Superfamily: cl25995, ProRule: PRU00159) [Table 9]

[0248] In some embodiments, the JM domain comprises the amino acid sequence of a wild-type ephrin receptor JM domain (e.g., an ephrin receptor JM domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 88-101), excluding one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, seven amino acid mutations, or more than seven amino acid mutations. The mutations can be substitutions, insertions, deletions, or any combination thereof.

[0249] In specific embodiments, the ephrin receptor JM domain comprises: (i) an (X1)-Ptyr-(X2) motif (wherein Ptyr is phosphotyrosine, X1 is Y, P, V, I, T, or F, and X2 is I, V, L, or A); (ii) an (X3)-Ptyr-(X4) motif (wherein Ptyr is phosphotyrosine, X3 is T, A, or S, and X4 is E or G); or (iii) both (i) and (ii).

[0250] In specific embodiments, the ephrin receptor JM domain comprises (i) a YX1DX2X3X4YEDP motif (wherein X1 is I or V, X2 is P or L, X3 is Q, H, F, D, E, or S, and X4 is A or T) (SEQ ID NO: 240); or (ii) a FX1DX2X3X4FEDP motif (wherein X1 is I or V, X2 is P or L, X3 is Q, H, F, D, E, or S, and X4 is A or T) (SEQ ID NO: 241).

[0251] (b) Kinase domain In one embodiment, the polypeptides described herein can comprise a kinase domain, a conserved protein domain family (for EphA, NCBI CDD accession number cd05066; for EphB, NCBI CDD accession number cd05033).

[0252] In specific embodiments, the polypeptides described herein comprise, from N-terminal to C-terminal, an ephrin receptor CR domain, two ephrin receptor FN III domains, a TM domain (e.g., an ephrin receptor TM domain), and further comprise an ephrin receptor JM domain at the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (e.g., fused to the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (with or without a linker, such as a peptide linker described herein)), and an ephrin receptor KD at the C-terminus of the ephrin receptor TM domain (e.g., fused to the C-terminus of the ephrin receptor TM domain (with or without a linker, such as a peptide linker described herein)).

[0253] In specific embodiments, the polypeptides described herein comprise, from N-terminal to C-terminal, an ephrin receptor CR domain, two ephrin receptor FN III domains, a TM domain (e.g., an ephrin receptor TM domain), and further comprise an ephrin receptor KD C-terminal to the TM domain (e.g., the ephrin receptor TM domain) (e.g., fused to the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (e.g., with or without a linker, such as a peptide linker described herein)).

[0254] In some embodiments, the endodomain of the parent Eph receptor is modified to abolish signal transduction ability. For example, the polypeptide can comprise a kinase domain with one or more amino acid mutations that inactivate kinase activity. A specific embodiment is a kinase-dead Eph receptor mutant in which the conserved lysines in the ATP-binding site are mutated by substituting lysine (L) with arginine (R) (e.g., at amino acid positions L656, L646, L653, L707, L663, L665, L667, L651, L654, L665, and L647 of parent EphA1-8 and EphB1-4, respectively), thereby inactivating the enzymatic activity of the kinase domain. In a further non-limiting example, kinase-dead Eph receptor mutants can be generated by introducing one or more point mutations affecting residues essential for kinase activity, e.g., by removing conserved tyrosine residues in the tyrosine kinase domain (e.g., positions 781, 772, 779, 779, 883, 831, 791, 793, 793, 778, 780, 792, and 774 in parent EphA1-8 and EphB1-4, respectively), rendering them unable to phosphorylate their substrates. For example, kinase-dead Eph receptors are described in Truitt L, Freywald A, "Dancing with the dead: Eph receptors and their kinase-null partners," Biochem Cell Biol. 2011 Apr;89(2):115-129. For example, mutant kinase-dead Eph receptors are described in Peter W. Janes et al., Eph receptor function is modulated by heterooligomerization of A and B type Eph receptors, J Cell Biol, 2011 December, 195(6): 1033-1045.

[0255] In some embodiments, the KD of the polypeptide has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the KD of a wild-type ephrin receptor (e.g., an ephrin receptor KD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 102-115 in Table 10). In some embodiments, the KD of the polypeptide is the KD of a wild-type ephrin receptor (e.g., an ephrin receptor KD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 102-115).

[0256] The polypeptides described herein can comprise a KD, which exhibits a three-dimensional structure that can be superimposed on the flexible domain structure of a wild-type Ephrin receptor. In certain embodiments, the polypeptides described herein can comprise a KD, which exhibits a three-dimensional structure that can be superimposed on the wild-type Eph receptor KD with a root mean square deviation (RMSD) of at most 1, 2, 4, 4, 5, 6, 7, 8, 9, or 10 Å between its equivalent Cα positions.

[0257] Table 10. KD (CDD Superfamily: cl21453, ProRule: PRU00159) [Table 10] TIFF2024514157000023.tif245170TIFF2024514157000024.tif225170

[0258] In some embodiments, the KD comprises the amino acid sequence of a wild-type ephrin receptor KD (e.g., an ephrin receptor KD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 102-115), excluding one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, seven amino acid mutations, or more than seven amino acid mutations. The mutations can be substitutions, insertions, deletions, or any combination thereof.

[0259] In a specific embodiment, the ephrin receptor KD comprises a (X7)-Ptyr-(X8) motif in the activation loop (wherein Ptyr is phosphotyrosine, X7 is T, V, or A, and X8 is E or T).

[0260] (c) SAM linker domain In certain embodiments, the polypeptides described herein comprise, from the N-terminus to the C-terminus, an ephrin receptor CR domain, two ephrin receptor FN III domains, a TM domain (e.g., an ephrin receptor TM domain), and further comprise an ephrin receptor JM domain at the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (e.g., fused to the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (with or without a linker, such as a peptide linker described herein)), an ephrin receptor KD at the C-terminus of the ephrin receptor TM domain (e.g., fused to the C-terminus of the ephrin receptor TM domain (with or without a linker, such as a peptide linker described herein)), and a SAM linker domain (e.g., an ephrin receptor SAM linker domain) at the C-terminus of the ephrin receptor KD (e.g., fused to the C-terminus of the ephrin receptor KD (with or without a linker, such as a peptide linker described herein)).

[0261] In specific embodiments, the polypeptides described herein comprise, from the N-terminus to the C-terminus, an ephrin receptor CR domain, two ephrin receptor FN III domains, a TM domain (e.g., an ephrin receptor TM domain), and further comprise an ephrin receptor KD at the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (e.g., fused to the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (with or without a linker, such as a peptide linker described herein)), and a SAM linker domain (e.g., an ephrin receptor SAM linker domain) at the C-terminus of the ephrin receptor KD (e.g., fused to the C-terminus of the ephrin receptor KD (with or without a linker, such as a peptide linker described herein)).

[0262] In specific embodiments, the polypeptides described herein comprise, from N-terminal to C-terminal, an ephrin receptor CR domain, two ephrin receptor FN III domains, a TM domain (e.g., an ephrin receptor TM domain), and further comprise a SAM linker domain (e.g., an ephrin receptor SAM linker domain) at the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (e.g., fused to the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (with or without a linker, such as a peptide linker described herein)).

[0263] In some embodiments, the SAM linker domain of the polypeptide has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the SAM linker domain of a wild-type ephrin receptor (e.g., an ephrin receptor SAM linker domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 116-129 in Table 11). In some embodiments, the SAM domain of the polypeptide is the SAM domain of a wild-type ephrin receptor (e.g., an ephrin receptor SAM linker domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 116-129).

[0264] In some embodiments, the SAM linker domain contains multiple amino acids that function as phosphorylation sites. In some embodiments, the SAM linker domain is a mutant that contains at least one, two, three, four, five, six, seven, or eight phosphorylation sites (e.g., tyrosine (Y), serine (S), and threonine (T) sites) that are substituted with phosphomimetic amino acids (e.g., glutamic acid or aspartic acid that mimics the negative charge of a phosphate group). In some embodiments, the SAM linker domain containing phosphomimetic amino acids results in a conformational change (extension of the C-terminus away from the kinase domain). In some embodiments, the SAM linker domain contains at least one, two, three, four, five, six, seven, or eight phosphorylation sites (e.g., tyrosine, serine, and threonine residues) that are substituted with non-phosphorylatable amino acids (e.g., alanine).

[0265] In a specific embodiment, the SAM linker domain described herein is an ephrin receptor SAM linker domain.

[0266] Table 11. SAM linker domains [Table 11]

[0267] (d) SAM domain In one embodiment, a polypeptide described herein can comprise a SAM domain, a conserved protein domain family (NCBI CDD Accession No. cl26516 or Simple Modular Architecture Research Tool (SMART) Accession No. smart00454). In certain embodiments, a polypeptide described herein comprises, from N- to C-terminal, an ephrin receptor CR domain, two ephrin receptor FN III domains, a TM domain (e.g., an ephrin receptor TM domain), and an ephrin receptor JM domain at the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (e.g., fused to the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (e.g., with or without a linker, such as a peptide linker described herein)), an ephrin receptor KD at the C-terminus of the ephrin receptor TM domain (e.g., fused to the C-terminus of the ephrin receptor TM domain (e.g., with or without a linker, such as a peptide linker described herein)), an ephrin receptor KD. It further comprises a SAM linker domain (e.g., an ephrin receptor SAM linker domain) at the C-terminus (e.g., fused to the C-terminus of the ephrin receptor KD (using a linker such as a peptide linker described herein or without a linker)), and a SAM domain (e.g., an ephrin receptor SAM linker domain) at the C-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain) (e.g., fused to the C-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain) (using a linker such as a peptide linker described herein or without a linker)).

[0268] In specific embodiments, the polypeptides described herein comprise, from N- to C-terminal, an ephrin receptor CR domain, two ephrin receptor FN domains, and one ephrin receptor CR domain. The nucleic acid sequence includes a III domain, a TM domain (e.g., an ephrin receptor TM domain), and further includes an ephrin receptor KD at the C-terminus of the TM domain (e.g., an ephrin receptor TM domain) (e.g., fused to the C-terminus of the TM domain (e.g., an ephrin receptor TM domain) (using a linker such as a peptide linker described herein or without a linker)), a SAM linker domain (e.g., an ephrin receptor SAM linker domain) at the C-terminus of the ephrin receptor KD (e.g., fused to the C-terminus of the ephrin receptor KD (using a linker such as a peptide linker described herein or without a linker)), and a SAM domain (e.g., an ephrin receptor SAM linker domain) at the C-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain) (e.g., fused to the C-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain) (using a linker such as a peptide linker described herein or without a linker)).

