Modified rhabdovirus glycoproteins and uses thereof

Abstract

The present disclosure provides a recombinant fusion protein, comprising rhabdovirus glycoprotein (G) and a targeting molecule linked to the N-terminus of rhabdovirus glycoprotein.In addition, related recombinant polynucleotides, host cells, and pharmaceutical compositions are provided.Recombinant viruses, such as recombinant pseudotyped viruses, and cell-derived nanovesicles comprising recombinant polynucleotides are also provided.In addition, methods are provided for using recombinant fusion protein, polynucleotides, viruses, and cell-derived nanovesicles, and / or pharmaceutical compositions thereof, including those used in cancer treatment.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 481,847, filed January 27, 2023, U.S. Provisional Application No. 63 / 466,931, filed May 16, 2023, and U.S. Provisional Application No. 63 / 597,831, filed November 10, 2023, the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy, created on January 19, 2024, is named 250298_000603_SL.xml and is 266,253 bytes in size.

[0003] The present disclosure provides a recombinant fusion protein, comprising rhabdovirus glycoprotein (G) and a targeting molecule linked to the N-terminus of rhabdovirus glycoprotein.In addition, related recombinant polynucleotides, host cells, and pharmaceutical compositions are provided.Recombinant viruses, such as recombinant pseudotyped viruses, and cell-derived nanovesicles comprising recombinant polynucleotides are also provided.In addition, methods are provided for using recombinant fusion protein, polynucleotides, viruses, and cell-derived nanovesicles, and / or pharmaceutical compositions thereof, including those used in cancer treatment. [Background technology]

[0004] A key challenge in the development of targeted vector therapies is the delivery of therapeutic agents to disease-specific cells / tissues. To achieve success, the delivered vector therapy must be specific to the target cells / tissues and reduce any off-target delivery that may cause toxicity. In addition, the therapeutic vector must be long enough to reach the target cells / tissues and overcome natural barriers, such as the patient's innate immune response.

[0005] Vesicular stomatitis virus (VSV) possesses oncolytic properties, and clinical trials are underway to determine its safety and efficacy as an anticancer therapy. Because the VSV-G glycoprotein is known to infect a very wide range of cells and tissues (Finkelshtein et al., 2013; Nikolic et al., 2018), an ongoing challenge is improving targeted delivery of VSV to tumor tissues to maximize therapeutic efficacy and minimize potential toxicity associated with infection of healthy tissues. One of the most concerning off-target effects associated with VSV is neurotoxicity. Viral loss to other tissues, including the liver and spleen, reduces the efficacy of oncolytic VSV. Strategies for directing VSV tropism to target tissues frequently negatively impact viral fitness, which also reduces the efficacy of VSV therapy.

[0006] Thus, there is an unmet need in the art for improved VSV-based therapies. Summary of the Invention [Means for solving the problem]

[0007] As specified in the Background section above, there is a significant need in the art for improved VSV-based therapies. The present application addresses these and other needs.

[0008] In one aspect, provided herein is a recombinant fusion protein, the fusion protein comprising: (i) a rhabdovirus glycoprotein (G), or a functional fragment or derivative thereof; (ii) a targeting molecule, which can be attached to the N-terminus of the rhabdovirus glycoprotein, or a functional fragment or derivative thereof, for example, via a linker, wherein the linker is susceptible to proteolytic cleavage by an endogenous protease or an exogenously added protease; and

[0009] In some embodiments, the linker comprises arginine (R) and / or lysine (K) residues.

[0010] In some embodiments, the linker is: KRAAASGGS(G4S)2GPK (SEQ ID NO: 174), KRAAASGGS(G4S)2 (SEQ ID NO: 2), (EAAAK)3 (SEQ ID NO: 3), KR(EAAAK)3 (SEQ ID NO: 4), AAARGSPK(G4S)3 (SEQ ID NO: 5), RAAARGSPK(G4S)3 (SEQ ID NO: 169), AAARGSPK(G4S)3K (SEQ ID NO: 19), K(G4S)3 (SEQ ID NO: 20), KR(G4S)3 (SEQ ID NO: 21), (G4S)3GPK (SEQ ID NO: 6), and AAA(G4S)3K (SEQ ID NO: 7).

[0011] In some embodiments, the N-terminus of the rhabdovirus glycoprotein, or functional fragment or derivative thereof, to which the targeting molecule is attached via a linker does not include one or more amino acids present at the N-terminus of the mature wild-type rhabdovirus glycoprotein.

[0012] In some embodiments, the rhabdovirus glycoprotein is vesicular stomatitis virus glycoprotein (VSV-G), or a functional fragment or derivative thereof.

[0013] In some embodiments, the VSV-G comprises the sequence of SEQ ID NO:8.

[0014] In some embodiments, the VSV-G consists of the sequence of SEQ ID NO:8.

[0015] In some embodiments, the targeting molecule can interfere with the ability of the VSV-G, or a functional fragment or derivative thereof, to interact with the low density lipoprotein receptor (LDLR).

[0016] In some embodiments, the VSV-G, or functional fragment or derivative thereof, comprises one or more mutations that reduce or eliminate binding of the VSV-G polypeptide, or functional fragment or derivative thereof, to the LDLR.

[0017] In some embodiments, the one or more mutations in the VSV-G, or functional fragment or derivative thereof, comprise one or more amino acid substitutions and / or deletions at positions corresponding to H8, K47, Y209, or R354 in SEQ ID NO:8.

[0018] In some embodiments, the VSV-G comprises or consists of SEQ ID NO: 8, and the one or more mutations are substitutions at positions K47 and R354.

[0019] In some embodiments, the VSV-G comprises or consists of SEQ ID NO: 8, and the one or more mutations are substitutions at positions K47, R354, and Y209.

[0020] In some embodiments, the VSV-G comprises or consists of SEQ ID NO: 8, and the one or more mutations are substitutions at position H8.

[0021] In some embodiments, the one or more mutations in the VSV-G, or functional fragment or derivative thereof, comprise one or more amino acid deletions at positions corresponding to H8, K47, Y209, or R354 in SEQ ID NO:8.

[0022] In some embodiments, the VSV-G comprises or consists of SEQ ID NO:8.

[0023] In some embodiments, the one or more deletions is a deletion at position K47.

[0024] In some embodiments, the one or more deletions is a deletion at position H8.

[0025] In some embodiments, the one or more deletions are at positions H8 and K47.

[0026] In some embodiments, the VSV-G, or functional fragment or derivative thereof, further comprises one or more viral titer-increasing mutations.

[0027] In some embodiments, the one or more viral titer-increasing mutations in the VSV-G, or functional fragment or derivative thereof, are M184T and / or F250L, as specified relative to their positions in SEQ ID NO:8.

[0028] In some embodiments, the rhabdovirus glycoprotein is a glycoprotein from Flanders virus (FLAV-G).

[0029] In some embodiments, the FLAV-G comprises the sequence of SEQ ID NO:9.

[0030] In some embodiments, the FLAV-G consists of the sequence of SEQ ID NO:9.

[0031] In some embodiments, the rhabdovirus glycoprotein is a glycoprotein from Chandipura virus (CHPV-G).

[0032] In some embodiments, the CHPV-G comprises the sequence of SEQ ID NO:10.

[0033] In some embodiments, the CHPV-G consists of the sequence of SEQ ID NO:10.

[0034] In some embodiments, the rhabdovirus glycoprotein is a glycoprotein from Perinet virus (PERV-G).

[0035] In some embodiments, the PERV-G comprises the sequence of SEQ ID NO:11.

[0036] In some embodiments, the PERV-G consists of the sequence of SEQ ID NO:11.

[0037] In some embodiments, the rhabdovirus glycoprotein is a glycoprotein from Piry virus (PIRYV-G).

[0038] In some embodiments, the PIRYV-G comprises the sequence of SEQ ID NO:12.

[0039] In some embodiments, the PIRYV-G consists of the sequence of SEQ ID NO:12.

[0040] In some embodiments, the rhabdovirus glycoprotein is a glycoprotein from Fukuoka virus (FUKV-G).

[0041] In some embodiments, the FUKV-G comprises the sequence of SEQ ID NO:13.

[0042] In some embodiments, the FUKV-G consists of the sequence of SEQ ID NO:13.

[0043] In some embodiments, the rhabdovirus glycoprotein is a glycoprotein from Joinjakaka virus (JOIV-G).

[0044] In some embodiments, the JOIV-G comprises the sequence of SEQ ID NO:14.

[0045] In some embodiments, the JOIV-G consists of the sequence of SEQ ID NO:14.

[0046] In some embodiments, the rhabdovirus glycoprotein is a glycoprotein from Kumasi virus (KRV-G).

[0047] In some embodiments, the KRV-G comprises the sequence of SEQ ID NO:15.

[0048] In some embodiments, the KRV-G consists of the sequence of SEQ ID NO:15.

[0049] In some embodiments, the rhabdovirus glycoprotein is a glycoprotein from Keuraliba virus (KEUV-G).

[0050] In some embodiments, the KEUV-G comprises the sequence of SEQ ID NO:17.

[0051] In some embodiments, the KEUV-G comprises the sequence of SEQ ID NO:17.

[0052] In some embodiments, the cytoplasmic tail of the rhabdovirus glycoprotein is removed or truncated and, optionally, replaced with another sequence.

[0053] In some embodiments, the cytoplasmic tail of the glycoprotein is truncated by up to 40 amino acids from the C-terminus.

[0054] In some embodiments, the cytoplasmic tail of the rhabdovirus glycoprotein is truncated 10 to 40 amino acids from the C-terminus.

[0055] In some embodiments, the cytoplasmic tail of the rhabdovirus glycoprotein is truncated 30 amino acids from the C-terminus.

[0056] In some embodiments of any of the above recombinant fusion proteins, the recombinant fusion protein may further comprise a cytoplasmic tail from VSV-G, or a functional fragment or derivative thereof.

[0057] In some embodiments, the cytoplasmic tail of VSV-G comprises the sequence CIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 16).

[0058] In another aspect, provided herein is a recombinant fusion protein, the fusion protein comprising a fusogen having at least 60% amino acid sequence identity to vesicular stomatitis virus glycoprotein (VSV-G) comprising SEQ ID NO: 8, or a functional fragment or derivative thereof, wherein the fusogen, or functional fragment or derivative thereof, comprises one or more amino acid deletions at positions corresponding to H8, K47, Y209, or R354 in SEQ ID NO: 8.

[0059] In some embodiments, the fusogen comprises the sequence of SEQ ID NO: 8, or a functional fragment or derivative thereof, having one or more amino acid deletions at positions H8, K47, Y209, or R354.

[0060] In some embodiments, the fusogen comprises or consists of the sequence of SEQ ID NO: 8, which has an amino acid deletion at position H8.

[0061] In some embodiments, the fusogen comprises or consists of the sequence of SEQ ID NO: 8, with amino acid deletions at positions H8 and K47.

[0062] In some embodiments, the fusogen comprises the sequence of SEQ ID NO: 8, with amino acid deletions at (i) position K47, (ii) position R354, and (iii) position H8 or Y209.

[0063] In some embodiments, the fusogen consists of the sequence of SEQ ID NO: 8, with amino acid deletions at (i) position K47, (ii) position R354, and (iii) position H8 or Y209.

[0064] In some embodiments, the fusogen comprises the sequence of SEQ ID NO: 8, which has an amino acid deletion at position K47.

[0065] In some embodiments, the fusogen consists of the sequence of SEQ ID NO: 8, with an amino acid deletion at position K47.

[0066] In another aspect, provided herein is a recombinant fusion protein comprising a fusogen having the sequence of SEQ ID NO: 8, with amino acid substitutions at (i) position K47, (ii) position R354, and (iii) position H8 or Y209.

[0067] In some embodiments, the fusogen consists of the sequence of SEQ ID NO: 8 with amino acid substitutions at (i) position K47, (ii) position R354, and (iii) position H8 or Y209.

[0068] In another aspect, provided herein is a recombinant fusion protein comprising a fusogen comprising the sequence of SEQ ID NO: 8, with amino acid substitutions at positions K47, R354, H8, and Y209.

[0069] In some embodiments, the fusogen consists of the sequence of SEQ ID NO: 8, with amino acid substitutions at positions K47, R354, H8, and Y209.

[0070] In some embodiments, the fusogen, or functional fragment or derivative thereof, further comprises one or more viral titer-increasing mutations.

[0071] In some embodiments, the one or more viral titer-increasing mutations are at one or more positions corresponding to positions M184 and / or F250 in SEQ ID NO:8.

[0072] In some embodiments of any of the above recombinant fusion proteins, the fusion protein may further comprise a targeting molecule positioned at the N-terminus of the fusogen, or functional fragment or derivative thereof.

[0073] In some embodiments, the targeting molecule is attached to the N-terminus of the fusogen, or functional fragment or derivative thereof, via a linker.

[0074] In some embodiments, the linker is susceptible to proteolytic cleavage by endogenous proteases or by exogenously added proteases.

[0075] In some embodiments, the linker comprises arginine (R) and / or lysine (K) residues.

[0076] In some embodiments, the linker is: KRAAASGGS(G4S)2GPK (SEQ ID NO: 174), KRAAASGGS(G4S)2 (SEQ ID NO: 2), (EAAAK)3 (SEQ ID NO: 3), KR(EAAAK)3 (SEQ ID NO: 4), AAARGSPK(G4S)3 (SEQ ID NO: 5), RAAARGSPK(G4S)3 (SEQ ID NO: 169), AAARGSPK(G4S)3K (SEQ ID NO: 19), K(G4S)3 (SEQ ID NO: 20), KR(G4S)3 (SEQ ID NO: 21), (G4S)3GPK (SEQ ID NO: 6), or AAA(G4S)3K (SEQ ID NO: 7).

[0077] In some embodiments, the linker is not susceptible to proteolytic cleavage by endogenous proteases or by exogenously added proteases.

[0078] In some embodiments, the N-terminus of the fusogen, or functional fragment or derivative thereof, to which the targeting molecule is attached does not include one or more amino acids present at the N-terminus of the mature wild-type fusogen.

[0079] In another aspect, provided herein is a recombinant fusion protein, the fusion protein comprising: (i) a glycoprotein from Flanders virus (FLAV-G), or a functional fragment or derivative thereof; (ii) a targeting molecule.

[0080] In some embodiments, the FLAV-G comprises the sequence of SEQ ID NO:9.

[0081] In some embodiments, the FLAV-G consists of the sequence of SEQ ID NO:9.

[0082] In another aspect, provided herein is a recombinant fusion protein, the fusion protein comprising: (i) a glycoprotein derived from Chandipura virus (CHPV-G), or a functional fragment or derivative thereof; (ii) a targeting molecule.