[0269] In specific embodiments, the polypeptides described herein comprise, from N-terminal to C-terminal, an ephrin receptor CR domain, two ephrin receptor FN III domains, a TM domain (e.g., an ephrin receptor TM domain), and further comprise a SAM linker domain (e.g., an ephrin receptor SAM linker domain) at the C-terminus of the TM domain (e.g., an ephrin receptor TM domain) (e.g., fused to the C-terminus of the TM domain (e.g., an ephrin receptor TM domain) (with a linker such as a peptide linker described herein or without a linker)), and a SAM domain (e.g., an ephrin receptor SAM linker domain) at the C-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain) (e.g., fused to the C-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain) (with a linker such as a peptide linker described herein or without a linker)).

[0270] In specific embodiments, the polypeptides described herein comprise, from N-terminal to C-terminal, an ephrin receptor CR domain, two ephrin receptor FN III domains, a TM domain (e.g., an ephrin receptor TM domain), and further comprise a SAM domain (e.g., an ephrin receptor SAM domain) C-terminal to the TM domain (e.g., the ephrin receptor TM domain) (e.g., fused to the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (with or without a linker, such as a peptide linker described herein)).

[0271] In some embodiments, the SAM domain of the polypeptide has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a SAM domain of a wild-type ephrin receptor (e.g., an ephrin receptor SAM domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130-143 in Table 12). In some embodiments, the ephrin receptor SAM domain comprising an amino acid sequence selected from the group consisting of is a SAM domain of a wild-type ephrin receptor (e.g., an ephrin receptor SAM domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130-143).

[0272] Table 12. SAM(CDD: cd09488, ProRule:RU00184) [Table 12]

[0273] In some embodiments, the SAM domain comprises the amino acid sequence of a wild-type ephrin receptor SAM domain (e.g., an ephrin receptor SAM domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130-143), excluding one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, seven amino acid mutations, or more than seven amino acid mutations. The mutations can be substitutions, insertions, deletions, or any combination thereof.

[0274] In some embodiments, the SAM domain is not derived from an ephrin receptor. In specific embodiments, the SAM domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 144-146 in Table 13. In certain embodiments, non-ephrin receptor SAM domains have a higher tendency to oligomerize than ephrin receptor SAM domains. In specific embodiments, non-ephrin receptor SAM domains with a higher tendency to oligomerize adopt a head-to-tail configuration, non-limiting examples of which include the TEL, TNKS1, or TNKS2 SAM domains. Some human SAM domains can oligomerize with varying self-affinities ranging from high micromolar to nanomolar concentrations, and methods for identifying and assaying such domains are known in the art (e.g., Knight et al. (2011). Protein Science, 20: 1697-1706).

[0275] In some embodiments, the polypeptides described herein can comprise a SAM domain, wherein the SAM domain exhibits a three-dimensional structure that can be superimposed on the flexible domain structure of a wild-type Ephrin receptor. In certain embodiments, the polypeptides described herein can comprise a SAM domain, wherein the SAM domain exhibits a three-dimensional structure that can be superimposed on the wild-type Eph receptor SAM domain with a root mean square deviation (rmds) of at most 1, 2, 4, 4, 5, 6, 7, 8, 9, or 10 Å between its equivalent Cα positions.

[0276] Table 13. Non-ephrin SAM domains [Table 13]

[0277] In a specific embodiment, the SAM domain comprises a phosphotyrosine in the α2 helix. In certain embodiments, the phosphotyrosine in the α2 helix of the SAM domain is in a (X5)-Ptyr-(X6) motif (wherein Ptyr is phosphotyrosine, X5 is C, R, Q, or H, and X6 is Q, I, E, K, R, or T).

[0278] In a specific embodiment, the SAM domain described herein is an ephrin receptor SAM domain.

[0279] (e) PBM domain In a specific embodiment, the polypeptide described herein comprises, from N-terminal to C-terminal direction, an ephrin receptor CR domain, two ephrin receptor FN III domains, a TM domain (e.g., an ephrin receptor TM domain), and an ephrin receptor JM domain at the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (e.g., fused to the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (with or without a linker, such as a peptide linker described herein)), an ephrin receptor KD at the C-terminus of the ephrin receptor TM domain (e.g., fused to the C-terminus of the ephrin receptor TM domain (with or without a linker, such as a peptide linker described herein)), and at the C-terminus of the ephrin receptor KD (e.g., at the C-terminus of the ephrin receptor KD (with or without a linker, such as a peptide linker described herein). and an ephrin receptor PBM domain at the C-terminus of the SAM domain (e.g., an ephrin receptor SAM domain) (e.g., fused to the C-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain) (e.g., fused to the C-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain) (with a linker such as a peptide linker described herein or without a linker)).

[0280] In a specific embodiment, the polypeptides described herein comprise, from N-terminal to C-terminal, an ephrin receptor CR domain, two ephrin receptor FN III domains, a TM domain (e.g., an ephrin receptor TM domain), and an ephrin receptor KD at the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (e.g., fused to the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (with or without a linker, such as a peptide linker described herein)), a SAM linker domain (e.g., an ephrin receptor SAM linker domain) at the C-terminus of the ephrin receptor KD (e.g., fused to the C-terminus of the ephrin receptor KD (with or without a linker, such as a peptide linker described herein)), a SAM linker domain The SAM domain (e.g., an ephrin receptor SAM linker domain) is C-terminal to the SAM linker domain (e.g., an ephrin receptor SAM linker domain) (e.g., fused to the C-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain) (using a linker such as a peptide linker described herein or without a linker)), and the SAM domain (e.g., an ephrin receptor SAM domain) is C-terminal to the SAM domain (e.g., an ephrin receptor SAM domain) (e.g., fused to the C-terminus of the SAM domain (e.g., an ephrin receptor SAM domain) (using a linker such as a peptide linker described herein or without a linker)).

[0281] In specific embodiments, the polypeptides described herein comprise, from N- to C-terminal, an ephrin receptor CR domain, two ephrin receptor FN domains, and one ephrin receptor CR domain. The SAM linker domain further comprises a III domain, a TM domain (e.g., an ephrin receptor TM domain) and a SAM linker domain (e.g., an ephrin receptor SAM linker domain) at the C-terminus of the TM domain (e.g., an ephrin receptor TM domain) (e.g., fused to the C-terminus of the TM domain (e.g., an ephrin receptor TM domain) (using a linker such as a peptide linker described herein or without a linker)), a SAM domain (e.g., an ephrin receptor SAM domain) at the C-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain) (e.g., fused to the C-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain) (using a linker such as a peptide linker described herein or without a linker)), and an ephrin receptor PBM domain at the C-terminus of the SAM domain (e.g., an ephrin receptor SAM domain) (e.g., fused to the C-terminus of the SAM domain (e.g., an ephrin receptor SAM domain) (using a linker such as a peptide linker described herein or without a linker)).

[0282] In specific embodiments, the polypeptides described herein comprise, from N-terminal to C-terminal, an ephrin receptor CR domain, two ephrin receptor FN III domains, a TM domain (e.g., an ephrin receptor TM domain), and further comprise a SAM domain (e.g., an ephrin receptor SAM domain) at the C-terminus of the TM domain (e.g., an ephrin receptor TM domain) (e.g., fused to the C-terminus of the TM domain (e.g., an ephrin receptor TM domain) (with or without a linker, such as a peptide linker described herein)), and an ephrin receptor PBM domain at the C-terminus of the SAM domain (e.g., an ephrin receptor SAM domain) (e.g., fused to the C-terminus of the SAM domain (e.g., an ephrin receptor SAM domain) (with or without a linker, such as a peptide linker described herein)).

[0283] In specific embodiments, the polypeptides described herein comprise, from N-terminal to C-terminal, an ephrin receptor CR domain, two ephrin receptor FN III domains, a TM domain (e.g., an ephrin receptor TM domain), and further comprise an ephrin receptor PBM domain C-terminal to the TM domain (e.g., the ephrin receptor TM domain) (e.g., fused to the C-terminus of the TM domain (e.g., the ephrin receptor TM domain) (e.g., with or without a linker, such as a peptide linker described herein)).

[0284] In some embodiments, the PBM domain of the polypeptide has at least 33%, at least 66%, or at least 99% sequence identity to a PBM domain of a wild-type ephrin receptor (e.g., an ephrin receptor PBM domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 147-159 and 256 in Table 14). In some embodiments, the PBM domain of the polypeptide is a PBM domain of a wild-type ephrin receptor (e.g., an ephrin receptor PBM domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 147-159 and 256).

[0285] Table 14. PDZ-binding motifs [Table 14]

[0286] In some embodiments, the PBM domain comprises the amino acid sequence of a wild-type Ephrin receptor PBM domain (e.g., an Ephrin receptor PBM domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 147-159 and 256), excluding one amino acid mutation or two amino acid mutations. The mutations can be substitutions, insertions, deletions, or any combination thereof.

[0287] 5.2.3 Cargo and Cargo-Binding Domains The polypeptides described herein can be used to deliver cargo (e.g., cargo proteins), for example, via extracellular vesicles (EVs) or hybridosomes, for example, for therapeutic or diagnostic uses. The cargo (e.g., cargo proteins) can be a portion of a polypeptide. That is, the cargo (e.g., cargo proteins) can be fused (e.g., via a linker) to the remainder of the polypeptide (see, e.g., Figure 9). Alternatively, the cargo (e.g., cargo proteins) can be bound (preferably reversibly bound) to the polypeptide via a cargo-binding domain. The cargo-binding domain can be bound directly to the cargo (e.g., cargo proteins) or indirectly via a scaffold-binding domain (SBD) linked to the cargo (e.g., cargo proteins). The cargo-binding domain can be either an ephrin receptor domain (e.g., an ephrin receptor JM domain, an ephrin receptor KD, an ephrin receptor SAM linker domain, an ephrin receptor SAM domain, or an ephrin receptor PBM domain; see, e.g., Figure 10), or a domain that can bind to a cargo but is not an ephrin receptor domain. Thus, in specific embodiments, the polypeptides described herein comprise a cargo (e.g., a cargo protein) or a cargo-binding domain. As used herein, the singular forms "a," "an," and "the" include plural referents. Thus, the polypeptides described herein can be used to deliver one or more (e.g., one, two, three, four, five, or more) cargoes, and the polypeptides described herein can comprise one or more (e.g., one, two, three, four, five, or more) cargo-binding domains.