[0083] In some embodiments, the CHPV-G comprises the sequence of SEQ ID NO:10.

[0084] In some embodiments, the CHPV-G consists of the sequence of SEQ ID NO:10.

[0085] In another aspect, provided herein is a recombinant fusion protein, the fusion protein comprising: (i) a glycoprotein derived from a perinetovirus (PERV-G), or a functional fragment or derivative thereof; (ii) a targeting molecule.

[0086] In some embodiments, the PERV-G comprises the sequence of SEQ ID NO:11.

[0087] In some embodiments, the PERV-G consists of the sequence of SEQ ID NO:11.

[0088] In another aspect, provided herein is a recombinant fusion protein, the fusion protein comprising: (i) a glycoprotein derived from a pyrivirus (PIRYV-G), or a functional fragment or derivative thereof; (ii) a targeting molecule.

[0089] In some embodiments, the PIRYV-G comprises the sequence of SEQ ID NO:12.

[0090] In some embodiments, the PIRYV-G consists of the sequence of SEQ ID NO:12.

[0091] In another aspect, provided herein is a recombinant fusion protein, the fusion protein comprising: (i) a glycoprotein derived from Fukuoka virus (FUKV-G), or a functional fragment or derivative thereof; (ii) a targeting molecule.

[0092] In some embodiments, the FUKV-G comprises the sequence of SEQ ID NO:13.

[0093] In some embodiments, the FUKV-G consists of the sequence of SEQ ID NO:13.

[0094] In another aspect, provided herein is a recombinant fusion protein, the fusion protein comprising: (i) a glycoprotein derived from Joint Jacaka virus (JOIV-G), or a functional fragment or derivative thereof; (ii) a targeting molecule.

[0095] In some embodiments, the JOIV-G comprises the sequence of SEQ ID NO:14.

[0096] In some embodiments, the JOIV-G consists of the sequence of SEQ ID NO:14.

[0097] In another aspect, provided herein is a recombinant fusion protein, the fusion protein comprising: (i) a glycoprotein derived from Kumasi virus (KRV-G), or a functional fragment or derivative thereof; (ii) a targeting molecule.

[0098] In some embodiments, the KRV-G comprises the sequence of SEQ ID NO:15.

[0099] In some embodiments, the KRV-G consists of the sequence of SEQ ID NO:15.

[0100] In another aspect, provided herein is a recombinant fusion protein, the fusion protein comprising: (i) a glycoprotein derived from Keurariba virus (KEUV-G), or a functional fragment or derivative thereof; (ii) a targeting molecule.

[0101] In some embodiments, the KEUV-G comprises the sequence of SEQ ID NO:17.

[0102] In some embodiments, the KEUV-G consists of the sequence of SEQ ID NO:17.

[0103] In some embodiments, the glycoprotein is a fragment, wherein the cytoplasmic tail of the glycoprotein has been removed or truncated and, optionally, replaced with another sequence.

[0104] In some embodiments, the cytoplasmic tail of the glycoprotein is truncated by up to 40 amino acids from the C-terminus.

[0105] In some embodiments, the cytoplasmic tail of the glycoprotein is truncated 10 to 40 amino acids from the C-terminus.

[0106] In some embodiments, the cytoplasmic tail of the glycoprotein is truncated 30 amino acids from the C-terminus.

[0107] In some embodiments of any of the above recombinant fusion proteins, the recombinant fusion protein may further comprise a cytoplasmic tail from VSV-G, or a functional fragment or derivative thereof.

[0108] In some embodiments, the cytoplasmic tail of VSV-G comprises the sequence CIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 16).

[0109] In some embodiments, the targeting molecule is located at the N-terminus of the glycoprotein, or a functional fragment or derivative thereof.

[0110] In some embodiments, the targeting molecule is attached to the N-terminus of the glycoprotein, or functional fragment or derivative thereof, via a linker.

[0111] In some embodiments, the linker is susceptible to proteolytic cleavage by endogenous proteases or by exogenously added proteases.

[0112] In some embodiments, the linker comprises arginine (R) and / or lysine (K) residues.

[0113] In some embodiments, the linker is: KRAAASGGS(G4S)2GPK (SEQ ID NO: 174), KRAAASGGS(G4S)2 (SEQ ID NO: 2), (EAAAK)3 (SEQ ID NO: 3), KR(EAAAK)3 (SEQ ID NO: 4), AAARGSPK(G4S)3 (SEQ ID NO: 5), RAAARGSPK(G4S)3 (SEQ ID NO: 169), AAARGSPK(G4S)3K (SEQ ID NO: 19), K(G4S)3 (SEQ ID NO: 20), KR(G4S)3 (SEQ ID NO: 21), (G4S)3GPK (SEQ ID NO: 6), and AAA(G4S)3K (SEQ ID NO: 7).

[0114] In some embodiments, the linker is not susceptible to proteolytic cleavage by endogenous proteases or by exogenously added proteases.

[0115] In some embodiments, the N-terminus of the glycoprotein, or functional fragment or derivative thereof, to which the targeting molecule is attached does not include one or more amino acids present at the N-terminus of the mature wild-type fusogen.

[0116] In some embodiments, the targeting molecule is an antibody or antigen-binding fragment thereof, an affibody, a darpin, a peptide, a natural or modified natural receptor ligand, a T cell receptor or a fragment or derivative thereof, or an MHC-peptide complex or a fragment or derivative thereof.

[0117] In some embodiments, the antibody or antigen-binding fragment thereof is a single-chain fragment variable (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a single heavy chain antibody.

[0118] In some embodiments, the targeting molecule targets EGFR, HER2, MUC16, cKit, αVβ3 integrin, IGF1R, BCMA, Nectin-4, MEK, CD44, CD3, CD4, CD28, stem cell factor, thrombopoietin, c-Met, CXCR4, IL2R, or IL-3.

[0119] In another aspect, provided herein are recombinant polynucleotides encoding the recombinant fusion proteins described herein.

[0120] In some embodiments, the polynucleotide comprises a sequence encoding a signal peptide sequence, such signal sequence being positioned at the extreme N-terminus of the encoded recombinant fusion protein.

[0121] In some embodiments, the polynucleotide is DNA.

[0122] In some embodiments, the polynucleotide is RNA.

[0123] In another aspect, provided herein is a recombinant polynucleotide, wherein the recombinant polynucleotide is an RNA molecule comprising a nucleotide sequence that is a template for a positive-sense transcription product that encodes a recombinant fusion protein described herein.

[0124] In some embodiments, the positive-sense transcript includes a sequence encoding a signal peptide sequence, with such signal sequence being positioned at the extreme N-terminus of the encoded recombinant fusion protein.

[0125] In some embodiments, the recombinant polynucleotide is an RNA molecule comprising a nucleotide sequence that is a template for a positive-sense transcript encoding a vesicular stomatitis virus (VSV) nucleoprotein (N) polypeptide or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive-sense transcript encoding a VSV phosphoprotein (P) polypeptide or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive-sense transcript encoding a VSV matrix (M) polypeptide or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive-sense transcript encoding a fusion protein described herein, and a nucleotide sequence that is a template for a positive-sense transcript encoding a VSV large protein (L) polypeptide or a functional fragment or derivative thereof.

[0126] In some embodiments, the VSV M polypeptide is a mutant VSV M polypeptide.

[0127] In some embodiments, the mutant VSV M polypeptide comprises a mutation at methionine (M)51.

[0128] In some embodiments, the mutation at methionine (M) 51 is a substitution of methionine (M) with arginine (R).

[0129] In some embodiments, the polynucleotide is optimized for expression in human cells.

[0130] In another aspect, provided herein is a composition comprising a recombinant polynucleotide described herein and a carrier and / or excipient.

[0131] In another aspect, provided herein is a host cell comprising a recombinant polynucleotide described herein.

[0132] In another aspect, provided herein are recombinant pseudotyped viral or cell-derived nanovesicles comprising a recombinant polynucleotide described herein.

[0133] In another aspect, provided herein are recombinant pseudotyped viral or cell-derived nanovesicles comprising one or more recombinant fusogenic proteins described herein.

[0134] In some embodiments, a recombinant pseudotyped virus or cell-derived nanovesicle described herein may comprise two or more different recombinant fusogenic proteins described herein.

[0135] In some embodiments, the recombinant fusogenic protein forms a chimeric trimer with one or two different fusogenic proteins on the surface of the recombinant pseudotyped virus or cell-derived nanovesicle.

[0136] In some embodiments, the chimeric trimer comprises (i) at least one fusion protein described herein and (ii) a fusion protein comprising a rhabdovirus glycoprotein, or a functional fragment or derivative thereof, without a targeting molecule.

[0137] In some embodiments, the fusogenic protein (ii) comprises a fusogen comprising the sequence of SEQ ID NO: 8, with amino acid substitutions and / or deletions at one or more positions selected from K47, R354, H8, and Y209.

[0138] In another aspect, the present invention provides recombinant pseudotype virus or cell-derived nanovesicle, comprising the chimeric trimer, comprising: (i) one or two monomers of the first fusion protein, wherein this first fusion protein comprises rhabdovirus glycoprotein or its functional fragment or derivative and targeting molecule or its functional fragment or derivative; and (ii) one or two monomers of the second fusion protein, wherein this second fusion protein comprises rhabdovirus glycoprotein or its functional fragment or derivative without targeting molecule.In some embodiments, for example, targeting molecule can be linked to the N-terminus of the rhabdovirus glycoprotein of functional fragment or its derivative.

[0139] In some embodiments, in the first fusogenic protein, the targeting molecule is attached to the rhabdovirus glycoprotein via a linker.

[0140] In some embodiments, the linker is not susceptible to proteolytic cleavage by endogenous proteases or by exogenously added proteases.

[0141] In some embodiments, the linker is susceptible to proteolytic cleavage by endogenous proteases or by exogenously added proteases.

[0142] In some embodiments, the first fusion protein and / or the second fusion protein comprises a rhabdovirus glycoprotein comprising the sequence of SEQ ID NO: 8, with amino acid substitutions and / or deletions at one or more positions selected from K47, R354, H8, and Y209.

[0143] In some embodiments, the first fusion protein and / or the second fusion protein comprises the rhabdovirus glycoprotein that comprises any of the various rhabdovirus glycoprotein sequences described herein.In some embodiments, the first fusion protein and / or the second fusion protein comprises the rhabdovirus glycoprotein that comprises one or more amino acid substitutions and / or deletions at the position corresponding to H8, K47, Y209 or R354 in SEQ ID NO: 8.In some embodiments, the first fusion protein and / or the second fusion protein comprises the rhabdovirus glycoprotein that comprises or consists of SEQ ID NO: 8, and the one or more amino acid mutations are the substitutions at K47 position and R354 position.In some embodiments, the one or more amino acid mutations are the substitutions at K47 position, R354 position and Y209 position.In some embodiments, the one or more amino acid mutations are the substitutions at H8 position.

[0144] In some embodiments, the first fusion protein and / or the second fusion protein comprises a rhabdovirus glycoprotein that comprises one or more amino acid deletions at positions corresponding to H8, K47, Y209 or R354 in SEQ ID NO: 8. In some embodiments, the first fusion protein and / or the second fusion protein comprises a rhabdovirus glycoprotein that comprises or consists of SEQ ID NO: 8, and the one or more amino acid deletions are at positions H8, K47, Y209 or R354. In some embodiments, the one or more amino acid deletions are at positions K47. In some embodiments, the one or more amino acid deletions are at positions H8. In some embodiments, the one or more amino acid deletions are at positions H8 and K47.

[0145] In another aspect, provided herein are recombinant pseudotyped virus or cell-derived nanovesicles comprising the glycoprotein from Flanders virus (FLAV-G), or a functional fragment or derivative thereof.

[0146] In some embodiments, the FLAV-G comprises the sequence of SEQ ID NO:9.

[0147] In some embodiments, the FLAV-G consists of the sequence of SEQ ID NO:9.

[0148] In another aspect, provided herein are recombinant pseudotyped virus or cell-derived nanovesicles comprising glycoprotein from Chandipura virus (CHPV-G), or a functional fragment or derivative thereof.

[0149] In some embodiments, the CHPV-G comprises the sequence of SEQ ID NO:10.

[0150] In some embodiments, the CHPV-G consists of the sequence of SEQ ID NO:10.

[0151] In another aspect, provided herein are recombinant pseudotyped virus or cell-derived nanovesicles comprising a glycoprotein from a Perinetovirus (PERV-G), or a functional fragment or derivative thereof.

[0152] In some embodiments, the PERV-G comprises the sequence of SEQ ID NO:11.

[0153] In some embodiments, the PERV-G consists of the sequence of SEQ ID NO:11.

[0154] In another aspect, provided herein are recombinant pseudotyped virus or cell-derived nanovesicles comprising a glycoprotein from a Piryvirus (PIRYV-G), or a functional fragment or derivative thereof.

[0155] In some embodiments, the PIRYV-G comprises the sequence of SEQ ID NO:12.

[0156] In some embodiments, the PIRYV-G consists of the sequence of SEQ ID NO:12.

[0157] In another aspect, provided herein are recombinant pseudotyped virus or cell-derived nanovesicles comprising glycoprotein from Fukuoka virus (FUKV-G), or a functional fragment or derivative thereof.

[0158] In some embodiments, the FUKV-G comprises the sequence of SEQ ID NO:13.

[0159] In some embodiments, the FUKV-G consists of the sequence of SEQ ID NO:13.

[0160] In another aspect, provided herein are recombinant pseudotyped virus or cell-derived nanovesicles comprising glycoprotein from Joint Jacaka virus (JOIV-G), or a functional fragment or derivative thereof.

[0161] In some embodiments, the JOIV-G comprises the sequence of SEQ ID NO:14.

[0162] In some embodiments, the JOIV-G consists of the sequence of SEQ ID NO:14.

[0163] In another aspect, provided herein are recombinant pseudotyped virus or cell-derived nanovesicles comprising the glycoprotein from Kumasi virus (KRV-G), or a functional fragment or derivative thereof.

[0164] In some embodiments, the KRV-G comprises the sequence of SEQ ID NO:15.

[0165] In some embodiments, the KRV-G consists of the sequence of SEQ ID NO:15.

[0166] In another aspect, provided herein are recombinant pseudotyped virus or cell-derived nanovesicles comprising a glycoprotein from Keurariba virus (KEUV-G), or a functional fragment or derivative thereof.

[0167] In some embodiments, the KEUV-G comprises the sequence of SEQ ID NO:17.

[0168] In some embodiments, the KEUV-G consists of the sequence of SEQ ID NO:17.

[0169] In some embodiments, the cytoplasmic tail of the glycoprotein is removed or truncated, and optionally replaced with another sequence.

[0170] In some embodiments, the cytoplasmic tail of the glycoprotein is truncated by up to 40 amino acids from the C-terminus.

[0171] In some embodiments, the cytoplasmic tail of the glycoprotein is truncated 10 to 40 amino acids from the C-terminus.

[0172] In some embodiments, the cytoplasmic tail of the glycoprotein is truncated 30 amino acids from the C-terminus.

[0173] In some embodiments, the glycoprotein further comprises a cytoplasmic tail from VSV-G, or a functional fragment or derivative thereof.

[0174] In some embodiments, the cytoplasmic tail of VSV-G comprises the sequence CIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 16).

[0175] In some embodiments, the virus is a rhabdovirus.

[0176] In some embodiments, the virus is a recombinant vesicular stomatitis virus (VSV).

[0177] In some embodiments, the virus is a retrovirus.

[0178] In some embodiments, the retrovirus is a lentivirus (LV).

[0179] In some embodiments, the virus is replication competent.

[0180] In some embodiments, the virus is non-replicating.

[0181] In some embodiments, the virus further comprises a molecular cargo.

[0182] In some embodiments, the molecular cargo is a transgene encoding a therapeutic protein, a suicide gene, a toxic protein or peptide, an antibody or fragment thereof, a chimeric antigen receptor (CAR), a T cell receptor (TCR), a gene editing system or component(s) thereof, an antisense oligonucleotide, a ribozyme, or an RNAi molecule.

[0183] In some embodiments, the molecular cargo is a therapeutic protein, a toxic protein or peptide, an antibody or fragment thereof, a chimeric antigen receptor (CAR), a T cell receptor (TCR), a gene editing system or component(s) thereof, an antisense oligonucleotide, a ribozyme, or an RNAi molecule.

[0184] In some embodiments, the molecular cargo is a gene-editing ribonucleoprotein complex or component(s) thereof.

[0185] In some embodiments, the molecular cargo is a Cas9 protein complexed with a guide RNA (gRNA) specific for a gene of interest.

[0186] In another aspect, provided herein is a composition comprising a recombinant pseudotyped virus or cell-derived nanovesicle as described herein and a carrier and / or excipient.

[0187] In another aspect, provided herein is a method of reducing the susceptibility of a recombinant virus or nanovesicle to serum neutralization in a subject in need thereof, the method comprising administering to the subject a recombinant pseudotyped virus or cell-derived nanovesicle described herein, or a composition described herein.

[0188] In another aspect, provided herein is a method of enhancing resistance to low-density lipoprotein (LDL) and / or very low-density lipoprotein (VLDL)-mediated neutralization in a subject in need thereof, the method comprising administering to the subject a recombinant pseudotyped virus or cell-derived nanovesicle described herein, or a composition described herein.

[0189] In another aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant pseudotyped virus or cell-derived nanovesicle described herein, or a composition described herein.

[0190] In some embodiments, the method does not include prior treatment with an LDL / VLDL-lowering medication.

[0191] In some embodiments, the method further comprises pretreatment with an LDL / VLDL lowering medication.

[0192] In another aspect, provided herein is a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of a recombinant pseudotyped virus or cell-derived nanovesicle described herein, or a composition described herein.

[0193] In another aspect, provided herein is a method for delivering a molecular cargo to a cell in a subject in need thereof, comprising administering to the subject an effective amount of a recombinant pseudotyped virus or cell-derived nanovesicle described herein, or a composition comprising the pseudotyped virus or cell-derived nanovesicle and a carrier and / or excipient, wherein the recombinant fusogenic protein within the recombinant pseudotyped virus or cell-derived nanovesicle comprises a targeting molecule that targets the cell.

[0194] In some embodiments, the subject is a human.

[0195] In another aspect, provided herein is a method for ex vivo delivery of molecular cargo to a cell, comprising administering to the cell an effective amount of a recombinant pseudotyped virus or cell-derived nanovesicle described herein, or a composition comprising the pseudotyped virus or cell-derived nanovesicle and a carrier and / or excipient, wherein the recombinant fusogenic protein within the recombinant pseudotyped virus or cell-derived nanovesicle comprises a targeting molecule that targets the cell. [Brief explanation of the drawings]