[0288] Exemplary cargoes (e.g., cargo proteins) include, but are not limited to, therapeutic molecules (e.g., therapeutic proteins), adjuvants, diagnostic proteins, and / or receptor proteins. The cargo may be a large polypeptide or peptide, such as RGD or an antimicrobial peptide.

[0289] A therapeutic molecule refers to any molecule that may have therapeutic use. A therapeutic molecule can be any inorganic or organic compound. A therapeutic molecule can reduce, inhibit, attenuate, reduce, arrest, or stabilize the occurrence or progression of a disease, disorder, or cell proliferation in an animal, e.g., a mammal or a human. Examples of therapeutic molecules that can be incorporated into nanovesicles containing Eph receptor-derived polypeptides include therapeutic agents, e.g., nucleic acids (e.g., DNA or mRNA molecules encoding polypeptides such as enzymes, mRNA molecules encoding polypeptides such as antigens, or RNA molecules with regulatory functions, e.g., miRNA, dsDNA, and lncRNA), amino acids (e.g., amino acids containing detectable moieties or toxins or amino acids that disrupt translation), polypeptides (e.g., enzymes, enzymes for gene editing, nucleic acid-binding proteins, antibodies, intrabodies, single-chain variable fragments (scFvs), affibodies, bi- and multispecific antibodies or binders, affibodies, darpins, receptors, ligands, or fragments thereof), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins). In certain embodiments, a therapeutic molecule can be a substance used in the diagnosis, treatment, or prevention of a disease or as a component of a pharmaceutical. In some embodiments, a "payload" can refer to a compound that facilitates obtaining diagnostic information about a targeted site within an organism, e.g., a mammal, or particularly a human. For example, an imaging agent can be classified as an active agent in the present disclosure, as it is a substance that provides the imaging information needed for diagnosis.

[0290] Further non-limiting examples of therapeutic nucleic acids contemplated for use in the present disclosure include siRNA, small or short hairpin RNA (shRNA), guide RNA (gRNA), single guide RNA (sgRNA), clustered regularly interspaced short palindromic repeat RNA (crRNA), trans-acting clustered regularly interspaced short palindromic repeat RNA (tracrRNA), immunostimulatory oligonucleotides, plasmids, antisense nucleic acids, and ribozymes. In certain embodiments, the therapeutic nucleic acid can be linear DNA, circular DNA, or an artificial chromosome. In some embodiments, the therapeutic DNA is maintained episomally. In some embodiments, the therapeutic DNA is integrated into the genome. The therapeutic RNA can be chemically modified RNA, for example, the therapeutic RNA can include one or more backbone modifications, sugar modifications, atypical bases, or caps. Examples of backbone modifications include phosphorothioate, N3' phosphoramidite, boranophosphate, phosphonoacetate, thio-PACE, morpholino phosphoramidite, or PNA. Examples of sugar modifications include 2'-0-Me, LNA, UNA, and 2'-0-MOE. Examples of non-standard bases include 5-bromo-U and 5-iodo-U, 2,6-diaminopurine, C-5 propynylpyrimidine, difluorotoluene, difluorobenzene, dichlorobenzene, 2-thiouridine, pseudouridine, and dihydrouridine. Examples of caps include ARCA. Further modifications are discussed, for example, in Deleavy et al., "Designing Chemically Modified Oligonucleotides for Targeted Gene Silencing," Chemistry & Biology, Vol. 19, Issue 8, August 24, 2012, pp. 937-954.

[0291] Other suitable therapeutic molecules include, but are not limited to, pharmacologically active drugs and genetically active molecules, including anti-neoplastic agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modulators, and neuroactive agents.Examples of suitable therapeutic payloads include those described in "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, NY, (1996), 9th Edition, under the following sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; Drugs Acting on the Central Nervous System; Autacoids; Drug Therapy of Inflammation; Water, Salts and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism; Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function; Drugs Affecting Uterine Motility; Affecting Uterine Motility; Chemotherapy of Parasitic Infections; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming Organs; Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology.Suitable payloads further include toxins and biological and chemical warfare agents, see, e.g., Somani, SM (ed.), Chemical Warfare Agents, Academic Press, New York (1992).

[0292] Further non-limiting examples of therapeutic molecules include antigen-binding molecules (e.g., therapeutic antibodies or antigen-binding fragments thereof), gene editors, transposases, enzymes or fragments thereof; ligands or fragments thereof, receptors or fragments thereof; antimicrobial peptides or fragments thereof, amino acids, and any combination thereof. In some embodiments, the therapeutic molecule is non-proteinaceous and is attached to the Eph receptor-derived polypeptide via a linker.

[0293] Antigen-binding molecules that function as therapeutic molecules can be monospecific, bispecific, or multispecific, i.e., they can target one or more epitopes of the same or different targets. The more specificities displayed on a nanovesicle, the more specific the targeting. In some embodiments, the antigen-binding molecule is i) a full-length antibody molecule (e.g., IgG, IgM, IgA, IgM, or IgE); ii) antibody fragments, such as CDR, Dab, Fab, Fab', F(ab)'2, Fd fragment, Fv fragment, disulfide-linked Fv, scFab, nanobody, minimal recognition unit, VHH, or V-NAR domains; iii) non-antibody scaffolds, such as affibodies, affilin molecules, affitins, adnectins, anticalins, avimers, centrins, lipocalin muteins, DARPins, phynomers, knottins, Kunitz-type domains, nanophytins, tetranectins, or trans-bodies; iv) fusion polypeptides comprising one or more antibody domains, such as bi-scFv, aBITE, diabody, bi-scFv, probody, tascFv (tandem scFv), triabody, tribody, tetrabody, IgGACH2, DVD-Ig, MATCH, minibody, scFv, scFv-Fc, bispecific F(ab')2, F(ab')3, monovalent IgG; v) Soluble T cell receptor (sTCR); vi) peptides, such as natural peptides, recombinant peptides, synthetic peptides; and / or vii) viral proteins, such as the receptor-binding domain of a viral spike protein (e.g., of a coronavirus) or influenza hemagglutinin (HA), or fragments thereof; : are each selected from the group consisting of:

[0294] Several reporter proteins are known in the art, such as green fluorescent protein (GFP) or luciferase, which are useful for monitoring intracellular trafficking and / or uptake of nanovesicles in recipient cells.

[0295] In certain embodiments, the diagnostic protein can be a fluorescent protein. In specific embodiments, the diagnostic protein can be a fusion protein comprising a moiety capable of binding to a biomarker of interest and a fluorescent protein (e.g., GFP).

[0296] (a) cargo fusion protein As described above, the polypeptides provided herein can contain one or more cargoes (e.g., cargo proteins), preferably biologically active molecules. Thus, when sorted into nanovesicles, the polypeptides function as scaffold proteins for such cargoes (e.g., cargo proteins). The polypeptides provide a protein scaffold suitable for loading any molecule of interest into nanovesicles in a predefined manner, for example, by genetic fusion. In certain embodiments, the cargo is a cargo protein (e.g., a cargo peptide or cargo polypeptide) fused in-frame to the remainder of the polypeptide. In certain embodiments, the cargo protein is fused to the remainder of the polypeptide via a linker. In some embodiments, the cargo protein is covalently fused to the remainder of the polypeptide via a linker. In specific embodiments, the linker is a peptide linker. In specific embodiments, the peptide linker comprises the amino acid sequence (GGGS)n (SEQ ID NO: 231), where n is an integer between 1 and 10. In specific embodiments, the peptide linker comprises the amino acid sequence GGGS. In a specific embodiment, the peptide linker comprises the amino acid sequence (GGGS)2 (SEQ ID NO: 232). In a specific embodiment, the peptide linker comprises the amino acid sequence (GGGS)3 (SEQ ID NO: 233).

[0297] Such one or more cargoes (e.g., cargo proteins) can be at the N-terminus and / or C-terminus of the remainder of the polypeptide (e.g., fused to the N-terminus and / or C-terminus), or can be located between different domains in the remainder of the polypeptide. In certain embodiments, one or more cargoes (e.g., cargo proteins) are presented toward the lumen of the nanovesicle. In some embodiments, one or more cargoes (e.g., cargo proteins) are at the C-terminus of (e.g., fused to the C-terminus of) the TM domain (e.g., an ephrin receptor TM domain). In some embodiments, one or more cargoes (e.g., cargo proteins) are at the C-terminus of (e.g., fused to the C-terminus of) the ephrin receptor JM domain. In some embodiments, one or more cargoes (e.g., cargo proteins) are at the C-terminus of (e.g., fused to the C-terminus of) the ephrin receptor KD. In some embodiments, one or more cargoes (e.g., cargo proteins) are at the C-terminus (e.g., fused to the C-terminus) of the SAM linker domain (e.g., an ephrin receptor SAM linker domain). In some embodiments, one or more cargoes (e.g., cargo proteins) are at the C-terminus (e.g., fused to the C-terminus) of the SAM domain (e.g., an ephrin receptor SAM domain). In some embodiments, one or more cargoes (e.g., cargo proteins) are at the C-terminus (e.g., fused to the C-terminus) of the ephrin receptor PBM domain. In certain embodiments, one or more cargoes (e.g., cargo proteins) are presented toward the external space of the nanovesicle. In some embodiments, one or more cargoes (e.g., cargo proteins) are at the N-terminus (e.g., fused to the N-terminus) of the ephrin ligand binding domain of the polypeptide. In some embodiments, one or more cargoes (e.g., cargo proteins) are at the N-terminus (e.g., fused to the N-terminus) of the ephrin receptor cysteine-rich domain of the polypeptide. In some embodiments, one or more cargoes (eg, cargo proteins) are N-terminal to (eg, fused to) the ephrin receptor FN1 domain.In some embodiments, one or more cargoes (e.g., cargo proteins) are N-terminal to (e.g., fused to) the ephrin receptor FN2 domain. In some embodiments, one or more cargoes (e.g., cargo proteins) are N-terminal to (e.g., fused to) the TM domain (e.g., ephrin receptor TM domain).