[0196] [Figure 1A] We demonstrate that serum inhibits wild-type (WT) vesicular stomatitis virus (VSV). Vero cells were infected with wild-type (WT) (glycoprotein [G]-containing) VSV (VSV-G) encoding a firefly luciferase (Fluc) reporter. Increasing concentrations of pooled human serum were added to the cells at various times during the infection course, including: 1) throughout the entire infection course (Figure 1A, left panel); 2) only during the first 4 hours of inoculation and then removed (Figure 1A, middle panel); or 3) only after 4 hours of inoculation (Figure 1A, right panel). Luciferase activity was measured 16 hours after the initial inoculation. In separate experiments, pooled serum was then left untreated or incubated at 56°C to inactivate complement in the serum. Serum (or medium alone) was then added to Vero cells. WT VSV encoding a green fluorescent protein (GFP) reporter (VSV-GFP) was added to the cells. After 16 hours, the plates were imaged by an imaging cytometer (Fig. 1B, left panel), and the number of GFP-positive cells was quantified (Fig. 1B, right panel). [Figure 1B]We demonstrate that serum inhibits wild-type (WT) vesicular stomatitis virus (VSV). Vero cells were infected with wild-type (WT) (glycoprotein [G]-containing) VSV (VSV-G) encoding a firefly luciferase (Fluc) reporter. Increasing concentrations of pooled human serum were added to the cells at various times during the infection course, including: 1) throughout the entire infection course (Figure 1A, left panel); 2) only during the first 4 hours of inoculation and then removed (Figure 1A, middle panel); or 3) only after 4 hours of inoculation (Figure 1A, right panel). Luciferase activity was measured 16 hours after the initial inoculation. In separate experiments, pooled serum was then left untreated or incubated at 56°C to inactivate complement in the serum. Serum (or medium alone) was then added to Vero cells. WT VSV encoding a green fluorescent protein (GFP) reporter (VSV-GFP) was added to the cells. After 16 hours, the plates were imaged by an imaging cytometer (Fig. 1B, left panel), and the number of GFP-positive cells was quantified (Fig. 1B, right panel). [Figure 2] We demonstrate that human serum inhibits WT VSV in human cell lines. Pooled human serum was mixed with VSV-Fluc virus and overlayed onto human cell lines, such as HT1080 human fibrosarcoma cells (Figure 2, left panel), human embryonic kidney (HEK) 293T cells (Figure 2, center panel), or SKOV3.ip1 human ovarian cancer cells (Figure 2, right panel). The graph shows luciferase activity measured by a standard luciferase assay in the three cell lines 16 hours postinfection. [Figure 3] We demonstrate that heat-inactivated serum inhibits wild-type VSV in K562 cells. VSV-GFP was mixed with medium, complement-activating serum (serum), or heat-inactivated serum (HI serum) and combined with K562 cells. After 24 hours, the plates were imaged using an imaging cytometer to determine the number of GFP-positive cells in each well. [Figure 4]This shows that heat-inactivated serum inhibits wild-type VSV in human cell lines. Medium alone, a complement-activated human serum pool (serum), or a complement-inactivated human serum pool (HI serum) was added to the human cell lines SKOV3.ip1, HT1080, and K562. VSV-GFP was then added to the cells, and the plates were imaged using an imaging cytometer. The number of GFP-positive cells in each well was determined 16 hours after infection. [Figure 5A] This indicates that lipoprotein-depleted serum has no inhibitory activity. Medium alone, heat-inactivated serum (HSA), human serum albumin (HSA), artificial serum (AF serum), intralipid, or lipoprotein-depleted serum (LD serum) was added to HT1080 or K562 human cell lines. VSV-GFP was then added to the cells. After 16 hours, the plates were imaged using an imaging cytometer to determine the number of GFP-positive cells in each well (Figure 5A). An example of a fluorescence microscopy image of HT1080 cells is shown in Figure 5B. [Figure 5B] This indicates that lipoprotein-depleted serum has no inhibitory activity. Medium alone, heat-inactivated serum (HSA), human serum albumin (HSA), artificial serum (AF serum), intralipid, or lipoprotein-depleted serum (LD serum) was added to HT1080 or K562 human cell lines. VSV-GFP was then added to the cells. After 16 hours, the plates were imaged using an imaging cytometer to determine the number of GFP-positive cells in each well (Figure 5A). An example of a fluorescence microscopy image of HT1080 cells is shown in Figure 5B. [Figure 6A]These results show that low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL), but not high-density lipoprotein (HDL), inhibit wild-type VSV. K562 cells were mixed with VSV-GFP and layered into wells containing various test conditions, including medium alone (M), human serum pool (S), lipoprotein-depleted human serum pool (D), or medium containing various concentrations of purified human LDL, HDL, or VLDL. After 16 hours (Figure 6A, upper panel and Figure 6B, left panel), cell photographs were taken using a fluorescence microscope. After 24 hours, the plates were imaged using an imaging cytometer, and the number of GFP-positive cells in each well was determined (Figure 6A, lower left panel and Figure 6B, right panel). The lower right panel of Figure 6A also shows a graph of luciferase activity (VSV-Fluc) in Vero cells previously infected with VSV-Fluc 16 hours earlier in the presence of various HDL concentrations. [Figure 6B] These results show that low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL), but not high-density lipoprotein (HDL), inhibit wild-type VSV. K562 cells were mixed with VSV-GFP and layered into wells containing various test conditions, including medium alone (M), human serum pool (S), lipoprotein-depleted human serum pool (D), or medium containing various concentrations of purified human LDL, HDL, or VLDL. After 16 hours (Figure 6A, upper panel and Figure 6B, left panel), cell photographs were taken using a fluorescence microscope. After 24 hours, the plates were imaged using an imaging cytometer, and the number of GFP-positive cells in each well was determined (Figure 6A, lower left panel and Figure 6B, right panel). The lower right panel of Figure 6A also shows a graph of luciferase activity (VSV-Fluc) in Vero cells previously infected with VSV-Fluc 16 hours earlier in the presence of various HDL concentrations. [Figure 7]The heat resistance of LDL and VLDL inhibitory activity is shown. Human LDL (Figure 7, left panel) or VLDL (Figure 7, right panel) was heat-inactivated (HI-LDL or HI-VLDL) or left untreated (LDL or VLDL). K562 cells were mixed with VSV-GFP and layered onto wells containing various test conditions, including medium alone (medium) or heat-inactivated (HI) or untreated LDL or VLDL. Plates were imaged using an imaging cytometer, and the number of GFP-positive cells in each well was determined. [Figure 8A] The mechanism of VSV activation by serum components is illustrated. Figure 8A shows a schematic diagram of pathogen (e.g., VSV) inactivation by IgM+ complement (heat-labile) in serum. The mechanism of VSV inactivation may be due to the binding of natural IgM to the G protein, followed by activation of the complement cascade, which leads to coating of the virus with C3b, irreversible destruction of viral infectivity, and subsequent viral lysis by the membrane attack complex. This mechanism of viral inactivation, although not immediate, can reduce the infectious titer of a VSV preparation or a preparation of a lentiviral vector incorporating the VSV-G protein by up to approximately 10,000-fold over a period of one hour. Figure 8B shows a schematic diagram of the mechanism of LDL / VLDL competition with VSV-G for binding to the LDL receptor. ApoB-100 binds to the LDL receptor at the same site as the VSV-G glycoprotein. ApoB-100-containing lipoproteins include LDL and VLDL, which compete with VSV-G for binding to the LDLR (Fig. 8B). [Figure 8B]The mechanism of VSV activation by serum components is illustrated. Figure 8A shows a schematic diagram of pathogen (e.g., VSV) inactivation by IgM+ complement (heat-labile) in serum. The mechanism of VSV inactivation may be due to the binding of natural IgM to the G protein, followed by activation of the complement cascade, which leads to coating of the virus with C3b, irreversible destruction of viral infectivity, and subsequent viral lysis by the membrane attack complex. This mechanism of viral inactivation, although not immediate, can reduce the infectious titer of a VSV preparation or a preparation of a lentiviral vector incorporating the VSV-G protein by up to approximately 10,000-fold over a period of one hour. Figure 8B shows a schematic diagram of the mechanism of LDL / VLDL competition with VSV-G for binding to the LDL receptor. ApoB-100 binds to the LDL receptor at the same site as the VSV-G glycoprotein. ApoB-100-containing lipoproteins include LDL and VLDL, which compete with VSV-G for binding to the LDLR (Fig. 8B). [Figure 9A] Figures A-B show the design of a retargeted VSV glycoprotein. The VSV-G protein contains a signal peptide (SP) that can be proteolytically cleaved after translation. The targeting molecule can be, for example, a single-chain fragment variable fragment (scFv) antibody raised against human epidermal growth factor receptor 2 (HER2) or epidermal growth factor receptor (EGFR), a natural ligand such as EGFm123 (modified EGF), and / or a nanobody or peptide, and can be added to the amino terminus of the G protein, for example, with or without a flexible linker. Mutations in the G protein can be incorporated at various amino acid positions, for example, amino acid positions 47 and 354 (K47Q / R354Q) (Figure 9A). The same targeting molecule can be added to the N-terminus of the alternative rhabdovirus Flanders G (FLAV-G), with or without a flexible linker (Figure 9B). [Figure 9B] Same as above. [Figure 10A]We demonstrate that VSV-G and anti-HER2 scFv carrying blinding mutations specifically bind HER2-expressing cell lines. SKOV3.ip1 cells (Figure 10A), HT1080 cells (Figure 10B), A549 (human lung adenocarcinoma) cells (Figure 10C), and BHK-21 (baby hamster kidney) cells (Figure 10D) stably expressing the double-split protein (DSP) DSP-1 or DSP-2 reporter were cocultured. The following day, cells were transfected with plasmids expressing GFP, WT VSV-G, or VSV-G carrying an N-terminal anti-HER2 scFv, as well as plasmids carrying either two mutations (K47Q / R354Q) or three mutations (K47Q / R354Q / E353A) aimed at eliminating glycoprotein interaction with the LDLR. After transfection, cells were treated with either pH 5.0 to mediate fusion or neutral phosphate-buffered saline (PBS) before the addition of fresh medium containing the luciferase substrate, EnduRen. Renilla luciferase activity was measured 4 hours after addition of the substrate. [Figure 10B] We demonstrate that VSV-G and anti-HER2 scFv carrying blinding mutations specifically bind HER2-expressing cell lines. SKOV3.ip1 cells (Figure 10A), HT1080 cells (Figure 10B), A549 (human lung adenocarcinoma) cells (Figure 10C), and BHK-21 (baby hamster kidney) cells (Figure 10D) stably expressing the double-split protein (DSP) DSP-1 or DSP-2 reporter were cocultured. The following day, cells were transfected with plasmids expressing GFP, WT VSV-G, or VSV-G carrying an N-terminal anti-HER2 scFv, as well as plasmids carrying either two mutations (K47Q / R354Q) or three mutations (K47Q / R354Q / E353A) aimed at eliminating glycoprotein interaction with the LDLR. After transfection, cells were treated with either pH 5.0 to mediate fusion or neutral phosphate-buffered saline (PBS) before the addition of fresh medium containing the luciferase substrate, EnduRen. Renilla luciferase activity was measured 4 hours after addition of the substrate. [Figure 10C]We demonstrate that VSV-G and anti-HER2 scFv carrying blinding mutations specifically bind HER2-expressing cell lines. SKOV3.ip1 cells (Figure 10A), HT1080 cells (Figure 10B), A549 (human lung adenocarcinoma) cells (Figure 10C), and BHK-21 (baby hamster kidney) cells (Figure 10D) stably expressing the double-split protein (DSP) DSP-1 or DSP-2 reporter were cocultured. The following day, cells were transfected with plasmids expressing GFP, WT VSV-G, or VSV-G carrying an N-terminal anti-HER2 scFv, as well as plasmids carrying either two mutations (K47Q / R354Q) or three mutations (K47Q / R354Q / E353A) aimed at eliminating glycoprotein interaction with the LDLR. After transfection, cells were treated with either pH 5.0 to mediate fusion or neutral phosphate-buffered saline (PBS) before the addition of fresh medium containing the luciferase substrate, EnduRen. Renilla luciferase activity was measured 4 hours after addition of the substrate. [Figure 10D] We demonstrate that VSV-G and anti-HER2 scFv carrying blinding mutations specifically bind HER2-expressing cell lines. SKOV3.ip1 cells (Figure 10A), HT1080 cells (Figure 10B), A549 (human lung adenocarcinoma) cells (Figure 10C), and BHK-21 (baby hamster kidney) cells (Figure 10D) stably expressing the double-split protein (DSP) DSP-1 or DSP-2 reporter were cocultured. The following day, cells were transfected with plasmids expressing GFP, WT VSV-G, or VSV-G carrying an N-terminal anti-HER2 scFv, as well as plasmids carrying either two mutations (K47Q / R354Q) or three mutations (K47Q / R354Q / E353A) aimed at eliminating glycoprotein interaction with the LDLR. After transfection, cells were treated with either pH 5.0 to mediate fusion or neutral phosphate-buffered saline (PBS) before the addition of fresh medium containing the luciferase substrate, EnduRen. Renilla luciferase activity was measured 4 hours after addition of the substrate. [Figure 11A]We demonstrate that infection with blinded and retargeted VSV-G correlates with HER2 receptor levels. HER2 receptor expression was determined in SKOV3.ip1, Vero, and Hela cells by flow cytometry (Figure 11A). Cells were infected with VSV-GFP (WT VSV), VSV-αHER2-VSV-G(K47Q / R354Q)-GFP, or VSV-αHER2-VSV-G(K47Q / R354Q / E353A)-GFP. Images were captured by fluorescence microscopy (Figure 11B). [Figure 11B] We demonstrate that infection with blinded and retargeted VSV-G correlates with HER2 receptor levels. HER2 receptor expression was determined in SKOV3.ip1, Vero, and Hela cells by flow cytometry (Figure 11A). Cells were infected with VSV-GFP (WT VSV), VSV-αHER2-VSV-G(K47Q / R354Q)-GFP, or VSV-αHER2-VSV-G(K47Q / R354Q / E353A)-GFP. Images were captured by fluorescence microscopy (Figure 11B). [Figure 12] We demonstrate that soluble αHER2 scFv selectively inhibits HER2-retargeted LDLR mutant virus infection and spread in SKOV3ip.1 cells. SKOV3ip.1 cells were infected with VSV-GFP, VSV-αHER2-VSV-G(K47Q / R354Q)-GFP, or VSV-αHER2-VSV-G(K47Q / R354Q / E353A)-GFP and treated with medium from cultures of cells transfected with a GFP-expressing plasmid (Mock-sup) or secreted anti-HER2 or anti-EGFR scFv. Viral infection and spread were monitored by imaging and counting the number of GFP-positive cells using an imaging cytometer. [Figure 13A]This demonstrates that VSV-G can target EGF or HER2 receptors in the same cell type. SK-BR-3 cells expressing both EGFR and HER2 were infected with control VSV-GFP or VSV containing two blinding mutations (K47Q / R354Q) and targeting molecules against EGFR or HER2 (scFvs raised against EGFm123 or HER2), and then treated with medium (control) or blocking molecules. 20 hours after infection, cells were imaged using an imaging cytometer (Figure 13A), and the number of GFP-positive cells was determined (Figure 13B). [Figure 13B] This demonstrates that VSV-G can target EGF or HER2 receptors in the same cell type. SK-BR-3 cells expressing both EGFR and HER2 were infected with control VSV-GFP or VSV containing two blinding mutations (K47Q / R354Q) and targeting molecules against EGFR or HER2 (scFvs raised against EGFm123 or HER2), and then treated with medium (control) or blocking molecules. 20 hours after infection, cells were imaged using an imaging cytometer (Figure 13A), and the number of GFP-positive cells was determined (Figure 13B). [Figure 14A] Figure 14 shows the specificity of retargeted VSV-αEGFR-G K47QR354Q-GFP in HT1080-EGFR knockout (KO) and HEK-293T-EGFR-KO cells. HT1080 WT or HT1080-EGFR-KO cells were infected with VSV-GFP (control) or VSV-αEGFR-G K47QR354Q-GFP. The GFP images shown in Figure 14A (left panel) were taken using a fluorescent microscope, and the number of GFP-positive cells was quantified using an imaging cytometer (Figure 14A, right panel). HEK-293T WT or HEK-293T-EGFR-KO cells were infected with VSV-GFP or VSV-αEGFR-G K47QR354Q-GFP. The GFP images shown in Figure 14B were taken using an imaging cytometer. The numbers above the images indicate the number of GFP-positive cells. [Figure 14B]Figure 14 shows the specificity of retargeted VSV-αEGFR-G K47QR354Q-GFP in HT1080-EGFR knockout (KO) and HEK-293T-EGFR-KO cells. HT1080 WT or HT1080-EGFR-KO cells were infected with VSV-GFP (control) or VSV-αEGFR-G K47QR354Q-GFP. The GFP images shown in Figure 14A (left panel) were taken using a fluorescent microscope, and the number of GFP-positive cells was quantified using an imaging cytometer (Figure 14A, right panel). HEK-293T WT or HEK-293T-EGFR-KO cells were infected with VSV-GFP or VSV-αEGFR-G K47QR354Q-GFP. The GFP images shown in Figure 14B were taken using an imaging cytometer. The numbers above the images indicate the number of GFP-positive cells. [Figure 15A] Figure 15 shows the specificity of retargeted VSV on a K562 cell panel. K562 parental cells or EGFR / HER2 receptor-expressing cells were infected with VSV-GFP or retargeted VSV with modified linker sequences (19 amino acid [aa] linker: RAAA(G4S)3 (SEQ ID NO: 170); 20 aa linker: KRAAASGGS(G4S)2GPK) (SEQ ID NO: 174)). GFP images shown in Figure 15A were taken using a fluorescent microscope. The number of GFP-positive cells was quantified using an imaging cytometer (Figure 15B). [Figure 15B] Figure 15 shows the specificity of retargeted VSV on a K562 cell panel. K562 parental cells or EGFR / HER2 receptor-expressing cells were infected with VSV-GFP or retargeted VSV with modified linker sequences (19 amino acid [aa] linker: RAAA(G4S)3 (SEQ ID NO: 170); 20 aa linker: KRAAASGGS(G4S)2GPK) (SEQ ID NO: 174)). GFP images shown in Figure 15A were taken using a fluorescent microscope. The number of GFP-positive cells was quantified using an imaging cytometer (Figure 15B). [Figure 16A]A to B show the incorporation of targeting molecules EGFm123 or hSCF in viral particles. Western blotting of EGFm123 showing viral particles is shown in Figure 16A. Western blotting of hSCF showing viral particles is shown in Figure 16B. [Figure 16B] Same as above. [Figure 17A] This shows the specificity of the retargeted VSV, which exhibits a smaller targeting molecule. K562 parental cells, K562-EGFR cells, or K562-HER2 cells were infected with VSV-GFP (as a control) or the indicated retargeted VSV. The GFP images shown in Figure 17A were taken using a fluorescent microscope. The number of GFP-positive cells was quantified using an imaging cytometer (Figure 17B). [Figure 17B] This shows the specificity of the retargeted VSV, which exhibits a smaller targeting molecule. K562 parental cells, K562-EGFR cells, or K562-HER2 cells were infected with VSV-GFP (as a control) or the indicated retargeted VSV. The GFP images shown in Figure 17A were taken using a fluorescent microscope. The number of GFP-positive cells was quantified using an imaging cytometer (Figure 17B). [Figure 18A] Figure 18 shows VSV-EGFm123 infection in EGFR KO cell lines. To generate the data shown in Figures 18A-18D, cells were infected with VSV-GFP or VSV-EGFm123 virus. GFP and phase microscopy contrast images of HeLa WT or HeLa-EGFR-KO cells were acquired as shown in Figure 18A. GFP images were captured using an imaging cytometer (Figure 18B), and infection levels were quantified as the number of GFP-positive cells for the HeLa cell panel (Figure 18C) or HT1080 cell panel (Figure 18D). The ability of VSV-EGFm123 to bind to its intended receptor was determined using HeLa or HT1080 WT or EGFR-KO cells (Figures 18E-18F). 1 x 106 cells were incubated with VSV-GFP or VSV-EGFm123 virus. Cells were fixed and then stained with PE-conjugated VSV-G antibody. Cells were analyzed by flow cytometry (FIGS. 18E-18F). [Figure 18B] Figure 18 shows VSV-EGFm123 infection in EGFR KO cell lines. To generate the data shown in Figures 18A-18D, cells were infected with VSV-GFP or VSV-EGFm123 virus. GFP and phase microscopy contrast images of HeLa WT or HeLa-EGFR-KO cells were acquired as shown in Figure 18A. GFP images were captured using an imaging cytometer (Figure 18B), and infection levels were quantified as the number of GFP-positive cells for the HeLa cell panel (Figure 18C) or HT1080 cell panel (Figure 18D). The ability of VSV-EGFm123 to bind to its intended receptor was determined using HeLa or HT1080 WT or EGFR-KO cells (Figures 18E-18F). 1 x 106 cells were incubated with VSV-GFP or VSV-EGFm123 virus. Cells were fixed and then stained with PE-conjugated VSV-G antibody. Cells were analyzed by flow cytometry (FIGS. 18E-18F). [Figure 18C] Figure 18 shows VSV-EGFm123 infection in EGFR KO cell lines. To generate the data shown in Figures 18A-18D, cells were infected with VSV-GFP or VSV-EGFm123 virus. GFP and phase microscopy contrast images of HeLa WT or HeLa-EGFR-KO cells were acquired as shown in Figure 18A. GFP images were captured using an imaging cytometer (Figure 18B), and infection levels were quantified as the number of GFP-positive cells for the HeLa cell panel (Figure 18C) or HT1080 cell panel (Figure 18D). The ability of VSV-EGFm123 to bind to its intended receptor was determined using HeLa or HT1080 WT or EGFR-KO cells (Figures 18E-18F). 1 x 106 cells were incubated with VSV-GFP or VSV-EGFm123 virus. Cells were fixed and then stained with PE-conjugated VSV-G antibody. Cells were analyzed by flow cytometry (FIGS. 18E-18F). [Figure 18D]Figure 18 shows VSV-EGFm123 infection in EGFR KO cell lines. To generate the data shown in Figures 18A-18D, cells were infected with VSV-GFP or VSV-EGFm123 virus. GFP and phase microscopy contrast images of HeLa WT or HeLa-EGFR-KO cells were acquired as shown in Figure 18A. GFP images were captured using an imaging cytometer (Figure 18B), and infection levels were quantified as the number of GFP-positive cells for the HeLa cell panel (Figure 18C) or HT1080 cell panel (Figure 18D). The ability of VSV-EGFm123 to bind to its intended receptor was determined using HeLa or HT1080 WT or EGFR-KO cells (Figures 18E-18F). 1 x 106 cells were incubated with VSV-GFP or VSV-EGFm123 virus. Cells were fixed and then stained with PE-conjugated VSV-G antibody. Cells were analyzed by flow cytometry (FIGS. 18E-18F). [Figure 18E] Figure 18 shows VSV-EGFm123 infection in EGFR KO cell lines. To generate the data shown in Figures 18A-18D, cells were infected with VSV-GFP or VSV-EGFm123 virus. GFP and phase microscopy contrast images of HeLa WT or HeLa-EGFR-KO cells were acquired as shown in Figure 18A. GFP images were captured using an imaging cytometer (Figure 18B), and infection levels were quantified as the number of GFP-positive cells for the HeLa cell panel (Figure 18C) or HT1080 cell panel (Figure 18D). The ability of VSV-EGFm123 to bind to its intended receptor was determined using HeLa or HT1080 WT or EGFR-KO cells (Figures 18E-18F). 1 x 106 cells were incubated with VSV-GFP or VSV-EGFm123 virus. Cells were fixed and then stained with PE-conjugated VSV-G antibody. Cells were analyzed by flow cytometry (FIGS. 18E-18F). [Figure 18F]Figure 18 shows VSV-EGFm123 infection in EGFR KO cell lines. To generate the data shown in Figures 18A-18D, cells were infected with VSV-GFP or VSV-EGFm123 virus. GFP and phase microscopy contrast images of HeLa WT or HeLa-EGFR-KO cells were acquired as shown in Figure 18A. GFP images were captured using an imaging cytometer (Figure 18B), and infection levels were quantified as the number of GFP-positive cells for the HeLa cell panel (Figure 18C) or HT1080 cell panel (Figure 18D). The ability of VSV-EGFm123 to bind to its intended receptor was determined using HeLa or HT1080 WT or EGFR-KO cells (Figures 18E-18F). 1 x 106 cells were incubated with VSV-GFP or VSV-EGFm123 virus. Cells were fixed and then stained with PE-conjugated VSV-G antibody. Cells were analyzed by flow cytometry (FIGS. 18E-18F). [Figure 19A] Panels A-B demonstrate that the VSV-G-QQ-EGFm123 virus is resistant to inhibition by LDL. VSV-GFP (VSV-G-wt) or VSV-GFP containing the K47Q / R354Q mutation and an additional G at EGFm123 (EGFR-retargeted virus) was incubated with medium alone, pooled human serum (complement-deficient lot), or pooled lipoprotein-depleted human serum. The mixture was layered onto HEK-293T plates. After 24 hours, the plates were imaged using an imaging cytometer, and the number of GFP-positive cells per well was determined (Figure 19A). VSV-GFP (VSV-G-WT) or VSV-GFP containing the K47Q / R354Q mutation and an additional G in EGFm123 (EGFm123-K47Q / R354Q) was mixed with K562-EGFR cells and added to wells containing medium alone (control) or increasing concentrations of purified human LDL. After 24 hours, plates were imaged using an imaging cytometer to determine the number of GFP-positive cells per well (Figure 19B). [Figure 19B] Same as above. [Figure 20]We demonstrate that the VSV-G-QQ-EGFm123 virus is resistant to inhibition by VLDL. VSV-GFP (WT-G) or VSV-GFP containing the K47Q / R354Q mutation and an additional G in EGFm123 (EGFm123-K47Q / R354Q) was mixed with K562-EGFR cells and added at the indicated concentrations to wells containing medium alone or purified human HDL, LDL, or VLDL. After 24 hours, plates were imaged using an inverted fluorescence microscope. [Figure 21] This shows that binding of wild-type VSV, but not EGFR-retargeted VSV, is reduced by LDL. VSV-GFP (VSV-G-WT) or VSV-GFP containing the K47Q / R354Q mutation and an additional G in EGFm123 (VSV-MC11-EGFm123-VSV-G(K47Q / R354Q)-GFP) was mixed with medium alone, pooled human serum (serum), or LDL and layered onto HT1080 cells. RNA was then extracted from the cells, and samples were subjected to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using primers specific for the VSV genome (IDT). Data represent the number of VSV genome copies relative to the copy number from a virus-only control medium. [Figure 22] The sensitivity of retargeted VSV-G to human serum and LDL is shown. VSV-GFP (VSV-G-WT) or VSV-GFP containing the K47Q / R354Q mutation and G appended to either human stem cell factor (hSCF) (VSV-GFP(hSCF-G-K47Q / R354Q)) or HER2 scFv (VSV-GFP(G-HER2)) was mixed with various cells from a panel of K562 cells, including parental K562, K562-HER2, or K562-cKit. The mixtures were added to wells containing medium alone, human pooled serum (serum), human pooled lipoprotein-depleted serum (LD serum), medium containing LDL (+LDL), or human pooled lipoprotein-depleted serum with LDL (LD serum + LDL). Fluorescence micrographs were taken 24 hours later. [Figure 23]The sensitivity of retargeted VSV-G to human serum and LDL in PC3 cells is shown. VSV-GFP (WT-G) or VSV-GFP containing the K47Q / R354Q mutation and G attached to either EGFm123 (G-QQ-EGFm123), human SCF (G-QQ-SCF), or HER2 scFv (G-QQ-HER2) was mixed with medium alone, fresh (complement-active) human pooled serum (serum), heat-inactivated pooled human serum (HI-serum), or human pooled lipoprotein-depleted serum containing LDL (+LDL). The mixture was layered onto PC3 prostate cancer cells. After 24 hours (WT) or 42 hours (retargeted virus), the cells were imaged using an imaging cytometer. [Figure 24] This figure shows that truncation of the cytoplasmic tail of FLAV-G enhances the fusion activity of FLAV-G. A panel of FLAV-G constructs containing anti-EGFR scFvs with successively shorter cytoplasmic tails was generated. The fusion activity shown in the graph was determined by DSP cell-cell fusion activity in SKOV3.ip1 cells. [Figure 25A] Figure 25A shows improved VSV targeting the HER2 receptor in PC3 cells. A schematic diagram of the FLAV-G construct design with a 23aa linker and VSV-G cytoplasmic tail, as well as a construct with a 19aa linker lacking the VSV-G cytoplasmic tail, is shown. Two scFvs targeting HER2 and one scFv targeting EGFR were cloned into the 19aa linker construct. To generate the data shown in Figures 25B-C, the specificity of the two HER2-targeting viruses was evaluated in a panel of PC3 cells, including parental PC3, PC3-EGFR, and PC3-HER2. Fluorescence microscopy images of infected cells using a microscope are shown in Figure 25B. The number of infected cells in each condition was determined using an imaging cytometer (Figure 25C). [Figure 25B]Figure 25A shows improved VSV targeting the HER2 receptor in PC3 cells. A schematic diagram of the FLAV-G construct design with a 23aa linker and VSV-G cytoplasmic tail, as well as a construct with a 19aa linker lacking the VSV-G cytoplasmic tail, is shown. Two scFvs targeting HER2 and one scFv targeting EGFR were cloned into the 19aa linker construct. To generate the data shown in Figures 25B-C, the specificity of the two HER2-targeting viruses was evaluated in a panel of PC3 cells, including parental PC3, PC3-EGFR, and PC3-HER2. Fluorescence microscopy images of infected cells using a microscope are shown in Figure 25B. The number of infected cells in each condition was determined using an imaging cytometer (Figure 25C). [Figure 25C] Figure 25A shows improved VSV targeting the HER2 receptor in PC3 cells. A schematic diagram of the FLAV-G construct design with a 23aa linker and VSV-G cytoplasmic tail, as well as a construct with a 19aa linker lacking the VSV-G cytoplasmic tail, is shown. Two scFvs targeting HER2 and one scFv targeting EGFR were cloned into the 19aa linker construct. To generate the data shown in Figures 25B-C, the specificity of the two HER2-targeting viruses was evaluated in a panel of PC3 cells, including parental PC3, PC3-EGFR, and PC3-HER2. Fluorescence microscopy images of infected cells using a microscope are shown in Figure 25B. The number of infected cells in each condition was determined using an imaging cytometer (Figure 25C). [Figure 26A]FLAV-G targets the insulin-like growth factor 1 (IGF1) receptor. To generate the data shown in Figures 26A-B, MCF7 breast cancer cells were infected with VSV-GFP (WT) or VSV containing the Flanders virus glycoprotein (FLAV-GΔ30) without any targeting molecule (non-targeted) or targeted to the insulin-like growth factor 1 (IGF1) receptor (IGF1R). Infected cells were imaged by fluorescence microscopy (Figure 26A, upper panel) and phase contrast microscopy (Figure 26A, lower panel). Infected cultures were also imaged using an imaging cytometer, and the number of GFP-positive cells was quantified (Figure 26B). Flow cytometry was used to determine IGF1 receptor expression in cells with fluorescently labeled anti-IGF1R antibody or isotype control (Figure 26C). [Figure 26B] FLAV-G targets the insulin-like growth factor 1 (IGF1) receptor. To generate the data shown in Figures 26A-B, MCF7 breast cancer cells were infected with VSV-GFP (WT) or VSV containing the Flanders virus glycoprotein (FLAV-GΔ30) without any targeting molecule (non-targeted) or targeted to the insulin-like growth factor 1 (IGF1) receptor (IGF1R). Infected cells were imaged by fluorescence microscopy (Figure 26A, upper panel) and phase contrast microscopy (Figure 26A, lower panel). Infected cultures were also imaged using an imaging cytometer, and the number of GFP-positive cells was quantified (Figure 26B). Flow cytometry was used to determine IGF1 receptor expression in cells with fluorescently labeled anti-IGF1R antibody or isotype control (Figure 26C). [Figure 26C]FLAV-G targets the insulin-like growth factor 1 (IGF1) receptor. To generate the data shown in Figures 26A-B, MCF7 breast cancer cells were infected with VSV-GFP (WT) or VSV containing the Flanders virus glycoprotein (FLAV-GΔ30) without any targeting molecule (non-targeted) or targeted to the insulin-like growth factor 1 (IGF1) receptor (IGF1R). Infected cells were imaged by fluorescence microscopy (Figure 26A, upper panel) and phase contrast microscopy (Figure 26A, lower panel). Infected cultures were also imaged using an imaging cytometer, and the number of GFP-positive cells was quantified (Figure 26B). Flow cytometry was used to determine IGF1 receptor expression in cells with fluorescently labeled anti-IGF1R antibody or isotype control (Figure 26C). [Figure 27A] Figure 27 shows the specificity of FLAV-G, which expresses modified epidermal growth factor (EGF). K562 cells stably transduced with the indicated receptors were infected with VSV-GFP (WT) or VSV (FLAV-GΔ30) containing the Flanders virus glycoprotein, which expresses EGFm123. Infected cells were imaged by fluorescence microscopy (Figure 27A, upper panel) and phase-contrast microscopy (Figure 27A, lower panel). Infected cultures were also imaged using a