[0298] In certain embodiments, one or more cargos (e.g., cargo proteins) are at the N-terminus (e.g., fused to the N-terminus) of a targeting domain described in this disclosure. In certain embodiments, one or more cargos (e.g., cargo proteins) are at the C-terminus (e.g., fused to the C-terminus) of a targeting domain described in this disclosure. In certain embodiments, one or more cargos (e.g., cargo proteins) are at the N-terminus (e.g., fused to the N-terminus) of a purification domain described in this disclosure. In certain embodiments, one or more cargos (e.g., cargo proteins) are at the C-terminus (e.g., fused to the C-terminus) of a purification domain described in this disclosure. In certain embodiments, one or more cargos (e.g., cargo proteins) are at the N-terminus (e.g., fused to the N-terminus) of a modified Fc domain described in this disclosure. In certain embodiments, one or more cargos (e.g., cargo proteins) are at the C-terminus (e.g., fused to the C-terminus) of a modified Fc domain described in this disclosure.

[0299] (b) cargo-binding domain The polypeptides described herein can be used to deliver cargo (e.g., cargo proteins) associated (preferably non-covalently bound) with the polypeptide via a cargo-binding domain. The cargo-binding domain can be either an ephrin receptor domain (e.g., an ephrin receptor JM domain, an ephrin receptor KD, an ephrin receptor SAM linker domain, an ephrin receptor SAM domain, or an ephrin receptor PBM domain; see, e.g., Figure 10 ), or a domain that can bind to cargo but is not an ephrin receptor domain (e.g., a SAM domain or SAM linker domain not derived from an ephrin receptor). Thus, in specific embodiments, the polypeptides described herein comprise a cargo-binding domain. The cargo-binding domain can bind directly to the cargo (e.g., a cargo protein) or indirectly via a scaffold-binding domain (SBD) linked to the cargo (e.g., a cargo protein). In specific embodiments, the cargo-binding domain can specifically bind to the cargo (e.g., a cargo protein). As used herein, the singular forms "a," "an," and "the" include plural referents. Thus, the polypeptides described herein can be used to deliver one or more (e.g., one, two, three, four, five, or more) cargoes, and the polypeptides described herein can include one or more (e.g., one, two, three, four, five, or more) cargo-binding domains.

[0300] In various embodiments, the cargo-binding domain can be fused in-frame to the remainder of the polypeptide. In certain embodiments, the cargo-binding domain is fused to the remainder of the polypeptide via a linker. In some embodiments, the cargo-binding domain is fused to the remainder of the polypeptide via a linker. In specific embodiments, the linker is a peptide linker. In specific embodiments, the peptide linker comprises the amino acid sequence (GGGS)n (SEQ ID NO: 231), where n is an integer from 1 to 10. In specific embodiments, the peptide linker comprises the amino acid sequence GGGS. In specific embodiments, the peptide linker comprises the amino acid sequence (GGGS)2 (SEQ ID NO: 232). In specific embodiments, the peptide linker comprises the amino acid sequence (GGGS)3 (SEQ ID NO: 233).

[0301] Such cargo-binding domains can be at the N-terminus or C-terminus of the remainder of the polypeptide (e.g., fused to the N-terminus and / or C-terminus), or can be located between different domains in the remainder of the polypeptide. In certain embodiments, the cargo-binding domain is presented toward the lumen of the nanovesicle. In some embodiments, the cargo-binding domain is at the C-terminus (e.g., fused to the C-terminus) of a TM domain (e.g., an ephrin receptor TM domain). In some embodiments, the cargo-binding domain is at the C-terminus (e.g., fused to the C-terminus) of an ephrin receptor JM domain. In some embodiments, the cargo-binding domain is at the C-terminus (e.g., fused to the C-terminus) of an ephrin receptor KD. In some embodiments, the cargo-binding domain is at the C-terminus (e.g., fused to the C-terminus) of a SAM linker domain (e.g., an ephrin receptor SAM linker domain). In some embodiments, the cargo-binding domain is at the C-terminus (e.g., fused to the C-terminus) of the SAM domain (e.g., an ephrin receptor SAM domain). In some embodiments, the cargo-binding domain is at the C-terminus (e.g., fused to the C-terminus) of the ephrin receptor PBM domain. In certain embodiments, the cargo-binding domain is presented toward the external space of the nanovesicle. In some embodiments, the cargo-binding domain is at the N-terminus (e.g., fused to the N-terminus) of the ephrin ligand-binding domain of the polypeptide. In some embodiments, the cargo-binding domain is at the N-terminus (e.g., fused to the N-terminus) of the ephrin receptor cysteine-rich domain of the polypeptide. In some embodiments, the cargo-binding domain is at the N-terminus (e.g., fused to the N-terminus) of the ephrin receptor FN1 domain. In some embodiments, the cargo-binding domain is at the N-terminus (e.g., fused to the N-terminus) of the ephrin receptor FN2 domain. In some embodiments, the cargo binding domain is N-terminal to (eg, fused to) the TM domain (eg, an ephrin receptor TM domain).

[0302] In some embodiments, the cargo-binding domain is at the N-terminus (e.g., fused to the N-terminus) of the targeting domain described in this disclosure. In some embodiments, the cargo-binding domain is at the C-terminus (e.g., fused to the C-terminus) of the targeting domain described in this disclosure. In some embodiments, the cargo-binding domain is at the N-terminus (e.g., fused to the N-terminus) of the purification domain described in this disclosure. In some embodiments, the cargo-binding domain is at the C-terminus (e.g., fused to the C-terminus) of the purification domain described in this disclosure. In some embodiments, the cargo-binding domain is at the N-terminus (e.g., fused to the N-terminus) of the modified Fc domain described in this disclosure. In some embodiments, the cargo-binding domain is at the C-terminus (e.g., fused to the C-terminus) of the modified Fc domain described in this disclosure.

[0303] In some embodiments, the bond between the cargo-binding domain and the cargo (e.g., cargo protein) is a covalent bond. In further embodiments, the association between the cargo-binding domain and the cargo (e.g., cargo protein) is a non-covalent bond (e.g., as shown in Figure 10). Preferably, the association between the cargo-binding domain and the cargo (e.g., cargo protein) is a reversible association.

[0304] In specific embodiments, the binding between the cargo-binding domain and the cargo (e.g., cargo protein) can be controlled. In specific embodiments, the binding between the cargo-binding domain and the cargo (e.g., cargo protein) can be controlled by a parameter whose value depends on the location of the polypeptide, for example, whether the polypeptide is in vitro or in vivo, or in which organ, tissue, cell, or subcellular compartment the polypeptide is located. In specific embodiments, the binding between the cargo-binding domain and the cargo (e.g., cargo protein) can be controlled by pH. In specific embodiments, the binding between the cargo-binding domain and the cargo (e.g., cargo protein) can be controlled by ionic strength. In specific embodiments, the binding between the cargo-binding domain and the cargo (e.g., cargo protein) can be controlled by the presence or absence of phosphatase. In specific embodiments, the binding between the cargo-binding domain and the cargo (e.g., cargo protein) can be controlled such that the cargo (e.g., cargo protein) binds to the cargo-binding domain in vitro but is released from the cargo-binding domain in vivo. For example, the binding between the cargo-binding domain and the cargo (e.g., cargo protein) can have a higher binding affinity (e.g., at least 2-fold higher, at least 5-fold higher, at least 10-fold higher, at least 20-fold higher, at least 50-fold higher, at least 100-fold higher, at least 200-fold higher, at least 500-fold higher, or at least 1000-fold higher) in an in vitro environment compared to an in vivo environment. In specific embodiments, the binding between the cargo-binding domain and the cargo (e.g., cargo protein) can be controlled such that the cargo (e.g., cargo protein) is released from the cargo-binding domain in a manner that depends on the intracellular compartment in which it is located. For example, in specific embodiments, the binding between the cargo-binding domain and the cargo (e.g., cargo protein) can be controlled such that the cargo (e.g., cargo protein) is released from the cargo-binding domain as it enters the cytosol.In a further non-limiting example, the binding between the cargo-binding domain and the cargo (e.g., cargo protein) can be controlled such that the cargo (e.g., cargo protein) is released from the cargo-binding domain when a nanovesicle containing the cargo protein in its lumen fuses with an endosomal membrane and contacts the cytosol.

[0305] In some embodiments, the cargo-binding domain of a polypeptide and the cargo, e.g., a cargo protein, are associated via an intermediary. In some embodiments, the cargo, e.g., a cargo protein, is linked to a scaffold-binding domain, and the scaffold protein comprises a cargo-binding domain, wherein the cargo-binding domain of the scaffold protein is associated with the scaffold-binding domain linked to the cargo (e.g., cargo protein). In some embodiments, the cargo protein has at least one scaffold-binding domain. The cargo protein can comprise or be linked to a scaffold-binding domain at any position of the protein, whether at the C- or N-terminus, or anywhere in between. In some embodiments, the binding between the cargo-binding domain and the scaffold-binding domain linked to the cargo protein can be controlled by considering the binding affinity, the binding kinetics (e.g., the intrinsic equilibrium dissociation constant), and the concentration of the binding partner. Methods for analyzing the binding affinity and binding kinetics between scaffold-binding domains and cargo-binding domains are known in the art (e.g., SPR, BLI, ELISA).

[0306] In certain embodiments, the bond between the cargo-binding domain and the cargo (e.g., a cargo protein) is a phosphotyrosine-based bond (e.g., a bond between phosphotyrosine and a phosphotyrosine-binding (PTB) domain, a bond between phosphotyrosine and a Src homology 2 (SH2) domain, or a bond between phosphotyrosine and a HYB domain, a GEP100 PH domain, a PKCδ domain, a PKCθ C2 domain, a catalytically inactive PTP domain, or a Raf-1 kinase inhibitory protein (RKIP) domain). In specific embodiments, the cargo-binding domain comprises a phosphotyrosine and a cargo (e.g., a cargo protein), or the scaffold-binding domain (SBD) comprises a domain capable of binding to phosphotyrosine, and the bond between the cargo-binding domain and the cargo (e.g., a cargo protein) is a bond between a phosphotyrosine and a domain capable of binding to phosphotyrosine.

[0307] In a specific embodiment, the domain capable of binding to phosphotyrosine is a PTB domain. In a specific embodiment, the PTB domain is derived from CBL (UniProt ID No. P22681) or comprises an amino acid sequence identical or similar to the amino acid sequence of SEQ ID NO: 160 listed in Table 15 below, and the cargo-binding domain comprises phosphotyrosine. In a specific embodiment, the PTB domain is derived from CBL (UniProt ID No. P22681) or comprises the amino acid sequence of SEQ ID NO: 160 listed in Table 15 below, and the cargo-binding domain comprises phosphotyrosine and is derived from or is derived from EphA2. In a specific embodiment, the domain capable of binding to phosphotyrosine is a functional variant of an SH2 domain (NCBI CDD Accession No. cl15255). In a specific embodiment, the domain capable of binding to phosphotyrosine is an SH2 domain derived from a protein listed in Table 16 below, or comprises an amino acid sequence identical or similar to the amino acid sequence listed in Table 16, and the cargo-binding domain comprises phosphotyrosine. In specific embodiments, the domain capable of binding phosphotyrosine is an SH2 domain derived from a protein listed in Table 16 below, or comprises an amino acid sequence identical or similar to an amino acid sequence listed in Table 16, and the cargo binding domain comprises phosphotyrosine and is from or derived from the corresponding parent Eph receptor listed in Table 16. In specific embodiments, the domain capable of binding phosphotyrosine is a HYB domain, a GEP100 PH domain, a PKCδ domain, a PKCθ C2 domain, a catalytically inactive PTP domain, or a Raf-1 kinase inhibitory protein (RKIP) domain.

[0308] Table 15. Exemplary phosphotyrosine and PTB domain-based binding [Table 15]

[0309] Table 16. Exemplary phosphotyrosine and SH2 domain-based binding [Table 16] TIFF2024514157000031.tif236170TIFF2024514157000032.tif137170

[0310] In some embodiments, the bond between the cargo-binding domain and the cargo (e.g., cargo protein) is a bond based on a SAM domain. In specific embodiments, the cargo-binding domain comprises a first SAM domain, or the cargo (e.g., cargo protein) or SBD comprises a domain capable of binding to the first SAM domain (e.g., a second SAM domain), and the bond between the cargo-binding domain and the cargo (e.g., cargo protein) is a bond between the first SAM domain and a domain capable of binding to the first SAM domain (e.g., a second SAM domain). In some embodiments, the second SAM domain belongs to CDD Accession No. cl15755. The first SAM domain and the second SAM domain can be the same or different SAM domains. In specific embodiments, the second SAM domain is derived from a protein listed in Table 17 below, or comprises an amino acid sequence identical or similar to an amino acid sequence listed in Table 17, and the cargo-binding domain comprises a first SAM domain. In a specific embodiment, the second SAM domain is derived from a protein listed in Table 17 below or comprises an amino acid sequence identical or similar to an amino acid sequence listed in Table 17, and the cargo binding domain comprises the first SAM domain and is derived from or from the corresponding parent Eph receptor listed in Table 17.

[0311] Table 17. Exemplary SAM domain-based binding [Table 17]

[0312] In certain embodiments, the linkage between the cargo-binding domain and the cargo (e.g., cargo protein) is a linkage based on a PDZ or PBM domain. In a specific embodiment, the cargo-binding domain comprises a PBM domain, and the cargo or SBD comprises a domain capable of binding to a PBM domain (e.g., a PDZ domain), wherein the linkage between the cargo-binding domain and the cargo (e.g., cargo protein) is a linkage between a PBM domain and a domain capable of binding to a PBM domain (e.g., a PDZ domain). In a specific embodiment, the PDZ domain is derived from a protein listed in Table 18 below, or comprises an amino acid sequence identical or similar to an amino acid sequence listed in Table 18, and the cargo-binding domain comprises a PBM domain. In a specific embodiment, the PDZ domain is derived from a protein listed in Table 18 below, or comprises an amino acid sequence identical or similar to an amino acid sequence listed in Table 18, and the cargo-binding domain comprises a PBM domain and is from or derived from the corresponding parent Eph receptor listed in Table 18. In some embodiments, the cargo-binding domain comprises a PDZ domain (CDD Accession No. cl00117), the cargo (e.g., cargo protein) or SBD comprises a domain capable of binding to a PDZ domain (e.g., a PBM domain), and the bond between the cargo-binding domain and the cargo (e.g., cargo protein) is a bond between the PDZ domain and a domain capable of binding to a PDZ domain (e.g., a PBM domain).

[0313] Table 18. Exemplary PDZ domain-based binding [Table 18]

[0314] The PDZ-binding motif at the C-terminus of Eph receptors can function as a phosphorylation-independent scaffold-binding domain site for PDZ domain-containing proteins (eg, cargo proteins).

[0315] In certain embodiments, the bond between the cargo-binding domain and the cargo (e.g., cargo protein) is a bond based on the Dbl-homology-pleckstrin homology (DH-PH) motif. The pleckstrin homology domain is characterized by NCBI CDD accession number cl17171. In specific embodiments, the cargo-binding domain comprises a DH-PH motif, the cargo or SBD comprises a domain capable of binding to the DH-PH motif, and the bond between the cargo-binding domain and the cargo (e.g., cargo protein) is a bond between the DH-PH motif and the domain capable of binding to the DH-PH motif. In specific embodiments, the cargo-binding domain comprises a domain capable of binding to the DH-PH motif, the cargo (e.g., cargo protein) or SBD comprises a DH-PH motif, and the bond between the cargo-binding domain and the cargo (e.g., cargo protein) is a bond between the domain capable of binding to the DH-PH motif and the DH-PH motif. In specific embodiments, the cargo (e.g., cargo protein) or SBD comprises a DH-PH motif derived from a protein listed in Table 19 below, or comprises an amino acid sequence identical or similar to an amino acid sequence listed in Table 19, and the cargo-binding domain comprises a domain capable of binding to a DH-PH motif. In specific embodiments, the cargo (e.g., cargo protein) or SBD comprises a DH-PH motif derived from a protein listed in Table 19 below, or comprises an amino acid sequence identical or similar to an amino acid sequence listed in Table 19, and the cargo-binding domain comprises a domain capable of binding to a DH-PH motif and is from or derived from the corresponding parent Eph receptor listed in Table 19.

[0316] Table 19. Exemplary DH-PH motif-based binding [Table 19]

[0317] In certain embodiments, the bond between the cargo-binding domain and the cargo (e.g., cargo protein) is a bond based on a Dbl-homology (DH) motif. In a specific embodiment, the cargo-binding domain comprises a DH motif, the cargo (e.g., cargo protein) or SBD comprises a domain capable of binding to a DH motif, and the bond between the cargo-binding domain and the cargo (e.g., cargo protein) is a bond between the DH motif and the domain capable of binding to the DH motif. In a specific embodiment, the cargo-binding domain comprises a domain capable of binding to a DH motif, the cargo (e.g., cargo protein) or SBD comprises a DH motif, and the bond between the cargo-binding domain and the cargo (e.g., cargo protein) is a bond between the domain capable of binding to a DH motif and the DH motif.

[0318] In certain embodiments, the bond between the cargo-binding domain and the cargo (e.g., cargo protein) is a bond based on a pleckstrin homology (PH) motif. In specific embodiments, the cargo-binding domain comprises a PH motif, the cargo (e.g., cargo protein) or SBD comprises a domain capable of binding to the PH motif, and the bond between the cargo-binding domain and the cargo (e.g., cargo protein) is a bond between the PH motif and the domain capable of binding to the PH motif. In specific embodiments, the cargo-binding domain comprises a domain capable of binding to a PH motif, the cargo (e.g., cargo protein) or SBD comprises a PH motif, and the bond between the cargo-binding domain and the cargo (e.g., cargo protein) is a bond between the domain capable of binding to the PH motif and the PH motif.

[0319] In certain embodiments, the cargo-binding domains described herein are derived from or are derived from ephrin receptors. In certain embodiments, the cargo-binding domains described herein are not derived from or are not derived from ephrin receptors. In certain embodiments, the polypeptides described herein comprise a cargo-binding domain derived from or derived from an ephrin receptor and a cargo-binding domain not derived from or are not derived from an ephrin receptor. The polypeptides described herein can comprise one or more additional domains derived from or derived from one or more ephrin receptors that do not function as cargo-binding domains, such as an ephrin receptor CR domain, two ephrin receptor FN III domains, an ephrin receptor TM domain, and optionally an ephrin receptor JM domain, an ephrin receptor KD, an ephrin receptor SAM linker domain, an ephrin receptor SAM domain, an ephrin receptor PBM domain, and / or preferably, an inactivated ephrin receptor LBD. The polypeptides described herein can further comprise a targeting domain, a purification domain, and / or a modified Fc domain.

[0320] In certain embodiments, the cargo-binding domain described herein is an ephrin receptor JM domain that can bind directly to a cargo (e.g., a cargo protein) or indirectly via an SBD linked to the cargo (e.g., a cargo protein). In a specific embodiment, the ephrin receptor JM domain is at the C-terminus of a TM domain (e.g., an ephrin receptor TM domain). In a specific embodiment, the ephrin receptor JM domain is fused to the C-terminus of a TM domain (e.g., an ephrin receptor TM domain) (with or without a linker, such as a peptide linker described herein). In a specific embodiment, the ephrin receptor JM domain comprises a phosphotyrosine, the cargo (e.g., a cargo protein) or the SBD comprises a domain capable of binding to phosphotyrosine, and the bond between the ephrin receptor JM domain and the cargo (e.g., a cargo protein) is a bond between the phosphotyrosine and the domain capable of binding to phosphotyrosine. In a specific embodiment, the domain capable of binding to phosphotyrosine is a PTB domain. In a specific embodiment, the domain capable of binding to phosphotyrosine is an SH2 domain. In a specific embodiment, the domain capable of binding to phosphotyrosine is a HYB domain, a GEP100 PH domain, a PKCδ domain, a PKCθ C2 domain, a catalytically inactive PTP domain, or a Raf-1 kinase inhibitory protein (RKIP) domain. In a specific embodiment, the ephrin receptor JM domain comprises: (i) a (X1)-Ptyr-(X2) motif (wherein Ptyr is phosphotyrosine, X1 is Y, P, V, I, T, or F, and X2 is I, V, L, or A); (ii) a (X3)-Ptyr-(X4) motif (wherein Ptyr is phosphotyrosine, X3 is T, A, or S, and X4 is E or G); or (iii) both (i) and (ii).When a polypeptide described herein comprises a cargo-binding domain that is an Ephrin receptor JM domain, in specific embodiments, the polypeptide further comprises one, two, three, or four of the following: (1) an Ephrin receptor KD, preferably at the C-terminus of the Ephrin receptor JM domain; (2) a SAM linker domain (e.g., an Ephrin receptor SAM linker domain), preferably at the C-terminus of the Ephrin receptor JM domain; (3) a SAM domain (e.g., an Ephrin receptor SAM domain), preferably at the C-terminus of the Ephrin receptor JM domain; and (4) an Ephrin receptor PBM domain, preferably at the C-terminus of the Ephrin receptor JM domain.