cytometer, and the number of GFP-positive cells was determined (Figure 27B). [Figure 27B] Figure 27 shows the specificity of FLAV-G, which expresses modified epidermal growth factor (EGF). K562 cells stably transduced with the indicated receptors were infected with VSV-GFP (WT) or VSV (FLAV-GΔ30) containing the Flanders virus glycoprotein, which expresses EGFm123. Infected cells were imaged by fluorescence microscopy (Figure 27A, upper panel) and phase-contrast microscopy (Figure 27A, lower panel). Infected cultures were also imaged using a cytometer, and the number of GFP-positive cells was determined (Figure 27B). [Figure 28A]This figure shows the design of retargeted VSV-G for lentivirus production. A schematic diagram of the generated construct is shown in Figure 28A. The VSV-G protein contained a signal peptide (SP) that can be proteolytically cleaved after translation. Exemplary targeting molecules include scFvs raised against HER2 or EGFR, natural ligands such as EGFm123 (modified EGF) or hSCF, or nanobodies against the EGF receptor, such as Nb 7D12, attached to the amino terminus of the G protein with or without a flexible linker. A construct without a targeting molecule was generated as a control. Blinding mutations in VSV-G were incorporated at K47 and R354 (Figure 28A). The figure discloses SEQ ID NOs: 35, 35, 35, and 236, respectively, in order of appearance. Lentivirus production in HEK-293T cells is outlined in Figure 28B. Western blotting of lentiviral particles is shown in Figure 28C. [Figure 28B] This figure shows the design of retargeted VSV-G for lentivirus production. A schematic diagram of the generated construct is shown in Figure 28A. The VSV-G protein contained a signal peptide (SP) that can be proteolytically cleaved after translation. Exemplary targeting molecules include scFvs raised against HER2 or EGFR, natural ligands such as EGFm123 (modified EGF) or hSCF, or nanobodies against the EGF receptor, such as Nb 7D12, attached to the amino terminus of the G protein with or without a flexible linker. A construct without a targeting molecule was generated as a control. Blinding mutations in VSV-G were incorporated at K47 and R354 (Figure 28A). The figure discloses SEQ ID NOs: 35, 35, 35, and 236, respectively, in order of appearance. Lentivirus production in HEK-293T cells is outlined in Figure 28B. Western blotting of lentiviral particles is shown in Figure 28C. [Figure 28C]This figure shows the design of retargeted VSV-G for lentivirus production. A schematic diagram of the generated construct is shown in Figure 28A. The VSV-G protein contained a signal peptide (SP) that can be proteolytically cleaved after translation. Exemplary targeting molecules include scFvs raised against HER2 or EGFR, natural ligands such as EGFm123 (modified EGF) or hSCF, or nanobodies against the EGF receptor, such as Nb 7D12, attached to the amino terminus of the G protein with or without a flexible linker. A construct without a targeting molecule was generated as a control. Blinding mutations in VSV-G were incorporated at K47 and R354 (Figure 28A). The figure discloses SEQ ID NOs: 35, 35, 35, and 236, respectively, in order of appearance. Lentivirus production in HEK-293T cells is outlined in Figure 28B. Western blotting of lentiviral particles is shown in Figure 28C. [Figure 29A] Validation of retargeted lentivirus in receptor-positive cell lines is shown. Lentivirus pseudotyped with VSV-G-WT or VSV-G carrying the K47Q / R354Q (G-QQ) mutation and attached to EGFR scFv, EGFR-E11 scFv, or EGFm123 ligand was transduced into parental Jurkat or K562 cells, or modified versions of cells overexpressing EGFR. Fluorescence microscopy images were taken 48 hours post-transfection (hpt) for Jurkat cells (Figure 29A). K562 cells were transduced with the viral supernatant, and fluorescence microscopy images were taken at 24 hpt (Figure 29B). [Figure 29B]Validation of retargeted lentivirus in receptor-positive cell lines is shown. Lentivirus pseudotyped with VSV-G-WT or VSV-G carrying the K47Q / R354Q (G-QQ) mutation and attached to EGFR scFv, EGFR-E11 scFv, or EGFm123 ligand was transduced into parental Jurkat or K562 cells, or modified versions of cells overexpressing EGFR. Fluorescence microscopy images were taken 48 hours post-transfection (hpt) for Jurkat cells (Figure 29A). K562 cells were transduced with the viral supernatant, and fluorescence microscopy images were taken at 24 hpt (Figure 29B). [Figure 30A] We demonstrate that EGFR-retargeting lentiviral vectors are less sensitive to serum and LDL inhibition. A fixed volume of GFP-expressing lentiviral vector pseudotyped with WT VSV-G (WT-G) or VSV-G harboring the K47Q / R354Q mutation and appended to EGFm123 (EGFm123 K47Q / R354Q) was used to transduce either parental K562 cells or modified EGFR-overexpressing K562 cells (K562-EGFR). Transduction was performed in the presence of medium alone, pooled human serum (complement-deficient), pooled lipoprotein-depleted human serum, or pooled lipoprotein-depleted human serum spiked with 150 mg / dL LDL. After 40 hours, cells were imaged both by fluorescence microscopy (Figure 30A) and imaging cytometer. The number of GFP-positive cells per well was determined using the cytometer software (Figure 30B). [Figure 30B]We demonstrate that EGFR-retargeting lentiviral vectors are less sensitive to serum and LDL inhibition. A fixed volume of GFP-expressing lentiviral vector pseudotyped with WT VSV-G (WT-G) or VSV-G harboring the K47Q / R354Q mutation and appended to EGFm123 (EGFm123 K47Q / R354Q) was used to transduce either parental K562 cells or modified EGFR-overexpressing K562 cells (K562-EGFR). Transduction was performed in the presence of medium alone, pooled human serum (complement-deficient), pooled lipoprotein-depleted human serum, or pooled lipoprotein-depleted human serum spiked with 150 mg / dL LDL. After 40 hours, cells were imaged both by fluorescence microscopy (Figure 30A) and imaging cytometer. The number of GFP-positive cells per well was determined using the cytometer software (Figure 30B). [Figure 31A] This shows that the VSV-GH8Q / K47Q / Y209Q / R354Q mutations enhanced the specificity of retargeted VSV with EGFm123. The construct design containing the LDLR-blinding mutations in VSV-G is shown in Figure 31A. For the data generated in Figures 31B-31C, K562 parental cells or K562-EGFR cells were infected with VSV-EGFm123 virus, and GFP images were acquired using a fluorescent microscope (Figure 31A, left panel). Quantification of GFP-positive cells by imaging cytometer is shown in the right panel of Figure 31A. GFP images captured by the imaging cytometer are shown in Figure 31D. These results indicated that combining the Y209Q mutation with VSV-G K47Q / R354Q enhanced the specificity of retargeted VSV expressing EGFm123, while combining the H8Q mutation with VSV-G K47Q / R354Q had little or no effect on enhancing retargeting specificity. [Figure 31B]This shows that the VSV-GH8Q / K47Q / Y209Q / R354Q mutations enhanced the specificity of retargeted VSV with EGFm123. The construct design containing the LDLR-blinding mutations in VSV-G is shown in Figure 31A. For the data generated in Figures 31B-31C, K562 parental cells or K562-EGFR cells were infected with VSV-EGFm123 virus, and GFP images were acquired using a fluorescent microscope (Figure 31A, left panel). Quantification of GFP-positive cells by imaging cytometer is shown in the right panel of Figure 31A. GFP images captured by the imaging cytometer are shown in Figure 31D. These results indicated that combining the Y209Q mutation with VSV-G K47Q / R354Q enhanced the specificity of retargeted VSV expressing EGFm123, while combining the H8Q mutation with VSV-G K47Q / R354Q had little or no effect on enhancing retargeting specificity. [Figure 31C] This shows that the VSV-GH8Q / K47Q / Y209Q / R354Q mutations enhanced the specificity of retargeted VSV with EGFm123. The construct design containing the LDLR-blinding mutations in VSV-G is shown in Figure 31A. For the data generated in Figures 31B-31C, K562 parental cells or K562-EGFR cells were infected with VSV-EGFm123 virus, and GFP images were acquired using a fluorescent microscope (Figure 31A, left panel). Quantification of GFP-positive cells by imaging cytometer is shown in the right panel of Figure 31A. GFP images captured by the imaging cytometer are shown in Figure 31D. These results indicated that combining the Y209Q mutation with VSV-G K47Q / R354Q enhanced the specificity of retargeted VSV expressing EGFm123, while combining the H8Q mutation with VSV-G K47Q / R354Q had little or no effect on enhancing retargeting specificity. [Figure 31D]This shows that the VSV-GH8Q / K47Q / Y209Q / R354Q mutations enhanced the specificity of retargeted VSV with EGFm123. The construct design containing the LDLR-blinding mutations in VSV-G is shown in Figure 31A. For the data generated in Figures 31B-31C, K562 parental cells or K562-EGFR cells were infected with VSV-EGFm123 virus, and GFP images were acquired using a fluorescent microscope (Figure 31A, left panel). Quantification of GFP-positive cells by imaging cytometer is shown in the right panel of Figure 31A. GFP images captured by the imaging cytometer are shown in Figure 31D. These results indicated that combining the Y209Q mutation with VSV-G K47Q / R354Q enhanced the specificity of retargeted VSV expressing EGFm123, while combining the H8Q mutation with VSV-G K47Q / R354Q had little or no effect on enhancing retargeting specificity. [Figure 32A] We demonstrate that deletion of the K47 residue in VSV-G eliminates VSV tropism and redirects VSV to EGF receptor (EGFR)-positive cells. K562 parental cells or K562-EGFR were infected with VSV-GFP or EGFm123, representing G-WT or G-ΔK47 VSV, and fluorescence microscopy images were acquired (Figure 32A, left panel). Quantification of GFP-positive cells by imaging cytometry is shown in Figure 32A, right panel. An example of a GFP image captured by imaging cytometry is shown in Figure 32B. Additional constructs containing deletions of residues H8, K47, Y209, and / or R354, intended to impair VSV LDLR tropism, are shown in Figure 32C. Further examples of constructs containing double-deletion mutations are shown in Figure 32D. [Figure 32B]We demonstrate that deletion of the K47 residue in VSV-G eliminates VSV tropism and redirects VSV to EGF receptor (EGFR)-positive cells. K562 parental cells or K562-EGFR were infected with VSV-GFP or EGFm123, representing G-WT or G-ΔK47 VSV, and fluorescence microscopy images were acquired (Figure 32A, left panel). Quantification of GFP-positive cells by imaging cytometry is shown in Figure 32A, right panel. An example of a GFP image captured by imaging cytometry is shown in Figure 32B. Additional constructs containing deletions of residues H8, K47, Y209, and / or R354, intended to impair VSV LDLR tropism, are shown in Figure 32C. Further examples of constructs containing double-deletion mutations are shown in Figure 32D. [Figure 32C] We demonstrate that deletion of the K47 residue in VSV-G eliminates VSV tropism and redirects VSV to EGF receptor (EGFR)-positive cells. K562 parental cells or K562-EGFR were infected with VSV-GFP or EGFm123, representing G-WT or G-ΔK47 VSV, and fluorescence microscopy images were acquired (Figure 32A, left panel). Quantification of GFP-positive cells by imaging cytometry is shown in Figure 32A, right panel. An example of a GFP image captured by imaging cytometry is shown in Figure 32B. Additional constructs containing deletions of residues H8, K47, Y209, and / or R354, intended to impair VSV LDLR tropism, are shown in Figure 32C. Further examples of constructs containing double-deletion mutations are shown in Figure 32D. [Figure 32D]We demonstrate that deletion of the K47 residue in VSV-G eliminates VSV tropism and redirects VSV to EGF receptor (EGFR)-positive cells. K562 parental cells or K562-EGFR were infected with VSV-GFP or EGFm123, representing G-WT or G-ΔK47 VSV, and fluorescence microscopy images were acquired (Figure 32A, left panel). Quantification of GFP-positive cells by imaging cytometry is shown in Figure 32A, right panel. An example of a GFP image captured by imaging cytometry is shown in Figure 32B. Additional constructs containing deletions of residues H8, K47, Y209, and / or R354, intended to impair VSV LDLR tropism, are shown in Figure 32C. Further examples of constructs containing double-deletion mutations are shown in Figure 32D. [Figure 33-1] Figure 1 shows sequence confirmation of VSV-G-ΔK47 residue in VSV1-409-0. The viral sequence shows the point mutation at F405I in VSV-G. The figure discloses SEQ ID NOs: 213 to 226, respectively, in order of appearance. [Figure 33-2] Same as above. [Figure 34A-1] Figure 34A shows the targeting by alternative rhabdovirus G protein. Figure 34A shows the selection of nine glycoproteins from nine different rhabdovirus genera (arrows). FLAV-G was selected as the lead for targeting. VSV containing a GFP reporter gene and FLAV GΔ30 carrying scFv targeting EGFR or HER2 were used to infect a panel of K562 cells that stably expressed (or did not express) EGFR or HER2. Cells were imaged using a fluorescent microscope (Figure 34B). [Figure 34A-2] Same as above. [Figure 34A-3] Same as above. [Figure 34A-4] Same as above. [Figure 34B]Figure 34A shows the targeting by alternative rhabdovirus G protein. Figure 34A shows the selection of nine glycoproteins from nine different rhabdovirus genera (arrows). FLAV-G was selected as the lead for targeting. VSV containing a GFP reporter gene and FLAV GΔ30 carrying scFv targeting EGFR or HER2 were used to infect a panel of K562 cells that stably expressed (or did not express) EGFR or HER2. Cells were imaged using a fluorescent microscope (Figure 34B). [Figure 35-1] Screening of additional alternative G proteins is shown. Twenty additional viral glycoproteins were selected for screening to identify glycoproteins capable of retargeting receptors of interest. A description of the viral species in the genus Vesiculovirus of the family Rhabdoviridae is shown in the table (Figure 35, left panel). Percent amino acid sequence identity to Vesicular Stomatitis Indiana virus (VSIV) (see, e.g., SEQ ID NO: 8) was calculated using Clustal Omega, with or without the signal peptide sequence. In the first stage of screening, additional glycoproteins from the genera Ledantevirus, Hapavirus, and Ephemelovirus were selected based on the favorable properties of Fukuoka (Ledantevirus), Flanders (Hapavirus), and Bovine Ephemeral Fever (Ephemerovirus) virus glycoproteins (Figure 35, right panel). [Figure 35-2] Same as above. [Figure 35-3] Same as above. [Figure 35-4] Same as above. [Figure 36-1]Screening of additional non-VSV rhabdovirus G proteins using an alternative format is shown. Twenty additional viral glycoproteins were selected for screening to identify glycoproteins capable of retargeting receptors of interest and potentially resistant to complement inactivation (Figure 36, left panel). Virus species from the genus Vesiculovirus in the family Rhabdoviridae were selected based on their percent amino acid sequence identity of less than 70% with Vesicular Stomatitis Indiana virus. In the first stage of screening, additional glycoproteins from the genera Redantevirus, Hapavirus, and Ephemelovirus were selected based on favorable properties of the Fukuoka (Redantevirus), Flanders (Hapavirus), and Bovine Ephemeral Fever (Ephemelovirus) virus glycoproteins. For each glycoprotein, a signal peptide sequence was predicted using SignalP 6.0, and a modified epidermal growth factor (EGFm123) was inserted immediately after the signal peptide sequence. These modified glycoprotein genes were synthesized and subcloned into a protein expression vector (pCG) (Figure 36, right panel). [Figure 36-2] Same as above. [Figure 36-3] Same as above. [Figure 36-4] Same as above. [Figure 37-1] Figure 37 shows the functional screening of alternative EGF-presenting rhabdovirus G protein for EGFR targeting.The schematic diagram of DSP cell-cell fusion assay described herein is shown in the left panel of Figure 37.The measurement of the fusion activity of various glycoproteins described herein in SKOV3.ip1 cell and EGFR KO SKOV3.ip1 cell is shown in Figure 37, center panel.The bar graph (upper) shows the cell surface expression of EGFm123-G protein measured by the median fluorescence intensity across EGFm123-G protein plasmid and the Western blot analysis (lower) of EGFm123 level for EGFm123-G protein plasmid is shown in Figure 37, right