[0321] In certain embodiments, the cargo-binding domain described herein is an ephrin receptor KD that can bind directly to a cargo (e.g., a cargo protein) or indirectly via an SBD linked to the cargo (e.g., a cargo protein). In a specific embodiment, the ephrin receptor KD is at the C-terminus of the TM domain (e.g., an ephrin receptor TM domain). In a specific embodiment, the ephrin receptor KD is fused to the C-terminus of the TM domain (e.g., an ephrin receptor TM domain) (using a linker such as a peptide linker described herein or without a linker). In a specific embodiment, the ephrin receptor KD is at the C-terminus of the ephrin receptor JM domain. In a specific embodiment, the ephrin receptor KD is fused to the C-terminus of the ephrin receptor JM domain (using a linker such as a peptide linker described herein or without a linker). In a specific embodiment, the ephrin receptor KD comprises a phosphotyrosine, the cargo (e.g., cargo protein) or SBD comprises a domain capable of binding to phosphotyrosine, and the bond between the ephrin receptor KD and the cargo (e.g., cargo protein) is a bond between the phosphotyrosine and the domain capable of binding to phosphotyrosine. In a specific embodiment, the domain capable of binding to phosphotyrosine is a PTB domain. In a specific embodiment, the domain capable of binding to phosphotyrosine is an SH2 domain. In a specific embodiment, the domain capable of binding to phosphotyrosine is a HYB domain, a GEP100 PH domain, a PKCδ domain, a PKCθ C2 domain, a catalytically inactive PTP domain, or a Raf-1 kinase inhibitory protein (RKIP) domain. In a specific embodiment, the ephrin receptor KD comprises a (X7)-Ptyr-(X8) motif in the activation loop, where Ptyr is phosphotyrosine, X7 is T, V, or A, and X8 is E or T.When the polypeptides described herein comprise a cargo-binding domain that is an ephrin receptor KD, in specific embodiments, the polypeptide further comprises one, two, three, or four of the following: (1) an ephrin receptor JM domain, preferably at the N-terminus of the ephrin receptor KD; (2) a SAM linker domain (e.g., an ephrin receptor SAM linker domain), preferably at the C-terminus of the ephrin receptor KD; (3) a SAM domain (e.g., an ephrin receptor SAM domain), preferably at the C-terminus of the ephrin receptor KD; and (4) an ephrin receptor PBM domain, preferably at the C-terminus of the ephrin receptor KD. In specific embodiments, the polypeptides provided herein further comprise an EphA2 KD that comprises an EphB2 CR domain, a first EphB2 FN III domain, and a second EphB2 FN III domain and functions as a cargo-binding domain. In a specific embodiment, the polypeptide provided herein comprises an EphB2 CR domain, a first EphB2 FN III domain, a second EphB2 FN III domain, and an EphB2 TM domain, and further comprises an EphA2 KD that functions as a cargo-binding domain. In a specific embodiment, the polypeptide provided herein comprises an EphB2 CR domain, a first EphB2 FN III domain, a second EphB2 FN III domain, an EphB2 TM domain, and an EphA2 JM domain, and further comprises an EphA2 KD that functions as a cargo-binding domain.

[0322] In certain embodiments, the cargo-binding domain described herein is a SAM linker domain (e.g., an ephrin receptor SAM linker domain) that can bind directly to a cargo (e.g., a cargo protein) or indirectly via an SBD linked to the cargo (e.g., a cargo protein). In a specific embodiment, the SAM linker domain (e.g., an ephrin receptor SAM linker domain) is at the C-terminus of a TM domain (e.g., an ephrin receptor TM domain). In a specific embodiment, the SAM linker domain (e.g., an ephrin receptor SAM linker domain) is fused to the C-terminus of a TM domain (e.g., an ephrin receptor TM domain) (using a linker such as a peptide linker described herein or without a linker). In a specific embodiment, the SAM linker domain (e.g., an ephrin receptor SAM linker domain) is at the C-terminus of an ephrin receptor JM domain. In a specific embodiment, the SAM linker domain (e.g., an ephrin receptor SAM linker domain) is fused to the C-terminus of the ephrin receptor JM domain (using a linker such as a peptide linker described herein, or without a linker). In a specific embodiment, the SAM linker domain (e.g., an ephrin receptor SAM linker domain) is at the C-terminus of the ephrin receptor KD. In a specific embodiment, the SAM linker domain (e.g., an ephrin receptor SAM linker domain) is fused to the C-terminus of the ephrin receptor KD (using a linker such as a peptide linker described herein, or without a linker). In a specific embodiment, the cargo (e.g., cargo protein) or SBD comprises a domain capable of binding to a SAM linker domain (e.g., an ephrin receptor SAM linker domain), and the bond between the SAM linker domain (e.g., an ephrin receptor SAM linker domain) and the cargo (e.g., cargo protein) is a bond between the SAM linker domain (e.g., an ephrin receptor SAM linker domain) and a domain capable of binding to a SAM linker domain (e.g., an ephrin receptor SAM linker domain).In a specific embodiment, the SAM linker domain (e.g., an ephrin receptor SAM linker domain) comprises a phosphorylated amino acid or a phosphomimetic amino acid, the cargo (e.g., a cargo protein) or SBD comprises a domain capable of binding to the phosphorylated amino acid or phosphomimetic amino acid, and the bond between the SAM linker domain (e.g., an ephrin receptor SAM linker domain) and the cargo (e.g., a cargo protein) is a bond between the phosphorylated amino acid or phosphomimetic amino acid and the domain capable of binding to the phosphorylated amino acid or phosphomimetic amino acid. When a polypeptide described herein comprises a cargo-binding domain that is a SAM linker domain (e.g., an ephrin receptor SAM linker domain), in specific embodiments, the polypeptide further comprises one, two, three, or four of the following: (1) an ephrin receptor JM domain, preferably located at the N-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain); (2) an ephrin receptor KD, preferably located at the N-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain); (3) a SAM domain (e.g., an ephrin receptor SAM domain), preferably located at the C-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain); and (4) an ephrin receptor PBM domain, preferably located at the C-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain).

[0323] In certain embodiments, the cargo-binding domain described herein is a SAM domain (e.g., an ephrin receptor SAM domain) that can bind directly to a cargo (e.g., a cargo protein) or indirectly via an SBD linked to the cargo (e.g., a cargo protein). In a specific embodiment, the SAM domain (e.g., an ephrin receptor SAM domain) is at the C-terminus of a TM domain (e.g., an ephrin receptor TM domain). In a specific embodiment, the SAM domain (e.g., an ephrin receptor SAM domain) is fused to the C-terminus of a TM domain (e.g., an ephrin receptor TM domain) (using a linker such as a peptide linker described herein or without a linker). In a specific embodiment, the SAM domain (e.g., an ephrin receptor SAM domain) is at the C-terminus of an ephrin receptor JM domain. In a specific embodiment, the SAM domain (e.g., an ephrin receptor SAM domain) is fused to the C-terminus of an ephrin receptor JM domain (using a linker such as a peptide linker described herein or without a linker). In a specific embodiment, the SAM domain (e.g., an ephrin receptor SAM domain) is at the C-terminus of the ephrin receptor KD. In a specific embodiment, the SAM domain (e.g., an ephrin receptor SAM domain) is fused to the C-terminus of the ephrin receptor KD (with or without a linker, such as a peptide linker described herein). In a specific embodiment, the SAM domain (e.g., an ephrin receptor SAM domain) is at the C-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain). In a specific embodiment, the SAM domain (e.g., an ephrin receptor SAM domain) is fused to the C-terminus of the SAM linker domain (e.g., an ephrin receptor SAM linker domain) (with or without a linker, such as a peptide linker described herein).In a specific embodiment, the cargo protein or SBD comprises a second SAM domain, and the bond between the SAM domain (e.g., an ephrin receptor SAM domain) and the cargo protein is the bond between the SAM domain (e.g., an ephrin receptor SAM domain) and the second SAM domain. The SAM domain (e.g., an ephrin receptor SAM domain) and the second SAM domain can be the same or different SAM domains. In a specific embodiment, the SAM domain (e.g., an ephrin receptor SAM domain) comprises phosphotyrosine, the cargo (e.g., a cargo protein) or SBD comprises a domain capable of binding to phosphotyrosine, and the bond between the SAM domain (e.g., an ephrin receptor SAM domain) and the cargo (e.g., a cargo protein) is the bond between the phosphotyrosine and the domain capable of binding to phosphotyrosine. In a specific embodiment, the domain capable of binding to phosphotyrosine is a PTB domain. In a specific embodiment, the domain capable of binding to phosphotyrosine is an SH2 domain (e.g., the SH2 domain of Grb2 or the SH2 domain of Grb7). In specific embodiments, the domain capable of binding to phosphotyrosine is a HYB domain, a GEP100 PH domain, a PKCδ domain, a PKCθ C2 domain, a catalytically inactive PTP domain, or a Raf-1 kinase inhibitory protein (RKIP) domain. In specific embodiments, the SAM domain (e.g., an ephrin receptor SAM domain) is a phosphotyrosine in the α2 helix. In certain embodiments, the phosphotyrosine in the α2 helix of the SAM domain is in a (X5)-Ptyr-(X6) motif (wherein Ptyr is phosphotyrosine, X5 is C, R, Q, or H, and X6 is Q, I, E, K, R, or T).When a polypeptide described herein comprises a cargo-binding domain that is a SAM domain (e.g., an ephrin receptor SAM domain), in specific embodiments, the polypeptide further comprises one, two, three, or four of the following: (1) an ephrin receptor JM domain, preferably at the N-terminus of the SAM domain (e.g., an ephrin receptor SAM domain); (2) an ephrin receptor KD, preferably at the N-terminus of the SAM domain (e.g., an ephrin receptor SAM domain); (3) a SAM linker domain (e.g., an ephrin receptor SAM linker domain), preferably at the N-terminus of the SAM domain (e.g., an ephrin receptor SAM domain); and (4) an ephrin receptor PBM domain, preferably at the C-terminus of the SAM domain (e.g., an ephrin receptor SAM domain).