panel. [Figure 37-2] Same as above. [Figure 37-3] Same as above. [Figure 37-4] Same as above. [Figure 38] Pseudotyping of lentiviruses with targeting G proteins is shown. Lentiviruses pseudotyped with a panel of targeting rhabdovirus glycoproteins (or non-targeting VSV G WT and EGFR-targeting FLAV-GΔ30 as controls) were generated by expressing a glycoprotein vector (pCG), a packaging plasmid (p8.91), and a GFP-expressing genome vector (pLV-SFFV-GFP) in HEK293T cells (Figure 38, left panel). The lentiviral supernatant was then added to a monolayer of SKOV3.ip1 or SKOV3.ip1 EGFR KO cells. The number of GFP-positive cells was determined using an imaging cytometer (Figure 38, right panel). [Figure 39] 1 shows a description of various features of the double split protein (DSP) screen described herein and the lentiviral screen described herein. [Figure 40] 1 shows an exemplary experimental time course of the VSV-G EGFR scFv transfection screen described herein. [Figure 41] 1 provides an example protocol for the EGFR scFv DSP transfection assay screen described herein. [Figure 42] 1 shows a flow chart of an exemplary analysis for EGFR scFv transfection screening. [Figure 43] 1 demonstrates that EGFR scFv screening in the SKOV3ip.1 DSP screen identified several scFv sequences with improved fusions. [Figure 44] Figure 1 shows that αEGFR-VSV-G exhibits a moderate preference for scFv orientation. [Figure 45] FIG. 1 is a schematic diagram of orientation preferences showing increased function when the scFv is N-terminal to the extracellular domain. [Figure 46] 1 shows an exemplary αEGFR lentivirus production protocol described herein. [Figure 47]1 shows a description of αEGFR titration by qPCR. [Figure 48] 1 shows a flow chart of an exemplary lentiviral screen described herein. [Figure 49] We show that several lentiviruses, including αEGFR scFv-VSV-G(QQ), demonstrate enhanced transduction efficiency in the presence of serum. [Figure 50] 1 shows a depiction of the transduction efficacy of exemplary example αEGFR scFv candidates. [Figure 51] Figure 1 shows a comparison of DSP and lentiviral screening for the αEGFR-VSV G construct, including comparison of scFv sequences using Multiple Sequence Comparison by Log-Expectation (MUSCLE) and graphical representation in a phylogenetic tree aligned using Jalvuew software and the BLOSUM62 neighbor algorithm. [Figure 52] Figure 1 shows the design of VSV constructs containing various linker sequences. Retargeted VSVs are included, each containing a 20-aa linker between the proteolytically cleaved EGFR scFv and VSV-G. Different linker sequences of variable length were cloned between the EGFR scFv and VSV-G. EIK, terminal amino acid sequence of the scFv; KFT, starting amino acid sequence of VSV-G. Linker sequences are indicated in red. F, flexible linker; R, rigid linker; Fm, moderately flexible linker; F-el, flexible elastin-like linker; IgG4 h, IgG4 hinge. VSV-G contains the K47Q and R354Q blinding mutations in the LDLR. The figure discloses SEQ ID NOs: 171-172, 36-39, 3, 173, and 40-41, respectively, in order of appearance. [Figure 53] This figure shows the rescue of EGFR-targeted VSV using an alternative linker sequence. The pVSV-MC11-EGFRscFv-VSV-G-GFP plasmid with an alternative linker sequence was rescued in SKOV3.ip1 cells using a vaccinia-based rescue system. The viral supernatant was collected and filtered (p0 supernatant). The p0 supernatant was then transferred to a monolayer of Vero-EGFR cells, and the GFP fluorescence microscope images shown in the figure were obtained. [Figure 54A] Characteristics of EGFR-targeted VSV are shown. The amplification steps, virus titers, and sequencing results are listed in the table shown in Figure 54A. Western blotting of virions is shown in Figure 54B. Bands represent intact EGFR scFv along with G and proteolytically cleaved G. The specificity of EGFR-targeted VSV is shown in Figure 54C. Photomicrographs show K562 parental or K562-EGFR after infection with EGFR-targeted VSV along with VSV-GFP, and fluorescent photomicrographs were taken. [Figure 54B] Characteristics of EGFR-targeted VSV are shown. The amplification steps, virus titers, and sequencing results are listed in the table shown in Figure 54A. Western blotting of virions is shown in Figure 54B. Bands represent intact EGFR scFv along with G and proteolytically cleaved G. The specificity of EGFR-targeted VSV is shown in Figure 54C. Photomicrographs show K562 parental or K562-EGFR after infection with EGFR-targeted VSV along with VSV-GFP, and fluorescent photomicrographs were taken. [Figure 54C] Characteristics of EGFR-targeted VSV are shown. The amplification steps, virus titers, and sequencing results are listed in the table shown in Figure 54A. Western blotting of virions is shown in Figure 54B. Bands represent intact EGFR scFv along with G and proteolytically cleaved G. The specificity of EGFR-targeted VSV is shown in Figure 54C. Photomicrographs show K562 parental or K562-EGFR after infection with EGFR-targeted VSV along with VSV-GFP, and fluorescent photomicrographs were taken. [Figure 55-1]We demonstrate that the 18aaL(F), 15aaL(R), and 16aaL(R) linkers exhibit proteolytic cleavage. To determine which linker provides specific targeting to EGFR, nine different linkers were selected for attachment to the EGFR scFv (Figure 55, left panel). The linker sequences were generated in a VSV backbone and then subcloned into a pCG vector backbone for use in lentivirus production. The constructs were transfected into HEK293T cells, and cell lysates were collected and run on an SDS-PAGE gel to analyze protein expression (Figure 55, right panel). The figure discloses SEQ ID NOs: 166-167, 26-30, 168, and 31-32, respectively, in order of appearance. [Figure 55-2] Same as above. [Figure 56] Figure 1 shows that the 18aaL(F), 15aaL(R), and 16aaL(R) linkers exhibit fusion activity. The fusion activity of nine constructs containing EGFR scFvs with variable linkers was measured in a mixed population of A549-EGFR expressing DSP1-7 and DSP8-11 cells. Half contain a double split protein (DSP) reporter gene with split GFP, and half contain Renilla luciferase. [Figure 57A]Figure 57A shows that the 18aaL(F), 15aaL(R), and 16aaL(R) linkers exhibit proteolytic cleavage in lentivirus-producing cell lysates and in lentivirus particles themselves. Figure 57A shows a schematic diagram of EGFR scFv with variable linker virus production. Lentivirus production plasmids were transfected into HEK293T cells to produce lentivirus. Western blots of cell lysates from the cells used to produce lentivirus were performed to detect EGFR scFv-linker-VSV-GQQ (full-length) or cleaved VSV-GQQ (Figure 57B, left panel). The supernatant containing the viral particles was collected and precipitated in a microcentrifuge. The supernatant was aspirated, and the cell pellet was lysed and reduced at 95°C for 5 minutes, then run on an SDS-PAGE gel to detect EGFR scFv-linker-VSV-GQQ (full-length) or cleaved VSV-GQQ (Figure 57B, center panel). Also included are titers of each virus as determined by p24 enzyme-linked immunosorbent assay (ELISA). [Figure 57B-1] Figure 57A shows that the 18aaL(F), 15aaL(R), and 16aaL(R) linkers exhibit proteolytic cleavage in lentivirus-producing cell lysates and in lentivirus particles themselves. Figure 57A shows a schematic diagram of EGFR scFv with variable linker virus production. Lentivirus production plasmids were transfected into HEK293T cells to produce lentivirus. Western blots of cell lysates from the cells used to produce lentivirus were performed to detect EGFR scFv-linker-VSV-GQQ (full-length) or cleaved VSV-GQQ (Figure 57B, left panel). The supernatant containing the viral particles was collected and precipitated in a microcentrifuge. The supernatant was aspirated, and the cell pellet was lysed and reduced at 95°C for 5 minutes, then run on an SDS-PAGE gel to detect EGFR scFv-linker-VSV-GQQ (full-length) or cleaved VSV-GQQ (Figure 57B, center panel). Also included are titers of each virus as determined by p24 enzyme-linked immunosorbent assay (ELISA). [Figure 57B-2] Same as above. [Figure 58A]Figure 58A shows that the 18aaL(F), 15aaL(R), and 16aaL(R) linkers, which exhibit proteolytic cleavage, demonstrate increased specificity for targeting K562-EGFR-expressing cells. K562 parental cells and K562-EGFR-expressing cells were transduced with each lentivirus, and GFP fluorescence microscopy and bright-field (BF) images were captured after transduction to determine the specificity of each retargeted lentivirus with different linkers (Figure 58A). A graph of the number of GFP-positive cells quantified in K562 and K562-EGFR cells transduced with each lentivirus is shown in Figure 58B. [Figure 58B] Figure 58A shows that the 18aaL(F), 15aaL(R), and 16aaL(R) linkers, which exhibit proteolytic cleavage, demonstrate increased specificity for targeting K562-EGFR-expressing cells. K562 parental cells and K562-EGFR-expressing cells were transduced with each lentivirus, and GFP fluorescence microscopy and bright-field (BF) images were captured after transduction to determine the specificity of each retargeted lentivirus with different linkers (Figure 58A). A graph of the number of GFP-positive cells quantified in K562 and K562-EGFR cells transduced with each lentivirus is shown in Figure 58B. [Figure 59] Targeting constructs used to assess virus specificity in the presence or absence of serum are shown. The presented ligands included human epidermal growth factor (EGF), human stem cell factor (hSCF), and scFvs against EGFR or Her2. The hSCF ligand was presented on the blinded (VSV-G-QQ) G protein, which poorly interacted with the LDLR, or the unblinded (VSV-G-WT) G protein. [Figure 60]Figure 59 shows fluorescent microscopy images of parental or stably expressing HER2, cKit (SCF receptor), or EGFR K562 cells infected with recombinant VSV incorporating the G protein shown in Figure 59 in the absence (left panel) or presence (right panel) of 25% heat-inactivated human serum. Infected cell monolayers were imaged under blue light 42 hours post-infection (hpi) (24 hpi for VSV-GFP with unmodified G protein). The left panel shows that ligand-presenting viruses specifically infected only target cells bearing the cognate receptor for their presented ligand. This was also evident for SCF-presenting viruses in which the G protein had not been mutated to remove LDLR tropism, indicating that the presented SCF domain may sterically interfere with G protein-LDLR interaction. The right panel shows that in the presence of 25% heat-inactivated human serum, entry of viruses with wild-type G proteins was blocked in all K562 clones, whereas entry of viruses targeted through alternative (i.e., non-LDLR) receptors was not blocked and in some cases could be enhanced. [Figure 61] We present proof-of-concept data supporting the retargeting of G-pseudotyped lentiviral vectors via presented domains containing EGFm123 and anti-epidermal growth factor receptor (EGFR) scFv (E11). [Figure 62] We demonstrate that increasing the amount of VSV G-QQ DNA proportionally increases the amount of VSV G-QQ protein. The first part of the figure shows a diagrammatic version of the lentivirus production protocol, along with a table with the amount of envelope glycoprotein DNA used. The bottom right contains a Western blot of VSV G (from the uncleaved chimera and VSV G-QQ). A rabbit anti-VSV G[8G5F11] antibody was used to probe the protein within the virion pellet. [Figure 63A]Figure 63A shows that increasing the ratio of VSV G-QQ to uncleaved EGFR scFv-17aaL(F)-VSV G-QQ improves targeting specificity. In Figure 63A, the upper panel shows transduction of K562 and K562-EGFR-expressing cells with lentiviruses containing a mixed ratio of EGFR scFv-17aaL(F)-VSV G-QQ and VSV G-QQ. Viruses were transduced at a multiplicity of infection (MOI) of 5. Brightfield (Figure 63A, lower panel) and GFP (Figure 63A, upper panel) expression of each lentivirus was imaged 72 hours post-transduction (hpt). The amount of DNA corresponding to each ratio listed at the top of the panel set is shown in the table below all panels. The first number indicates the amount of VSV G-QQ DNA used, and the second number indicates the amount of EGFR scFv-17aaL(F)-VSV G-QQ DNA used. Decreasing the amount of EGFR scFv-17aaL(F)-VSV G-QQ DNA and increasing the amount of VSV G-QQ DNA resulted in improved targeting specificity. Celigo quantification of the number of GFP-positive cells for each virus mixture ratio is shown in Figure 63B. [Figure 63B]Figure 63A shows that increasing the ratio of VSV G-QQ to uncleaved EGFR scFv-17aaL(F)-VSV G-QQ improves targeting specificity. In Figure 63A, the upper panel shows transduction of K562 and K562-EGFR-expressing cells with lentiviruses containing a mixed ratio of EGFR scFv-17aaL(F)-VSV G-QQ and VSV G-QQ. Viruses were transduced at a multiplicity of infection (MOI) of 5. Brightfield (Figure 63A, lower panel) and GFP (Figure 63A, upper panel) expression of each lentivirus was imaged 72 hours post-transduction (hpt). The amount of DNA corresponding to each ratio listed at the top of the panel set is shown in the table below all panels. The first number indicates the amount of VSV G-QQ DNA used, and the second number indicates the amount of EGFR scFv-17aaL(F)-VSV G-QQ DNA used. Decreasing the amount of EGFR scFv-17aaL(F)-VSV G-QQ DNA and increasing the amount of VSV G-QQ DNA resulted in improved targeting specificity. Celigo quantification of the number of GFP-positive cells for each virus mixture ratio is shown in Figure 63B. [Figure 64] Possible mixed trimer models for uncleaved retargeted VSV G and blinded VSV G are shown. The model on the left shows a homotrimeric VSV G. Each monomer contains an EGFR scFv retargeting domain, a linker, and blinded VSV G (mutations at K47Q and R354Q-QQ). For lentiviruses that are not specifically targeted or cleaved initially, mixing uncleaved EGFR scFv retargeting monomers with blinded VSV G-QQ can result in heterotrimers that allow targeting specificity. [Figure 65A]Figure 65A shows the in vivo targeting of EGFR+ (epidermal growth factor receptor-positive) tumors by intravenously (iv) administered VSV expressing EGF (epidermal growth factor) in a SCID (severe combined immunodeficiency) mouse model. In the study design (Figure 65A), female CB17 SCID mice were subcutaneously implanted with mouse myeloma 5TGM1 cells expressing human EGF receptor (hEGFR). When the mean tumor volume was approximately 150 mm3, the mice were randomized and divided into groups. Mice were intravenously treated with either saline or 1 x 108 TCID50 VSV-M-RFP-GFP or 1 x 108 TCID50 VSV-M-GqqEGFm123-GFP virus. Mice were observed for adverse clinical signs, body weight, and tumor growth until D40 (day 40) after treatment. The 5TGM1-hEGFR+ tumor growth profile is shown in Figure 65B. Mice treated with VSV-M-RFP-GFP developed adverse clinical signs and were either found dead or sacrificed. By D21 post-infection, no mice were alive (Figures 65B-C). In contrast to mice treated with VSV-M-RFP-GFP, the VSV-M-GqqEGFm123-GFP virus completely blocked tumor growth in all six treated mice. None of the mice developed adverse clinical signs (Figures 65B-C). [Figure 65B]Figure 65A shows the in vivo targeting of EGFR+ (epidermal growth factor receptor-positive) tumors by intravenously (iv) administered VSV expressing EGF (epidermal growth factor) in a SCID (severe combined immunodeficiency) mouse model. In the study design (Figure 65A), female CB17 SCID mice were subcutaneously implanted with mouse myeloma 5TGM1 cells expressing human EGF receptor (hEGFR). When the mean tumor volume was approximately 150 mm3, the mice were randomized and divided into groups. Mice were intravenously treated with either saline or 1 x 108 TCID50 VSV-M-RFP-GFP or 1 x 108 TCID50 VSV-M-GqqEGFm123-GFP virus. Mice were observed for adverse clinical signs, body weight, and tumor growth until D40 (day 40) after treatment. The 5TGM1-hEGFR+ tumor growth profile is shown in Figure 65B. Mice treated with VSV-M-RFP-GFP developed adverse clinical signs and were either found dead or sacrificed. By D21 post-infection, no mice were alive (Figures 65B-C). In contrast to mice treated with VSV-M-RFP-GFP, the VSV-M-GqqEGFm123-GFP virus completely blocked tumor growth in all six treated mice. None of the mice developed adverse clinical signs (Figures 65B-C). [Figure 65C]Figure 65A shows the in vivo targeting of EGFR+ (epidermal growth factor receptor-positive) tumors by intravenously (iv) administered VSV expressing