[0324] In certain embodiments, the cargo-binding domain described herein is an ephrin receptor PBM domain that can bind directly to a cargo (e.g., a cargo protein) or indirectly via an SBD linked to the cargo (e.g., a cargo protein). In a specific embodiment, the ephrin receptor PBM domain is at the C-terminus of a TM domain (e.g., an ephrin receptor TM domain). In a specific embodiment, the ephrin receptor PBM domain is fused to the C-terminus of a TM domain (e.g., an ephrin receptor TM domain) (with or without a linker, such as a peptide linker described herein). In a specific embodiment, the ephrin receptor PBM domain is at the C-terminus of an ephrin receptor JM domain. In a specific embodiment, the ephrin receptor PBM domain is fused to the C-terminus of an ephrin receptor JM domain (with or without a linker, such as a peptide linker described herein). In a specific embodiment, the ephrin receptor PBM domain is at the C-terminus of an ephrin receptor KD. In a specific embodiment, the ephrin receptor PBM domain is fused to the C-terminus of the ephrin receptor KD (using a linker, such as a peptide linker described herein, or without a linker). In a specific embodiment, the ephrin receptor PBM domain is at the C-terminus of a SAM linker domain (e.g., an ephrin receptor SAM linker domain). In a specific embodiment, the ephrin receptor PBM domain is fused to the C-terminus of a SAM linker domain (e.g., an ephrin receptor SAM linker domain) (using a linker, such as a peptide linker described herein, or without a linker). In a specific embodiment, the ephrin receptor PBM domain is at the C-terminus of a SAM domain (e.g., an ephrin receptor SAM domain). In a specific embodiment, the ephrin receptor PBM domain is fused to the C-terminus of a SAM domain (e.g., an ephrin receptor SAM domain) (using a linker, such as a peptide linker described herein, or without a linker).In specific embodiments, the cargo (e.g., cargo protein) or SBD comprises a PDZ domain, and the binding between the Ephrin receptor PBM domain and the cargo (e.g., cargo protein) is the binding between the Ephrin receptor PBM domain and the PDZ domain. When a polypeptide described herein comprises a cargo-binding domain that is an Ephrin receptor PBM domain, in specific embodiments, the polypeptide further comprises one, two, three, or four of the following: (1) an Ephrin receptor JM domain, preferably at the N-terminus of the Ephrin receptor PBM domain; (2) an Ephrin receptor KD, preferably at the N-terminus of the Ephrin receptor PBM domain; (3) a SAM linker domain (e.g., an Ephrin receptor SAM linker domain), preferably at the N-terminus of the Ephrin receptor PBM domain; and (4) a SAM domain (e.g., an Ephrin receptor SAM domain), preferably at the N-terminus of the Ephrin receptor PBM domain.

[0325] (c) Adaptor proteins and dimerizers In one aspect, the polypeptides described herein can be further controlled spatially and temporally to be brought into close proximity (e.g., clustered) with one another by adaptor proteins (see, e.g., FIG. 12). The propensity of a transmembrane scaffold protein to form multimers and the size of the resulting clusters depend on the total number of scaffold proteins in a membrane (e.g., a cell membrane or nanovesicle membrane); however, large clusters do not readily form because new scaffold proteins typically require time to be synthesized and incorporated into the membrane, and there is an equilibrium between scaffold protein synthesis and degradation. In some embodiments, the use of adaptor proteins that bind to the cytoplasmic domain of a scaffold protein can modify (e.g., increase) the interaction between the scaffold protein and the scaffold protein. In some embodiments, the binding of an adaptor protein to a scaffold protein can be controlled spatially and temporally through processes including adapter protein synthesis, buffering, or enzymatic modification, such as phosphorylation, methylation, or cleavage, of the adaptor protein.

[0326] In some embodiments, functional fragments of adaptor proteins are synthesized (e.g., by source cells), the fragments comprising a scaffold-binding domain linked to an inducible dimerizer (e.g., a chemically inducible dimerizer), and the fragments form adaptor proteins upon addition of a dimerizing chemical (e.g., rapamycin), thereby bringing the scaffold proteins into close proximity with each other.

[0327] In certain embodiments, the adaptor protein comprises several covalently linked scaffold-binding domains as described in Section 5.2.3(b). In certain embodiments, the adaptor protein comprises several identical (e.g., repeated) covalently linked scaffold-binding domains as described in Section 5.2.3(b). In certain embodiments, the adaptor protein comprises several covalently linked heterologous scaffold binding domains as described in Section 5.2.3(b).

[0328] In certain embodiments, the adaptor protein comprises one or more scaffold-binding domains capable of binding to phosphotyrosine, e.g., an SBD comprising a PTB domain. In a specific embodiment, the adaptor protein comprises a PTB domain from CBL (UniProt ID No. P22681) or comprises the amino acid sequence of SEQ ID NO: 160 listed in Table 15, and the scaffold protein comprises a phosphotyrosine.

[0329] In certain embodiments, the adaptor protein comprises one or more scaffold-binding domains comprising a domain capable of binding to phosphotyrosine, e.g., an SH domain or variant thereof. In a specific embodiment, the adaptor protein comprises one or more scaffold-binding domains comprising a functional variant of an SH2 domain (NCBI CDD Accession No. cl15255). In a specific embodiment, the adaptor protein comprises an SH2 domain from one or more proteins listed in Table 16, or comprises one or more amino acid sequences identical or similar to the amino acid sequences listed in Table 16.

[0330] In certain embodiments, the adaptor protein comprises one or more scaffold binding domains that comprise a domain capable of binding to a SAM domain. In specific embodiments, the adaptor protein comprises one or more SAM domains from one or more proteins listed in Table 17, or comprises one or more amino acid sequences that are identical or similar to an amino acid sequence listed in Table 17, and the scaffold protein also comprises a SAM domain.

[0331] In some embodiments, the bond between the adaptor protein and the scaffold protein is a bond based on a PDZ domain. In a specific embodiment, the adaptor protein comprises a scaffold binding domain comprising a PDZ domain, the scaffold protein comprises a PBM domain, and the bond between the adaptor protein and the scaffold protein is a bond between the PDZ domain and the PBM domain. In a specific embodiment, the adaptor protein comprises a PDZ domain derived from a protein listed in Table 18, or comprises an amino acid sequence identical or similar to the amino acid sequence listed in Table 18, and the scaffold protein comprises a PBM domain.

[0332] In some embodiments, the adaptor protein comprises two, three, four, or five scaffold binding domains as described above. In certain embodiments, the adaptor protein comprises two or more linked heterodomain scaffold binding domains. In some embodiments, the adaptor protein comprises one, two, three, four, and / or five scaffold binding domains, wherein each scaffold domain interacts with one scaffold protein.

[0333] 5.2.4 Targeting and Purification Domains As described above, the polypeptides provided herein can also comprise one or more functional moieties (e.g., fusion moieties, see, e.g., Figures 4-7), preferably a targeting domain capable of targeting nanovesicles (e.g., EVs or hybridosomes) comprising the polypeptide to a specific organ, tissue, or cell type, and / or a purification domain capable of facilitating the purification of such nanovesicles (e.g., EVs or hybridosomes). See Figure 8 for a schematic diagram of an exemplary Eph receptor-derived polypeptide having a targeting domain. In preferred embodiments, the one or more functional moieties are proteins (e.g., peptides or polypeptides). In preferred embodiments, the one or more functional moieties are fused in-frame to the remainder of the polypeptide. In certain embodiments, the one or more functional moieties are covalently fused to the remainder of the polypeptide via a linker. In a specific embodiment, the linker is a peptide linker. In a specific embodiment, the peptide linker comprises the amino acid sequence (GGGS)n (SEQ ID NO: 231), where n is an integer from 1 to 10. In a specific embodiment, the peptide linker comprises the amino acid sequence GGGS. In a specific embodiment, the peptide linker comprises the amino acid sequence (GGGS)2 (SEQ ID NO: 232). In a specific embodiment, the peptide linker comprises the amino acid sequence (GGGS)3 (SEQ ID NO: 233).

[0334] Such one or more functional moieties can be at the N-terminus or C-terminus of the remainder of the polypeptide (e.g., fused to the N-terminus and / or C-terminus), or can be located between different domains of the remainder of the polypeptide. In certain embodiments, one or more functional moieties are presented toward the external space of the nanovesicle. In some embodiments, one or more functional moieties are at the N-terminus of the ephrin ligand binding domain of the polypeptide (e.g., fused to the N-terminus). In some embodiments, one or more functional moieties are at the N-terminus of the ephrin receptor cysteine-rich domain of the polypeptide (e.g., fused to the N-terminus). In some embodiments, one or more functional moieties are at the N-terminus of the ephrin receptor FN1 domain (e.g., fused to the N-terminus). In some embodiments, one or more functional moieties are at the N-terminus of the ephrin receptor FN2 domain (e.g., fused to the N-terminus). In some embodiments, one or more functional moieties are at the N-terminus of the TM domain (e.g., ephrin receptor TM domain) (e.g., fused to the N-terminus). In some embodiments, one or more functional moieties are presented toward the lumen of the nanovesicle. In some embodiments, one or more functional moieties are at the C-terminus (e.g., fused to the C-terminus) of the TM domain (e.g., ephrin receptor TM domain). In some embodiments, one or more functional moieties are at the C-terminus (e.g., fused to the C-terminus) of the Ephrin receptor JM domain. In some embodiments, one or more functional moieties are at the C-terminus (e.g., fused to the C-terminus) of the Ephrin receptor KD. In some embodiments, one or more functional moieties are at the C-terminus (e.g., fused to the C-terminus) of the SAM linker domain (e.g., ephrin receptor SAM linker domain). In some embodiments, one or more functional moieties are at the C-terminus (e.g., fused to the C-terminus) of the SAM domain (e.g., ephrin receptor SAM domain). In some embodiments, one or more functional moieties are at the C-terminus (e.g., fused to the C-terminus) of the Ephrin receptor PBM domain.

[0335] In certain embodiments, one or more functional moieties are at the N-terminus (e.g., fused to the N-terminus) of a cargo (e.g., cargo protein) described in this disclosure. In certain embodiments, one or more functional moieties are at the C-terminus (e.g., fused to the C-terminus) of a cargo (e.g., cargo protein) described in this disclosure. In certain embodiments, one or more functional moieties are at the N-terminus (e.g., fused to the N-terminus) of a cargo binding domain described in this disclosure. In certain embodiments, one or more functional moieties are at the C-terminus (e.g., fused to the C-terminus) of a cargo binding domain described in this disclosure. In certain embodiments, one or more functional moieties are at the N-terminus (e.g., fused to the N-terminus) of a modified Fc domain described in this disclosure. In certain embodiments, one or more functional moieties are at the C-terminus (e.g., fused to the C-terminus) of a modified Fc domain described in this disclosure.

[0336] Exemplary functional moieties include, but are not limited to, targeting domains and purification domains, such as affinity tags. A functional moiety may be a large polypeptide or peptide.

[0337] In some embodiments, the targeting domain described herein is at the N-terminus (e.g., fused to the N-terminus) of the purification domain described herein. In some embodiments, the targeting domain described herein is at the C-terminus (e.g., fused to the C-terminus) of the purification domain described herein.

[0338] In some embodiments, the targeting domain is preferably located on the surface of the nanovesicle. Thus, in some embodiments, the targeting domain is fused to the N-terminus of the scaffold. The targeting domain helps direct the nanovesicle to a particular organ, tissue, or cell, and is preferably organ, tissue, or cell-specific. One or more targeting domains can be fused to the...

Claims

1. From N-terminus to C-terminus: (a) ephrin receptor cysteine-rich (CR) domain; (b) a first ephrin receptor fibronectin type III (FN III) domain and a second ephrin receptor FN III domain; and (c) transmembrane (TM) domain; A polypeptide comprising: (i) lacking ephrin-binding activity, (ii) lacking ephrin receptor kinase activity, or (iii) lacking both ephrin-binding activity and ephrin receptor kinase activity; The polypeptide.

2. The polypeptide of claim 1, further comprising a targeting domain at the N-terminus of the ephrin receptor CR domain.

3. 3. The polypeptide of claim 2, wherein the targeting domain is selected from the group consisting of: scFv, (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Fd fragment, diabody, F(ab')3, disulfide-linked Fv, sdAb (VHH or nanobody), CDR, di-scFv, bi-scFv, tascFv (tandem scFv), triabody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, DARPin, centirin, affibody, affilin, affitin, anticalin, avimer, finomer, Kunitz domain peptide, monobody (or adnectin), tribody, and nanophytin.

4. The targeting domain specifically binds to a marker, wherein the marker is a tumor-associated antigen, and the tumor-associated antigen is selected from the group consisting of human epidermal growth factor receptor 2 (HER2), CD20, CD33, B-cell maturation antigen (BCMA), prostate-specific membrane antigen (PSMA), DLL3, ganglioside GD2 (GD2), CD123, anoctamin-1 (Ano1), mesothelin, carbonic anhydrase IX (CAIX), tumor-associated calcium signaling agent 2 (TROP2), carcinoembryonic antigen (CEA), claudin-18.2, receptor tyrosine kinase-like orphan receptor 1 (ROR1), trophoblast glycoprotein (5T4), glycoprotein nonmetastatic melanoma protein B (GPNMB), folate receptor-alpha (FR-α), pregnancy-associated plasma protein A (PAPP-A), CD37, epithelial cell adhesion molecule (EpCAM), CD2, CD 19, CD30, CD38, CD40, CD52, CD70, CD79b, fms-like tyrosine kinase 3 (FLT3), glypican 3 (GPC3), B7 homolog 6 (B7H6), CC chemokine receptor type 4 (CCR4), C-X-C motif chemokine receptor 4 (CXCR4), receptor tyrosine kinase-like orphan receptor 2 (ROR2), CD133, HLA class I histocompatibility antigen, alpha chain E (HLA-E), epidermal growth factor receptor (EGFR / ERBB-1), insulin-like growth factor 1-receptor (IGF1R), and human epidermal growth factor receptor 3.

5. 2. The polypeptide of claim 1, further comprising a cargo protein or cargo binding domain at the C-terminus of the TM domain, wherein the cargo protein or cargo binding domain is fused to the remainder of the polypeptide via a linker.

6. The polypeptide of claim 5, wherein the linker is a peptide linker.

7. 6. The polypeptide of claim 5, wherein the cargo-binding domain binds directly to the cargo protein or indirectly via a scaffold-binding domain (SBD) linked to the cargo protein, and wherein the binding between the cargo-binding domain and the cargo protein (i) can be controlled by pH or ionic strength, and / or (ii) can be controlled such that the cargo protein binds to the cargo-binding domain in vitro but is released from the cargo-binding domain in vivo, or the cargo protein is released from the cargo-binding domain in a manner that depends on the subcellular compartment in which they are located.

8. The cargo binding domain: (a)(i)(X 1 )-Ptyr-(X 2 ) motif (where Ptyr is phosphotyrosine and X 1 is Y, P, V, I, T, or F, and X 2 is I, V, L, or A); (ii) (X 3 )-Ptyr-(X 4 ) motif (where Ptyr is phosphotyrosine and X 3 is T, A, or S, and X 4 is E or G); or (iii) an ephrin receptor JM domain that contains both (i) and (ii); (b) During the activation loop, (X 7 )-Ptyr-(X 8 ) motif (where Ptyr is phosphotyrosine and X 7 is T, V, or A, and X 8 is E or T); (c) sterile alpha motif (SAM) linker domain, including the ephrin receptor SAM linker domain; (d) a sterile alpha motif (SAM) domain, including an ephrin receptor SAM domain; and (e) Ephrin receptor PDZ-binding motif (PBM) domain The polypeptide of claim 5, comprising any one or more of:

9. The polypeptide of claim 5, wherein the cargo protein is a therapeutic or diagnostic protein.

10. (a) the polypeptide lacks an ephrin receptor ligand binding domain (LBD) or contains a mutated ephrin receptor LBD; (b) the polypeptide comprises two distinct domains, preferably in a head-to-tail arrangement, that enable the polypeptide to undergo heterodomain dimerization with another polypeptide identical to the polypeptide; (c) the polypeptide comprises a transmembrane (TM) domain that is an ephrin receptor TM domain; (d) any one or more of the ephrin receptor domains of the polypeptide are or are derived from EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, or a combination thereof; (e) the first Ephrin receptor FN III domain and the second Ephrin receptor FN III domain of the polypeptide comprise different amino acid sequences; and / or (f) the polypeptide further comprises a modified Fc domain of an immunoglobulin; (i) the modified Fc domain is N-terminal to the ephrin receptor CR domain, and the modified Fc domain is fused to the remainder of the polypeptide by a linker sequence; (ii) the modified Fc domain comprises, from the N-terminus to the C-terminus, a modified CH2 domain that has been modified to have reduced effector function compared to an unmodified CH2 domain, and a modified CH3 domain that has been modified to lack the ability to form homodimers; (iii) the modified Fc domain is capable of specifically binding to the Fc binding site of the neonatal Fc receptor (FcRn); and lacks the ability to form homodimers; (iv) the dissociation constant of the modified Fc domain bound to the FcRn at a pH of 6.5 is at most 10 -4 M value of the modified Fc domain bound to the FcRn at a pH of 7.4 or a dissociation constant of at least 10 -4 having a value of M; (v) the modified Fc domain has the amino acid sequence 【Chemistry 1】 (where X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , and X 8 each is any amino acid) capable of specifically binding to; (vi) the modified Fc domain is capable of specifically binding to an amino acid sequence between positions 135 and 158 of human FcRn (SEQ ID NO: 228) and / or mouse FcRn (SEQ ID NO: 227); (vii) the polypeptide comprising the modified Fc domain does not substantially bind to C1q, FcγRI, FcγRII, or FcγRIII; and / or (viii) a. the complement-dependent cytotoxicity (CDC) activity of the modified Fc domain; b. antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the modified Fc domain; c. antibody-dependent cell-mediated phagocytosis (ADCP) activity of the modified Fc domain; and / or d. Antibody-dependent intracellular neutralization (ADIN) activity of the modified Fc domain is reduced by at least 10%, 20%, 30%, 40%, or 50% compared to the unmodified Fc domain; or The polypeptide of claim 1, which is reduced by at least 1.5-fold, 2-fold, 3-fold, 4-fold, or 5-fold compared to an unmodified Fc domain.

11. A nucleic acid encoding the polypeptide of any one of claims 1 to 10.

12. 12. An expression vector comprising the nucleic acid of claim 11.

13. A cell comprising the nucleic acid of claim 11.

14. An extracellular vesicle (EV) or hybridosome comprising a polypeptide according to any one of claims 1 to 10.

15. 1. A method for purifying EVs or hybridosomes, comprising: (a) providing said EV or hybridosome, wherein said EV or hybridosome comprises a first binding partner, said first binding partner being capable of binding to an Fc binding site of a neonatal Fc receptor (FcRn) in a pH-dependent manner and comprising or consisting of a polypeptide according to any one of claims 1 to 10; (b) contacting the EVs or hybridosomes containing the first binding partner with a second binding partner at a first pH, where the second binding partner comprises an Fc binding site of the FcRn and is associated with a solid matrix; and (c) eluting the EVs or hybridosomes comprising the first binding partner from the solid matrix at a second pH.

16. 16. The method of claim 15, wherein the first pH is less than 6.5 and the second pH is greater than 7.4.

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