EGF (epidermal growth factor) in a SCID (severe combined immunodeficiency) mouse model. In the study design (Figure 65A), female CB17 SCID mice were subcutaneously implanted with mouse myeloma 5TGM1 cells expressing human EGF receptor (hEGFR). When the mean tumor volume was approximately 150 mm3, the mice were randomized and divided into groups. Mice were intravenously treated with either saline or 1 x 108 TCID50 VSV-M-RFP-GFP or 1 x 108 TCID50 VSV-M-GqqEGFm123-GFP virus. Mice were observed for adverse clinical signs, body weight, and tumor growth until D40 (day 40) after treatment. The 5TGM1-hEGFR+ tumor growth profile is shown in Figure 65B. Mice treated with VSV-M-RFP-GFP developed adverse clinical signs and were either found dead or sacrificed. By D21 post-infection, no mice were alive (Figures 65B-C). In contrast to mice treated with VSV-M-RFP-GFP, the VSV-M-GqqEGFm123-GFP virus completely blocked tumor growth in all six treated mice. None of the mice developed adverse clinical signs (Figures 65B-C). [Figure 66A]Optimization of the KRV-G cytoplasmic tail. Fusion activity of KRV-G cytoplasmic tail truncation mutants. SKOV3.ip1 cells stably transduced with either the N- or C-terminal half of a dual-split protein GFP-Renilla luciferase reporter were seeded in equal proportions. The following day, cells were transfected with plasmids expressing VSV-G WT or EGFm123-modified (at the N-terminus), FLAV-G with a 30-amino acid deletion at the C-terminus (Δ30), or KRV-G with cytoplasmic tail truncations (e.g., 10-amino acid deletion (Δ10), 20-amino acid deletion (Δ20), or 30-amino acid deletion (Δ30)). The day after transfection, cells were treated with phosphate-buffered saline (PBS) at pH 5.0 or pH 7.4 (as a control) for 2 min, and then the saline was replaced with fresh medium containing EnduRen substrate. The resulting luminescence was measured after 2 hours (Figure 66A). GFP-encoding VSV carrying KRV-G or KRV-G with 10 C-terminal amino acids deleted (Δ10) and N-terminally modified with an EGFm123 molecule were rescued and amplified. The infectious titers of the resulting viruses were determined by TCID50 assay (Figure 66B). The specificity of full-length or Δ10 KRV-G was determined by monitoring the infection course of recombinant VSV carrying these EGFm123-tagged glycoproteins and green fluorescent protein (GFP) (Figures 66C-66D). The recombinant viruses were used to infect wild-type (WT) or EGF receptor (EGFR) knockout (KO) versions of SKOV3.ip1 (Figure 66C) or HEK293T (Figure 66D) cells at an MOI of 1. One day postinfection, the number of infected cells expressing the virus-encoded GFP was measured using a Celigo image cytometer. [Figure 66B]Optimization of the KRV-G cytoplasmic tail. Fusion activity of KRV-G cytoplasmic tail truncation mutants. SKOV3.ip1 cells stably transduced with either the N- or C-terminal half of a dual-split protein GFP-Renilla luciferase reporter were seeded in equal proportions. The following day, cells were transfected with plasmids expressing VSV-G WT or EGFm123-modified (at the N-terminus), FLAV-G with a 30-amino acid deletion at the C-terminus (Δ30), or KRV-G with cytoplasmic tail truncations (e.g., 10-amino acid deletion (Δ10), 20-amino acid deletion (Δ20), or 30-amino acid deletion (Δ30)). The day after transfection, cells were treated with phosphate-buffered saline (PBS) at pH 5.0 or pH 7.4 (as a control) for 2 min, and then the saline was replaced with fresh medium containing EnduRen substrate. The resulting luminescence was measured after 2 hours (Figure 66A). GFP-encoding VSV carrying KRV-G or KRV-G with 10 C-terminal amino acids deleted (Δ10) and N-terminally modified with an EGFm123 molecule were rescued and amplified. The infectious titers of the resulting viruses were determined by TCID50 assay (Figure 66B). The specificity of full-length or Δ10 KRV-G was determined by monitoring the infection course of recombinant VSV carrying these EGFm123-tagged glycoproteins and green fluorescent protein (GFP) (Figures 66C-66D). The recombinant viruses were used to infect wild-type (WT) or EGF receptor (EGFR) knockout (KO) versions of SKOV3.ip1 (Figure 66C) or HEK293T (Figure 66D) cells at an MOI of 1. One day postinfection, the number of infected cells expressing the virus-encoded GFP was measured using a Celigo image cytometer. [Figure 66C]Optimization of the KRV-G cytoplasmic tail. Fusion activity of KRV-G cytoplasmic tail truncation mutants. SKOV3.ip1 cells stably transduced with either the N- or C-terminal half of a dual-split protein GFP-Renilla luciferase reporter were seeded in equal proportions. The following day, cells were transfected with plasmids expressing VSV-G WT or EGFm123-modified (at the N-terminus), FLAV-G with a 30-amino acid deletion at the C-terminus (Δ30), or KRV-G with cytoplasmic tail truncations (e.g., 10-amino acid deletion (Δ10), 20-amino acid deletion (Δ20), or 30-amino acid deletion (Δ30)). The day after transfection, cells were treated with phosphate-buffered saline (PBS) at pH 5.0 or pH 7.4 (as a control) for 2 min, and then the saline was replaced with fresh medium containing EnduRen substrate. The resulting luminescence was measured after 2 hours (Figure 66A). GFP-encoding VSV carrying KRV-G or KRV-G with 10 C-terminal amino acids deleted (Δ10) and N-terminally modified with an EGFm123 molecule were rescued and amplified. The infectious titers of the resulting viruses were determined by TCID50 assay (Figure 66B). The specificity of full-length or Δ10 KRV-G was determined by monitoring the infection course of recombinant VSV carrying these EGFm123-tagged glycoproteins and green fluorescent protein (GFP) (Figures 66C-66D). The recombinant viruses were used to infect wild-type (WT) or EGF receptor (EGFR) knockout (KO) versions of SKOV3.ip1 (Figure 66C) or HEK293T (Figure 66D) cells at an MOI of 1. One day postinfection, the number of infected cells expressing the virus-encoded GFP was measured using a Celigo image cytometer. [Figure 66D]Optimization of the KRV-G cytoplasmic tail. Fusion activity of KRV-G cytoplasmic tail truncation mutants. SKOV3.ip1 cells stably transduced with either the N- or C-terminal half of a dual-split protein GFP-Renilla luciferase reporter were seeded in equal proportions. The following day, cells were transfected with plasmids expressing VSV-G WT or EGFm123-modified (at the N-terminus), FLAV-G with a 30-amino acid deletion at the C-terminus (Δ30), or KRV-G with cytoplasmic tail truncations (e.g., 10-amino acid deletion (Δ10), 20-amino acid deletion (Δ20), or 30-amino acid deletion (Δ30)). The day after transfection, cells were treated with phosphate-buffered saline (PBS) at pH 5.0 or pH 7.4 (as a control) for 2 min, and then the saline was replaced with fresh medium containing EnduRen substrate. The resulting luminescence was measured after 2 hours (Figure 66A). GFP-encoding VSV carrying KRV-G or KRV-G with 10 C-terminal amino acids deleted (Δ10) and N-terminally modified with an EGFm123 molecule were rescued and amplified. The infectious titers of the resulting viruses were determined by TCID50 assay (Figure 66B). The specificity of full-length or Δ10 KRV-G was determined by monitoring the infection course of recombinant VSV carrying these EGFm123-tagged glycoproteins and green fluorescent protein (GFP) (Figures 66C-66D). The recombinant viruses were used to infect wild-type (WT) or EGF receptor (EGFR) knockout (KO) versions of SKOV3.ip1 (Figure 66C) or HEK293T (Figure 66D) cells at an MOI of 1. One day postinfection, the number of infected cells expressing the virus-encoded GFP was measured using a Celigo image cytometer. [Figure 67A]We demonstrate that epidermal growth factor receptor (EGFR)-targeted KRV-G and KEUV-G are more resistant to serum inhibitors and have comparable specificity to blinded VSV-G. GFP-encoding VSVs were generated with either the WT VSV glycoprotein (VSV-G) or one of the following modified glycoproteins replacing the native VSV-G: VSV-G containing LDLR receptor-targeting glutamine substitutions at K47 and R354 (QQ) or K47, Y209, and R354 (QQQ), Kumasi rhabdovirus G (KRV-G), KRV-G with the C-terminal 10 amino acids removed (Δ10), Keuraribavirus G (KEUV-G), Perinetovirus G (PERV-G), Piryvirus G (PIRYV-G), Fukuokavirus G (FUKV-G), or Cliopolisvirus G (CURV-G). The indicated viruses contained a modified epidermal growth factor (EGF) molecule added to the N-terminus of the encoded glycoprotein. The susceptibility of the modified VSV to complement-activating serum was determined by incubating 1 x 10 infectious units of VSV in OptiMEM medium alone, OptiMEM containing 50% heat-inactivated human serum, or OptiMEM containing 50% complement-activating serum at 37°C for 1 hour. The incubated virus was serially diluted and used to inoculate Vero cells stably expressing human EGFR in a standard TCID50 titration assay. Titration plates were scored 3 days after inoculation (Figure 67A). The specificity of VSV retargeted to EGFR using various rhabdovirus glycoproteins was determined by monitoring the infection course of a recombinant VSV encoding the GFP gene over a 3-day period. HT1080 cells (WT) or HT0180 cells (KO) in which the EGFR gene was knocked out were infected with the indicated VSV at an MOI of 1, and the number of infected cells expressing virus-encoded GFP was measured 1, 2, and 3 days after infection using a Celigo image cytometer (Figure 67B). [Figure 67B]We demonstrate that epidermal growth factor receptor (EGFR)-targeted KRV-G and KEUV-G are more resistant to serum inhibitors and have comparable specificity to blinded VSV-G. GFP-encoding VSVs were generated with either the WT VSV glycoprotein (VSV-G) or one of the following modified glycoproteins replacing the native VSV-G: VSV-G containing LDLR receptor-targeting glutamine substitutions at K47 and R354 (QQ) or K47, Y209, and R354 (QQQ), Kumasi rhabdovirus G (KRV-G), KRV-G with the C-terminal 10 amino acids removed (Δ10), Keuraribavirus G (KEUV-G), Perinetovirus G (PERV-G), Piryvirus G (PIRYV-G), Fukuokavirus G (FUKV-G), or Cliopolisvirus G (CURV-G). The indicated viruses contained a modified epidermal growth factor (EGF) molecule added to the N-terminus of the encoded glycoprotein. The susceptibility of the modified VSV to complement-activating serum was determined by incubating 1 x 10 infectious units of VSV in OptiMEM medium alone, OptiMEM containing 50% heat-inactivated human serum, or OptiMEM containing 50% complement-activating serum at 37°C for 1 hour. The incubated virus was serially diluted and used to inoculate Vero cells stably expressing human EGFR in a standard TCID50 titration assay. Titration plates were scored 3 days after inoculation (Figure 67A). The specificity of VSV retargeted to EGFR using various rhabdovirus glycoproteins was determined by monitoring the infection course of a recombinant VSV encoding the GFP gene over a 3-day period. HT1080 cells (WT) or HT0180 cells (KO) in which the EGFR gene was knocked out were infected with the indicated VSV at an MOI of 1, and the number of infected cells expressing virus-encoded GFP was measured 1, 2, and 3 days after infection using a Celigo image cytometer (Figure 67B). [Figure 68A]This shows that the Coomassie rhabdovirus glycoprotein (KRV-G) can be retargeted to EGFR. The infection course of a recombinant VSV encoding the GFP gene was monitored over a 3-day period. For the virus shown in Figure 68A, the glycoprotein gene was replaced with KRV-G (Δ10), in which 10 amino acids from the C-terminus were deleted. Where indicated, a targeting moiety (either a modified epidermal growth factor (EGF) or an scFv targeting the EGF receptor (EGFR)) was added to the N-terminus of KRV-G. SKOV3.ip1 cells (WT) or SKOV3.ip1 cells in which the EGFR gene was knocked out (KO) were infected with the indicated VSV at an MOI of 1, and the number of infected cells expressing the virus-encoded GFP was measured 1, 2, and 3 days postinfection using a Celigo image cytometer. For the viruses depicted in Figure 68B, the VSV-G gene was mutated to contain either two (QQ) or three (QQQ) mutations to ablate LDLR binding, or replaced with the glycoprotein from Coomassie rhabdovirus (KRV-G) (Δ10) with the C-terminal 10 amino acids deleted. Where indicated, a targeting moiety (either a modified epidermal growth factor (EGF), an scFv targeting the EGF receptor (EGFR), or an scFv targeting Her2) was appended to the N-terminus of KRV-G or VSV-G. SKOV3.ip1 cells (WT) or SKOV3.ip1 cells in which the EGFR gene had been knocked out (KO) were infected with the indicated KRV or VSV at an MOI of 1, and the number of infected cells expressing virus-encoded GFP was measured 1, 2, and 3 days postinfection using a Celigo image cytometer. [Figure 68B]This shows that the Coomassie rhabdovirus glycoprotein (KRV-G) can be retargeted to EGFR. The infection course of a recombinant VSV encoding the GFP gene was monitored over a 3-day period. For the virus shown in Figure 68A, the glycoprotein gene was replaced with KRV-G (Δ10), in which 10 amino acids from the C-terminus were deleted. Where indicated, a targeting moiety (either a modified epidermal growth factor (EGF) or an scFv targeting the EGF receptor (EGFR)) was added to the N-terminus of KRV-G. SKOV3.ip1 cells (WT) or SKOV3.ip1 cells in which the EGFR gene was knocked out (KO) were infected with the indicated VSV at an MOI of 1, and the number of infected cells expressing the virus-encoded GFP was measured 1, 2, and 3 days postinfection using a Celigo image cytometer. For the viruses depicted in Figure 68B, the VSV-G gene was mutated to contain either two (QQ) or three (QQQ) mutations to ablate LDLR binding, or replaced with the glycoprotein from Coomassie rhabdovirus (KRV-G) (Δ10) with the C-terminal 10 amino acids deleted. Where indicated, a targeting moiety (either a modified epidermal growth factor (EGF), an scFv targeting the EGF receptor (EGFR), or an scFv targeting Her2) was appended to the N-terminus of KRV-G or VSV-G. SKOV3.ip1 cells (WT) or SKOV3.ip1 cells in which the EGFR gene had been knocked out (KO) were infected with the indicated KRV or VSV at an MOI of 1, and the number of infected cells expressing virus-encoded GFP was measured 1, 2, and 3 days postinfection using a Celigo image cytometer. [Figure 69] This figure shows an example of the design of a retargeted mutant VSV-G construct. The VSV-G protein contains a signal peptide (SP) that is proteolytically cleaved after translation. The targeting molecule is a natural ligand, such as, but not limited to, EGFm123 (modified EGF), which can be added to the amino terminus of the G protein without a flexible linker. The LDLR-binding residues (H8, K47, R354, and Y209) are deleted in the G protein in either single, double, triple, or quadruple combinations. [Figure 70] Protein expression of VSV-G deletion mutant constructs is shown. Plasmid DNA constructs were transfected with lentiviral transfer and packaging constructs to produce lentivirus in HEK-293T cells. For comparison, triple or quadruple substitution mutants combined with G-QQ (K47QR354Q) were included in this experiment. After collection of lentiviral supernatant, cell lysates were collected 72 hours post-transfection and subsequently subjected to Western blotting with anti-VSV-G and anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase) antibodies. [Figure 71] We demonstrate the incorporation of VSV-G deletion mutants into lentiviral particles. Lentiviral supernatants collected 72 hours post-transfection were titrated by p24 ELISA. A total of 5 x 105 physical particles were lysed and loaded onto SDS-PAGE, followed by Western blotting with anti-VSV-G and anti-p24 antibodies. [Figure 72A] Screening of deletion mutants that ablate VSV-G tropism is shown. Lentiviral transduction on a panel of K562 cells is shown in Figures 72A-72B. K562 parental cells or K562-EGFR cells were transduced with the indicated retargeted lentiviruses presenting EGFm123 at an MOI of 20. Lentivirus pseudotyped with WT-G was used as a control. Celigo images (Figures 72A-72B) and Nikon images (Figure 72C) were taken 72 hours post-transduction, and transduced GFP-positive cells were quantified (Figure 72D). [Figure 72B] Screening of deletion mutants that ablate VSV-G tropism is shown. Lentiviral transduction on a panel of K562 cells is shown in Figures 72A-72B. K562 parental cells or K562-EGFR cells were transduced with the indicated retargeted lentiviruses presenting EGFm123 at an MOI of 20. Lentivirus pseudotyped with WT-G was used as a control. Celigo images (Figures 72A-72B) and Nikon images (Figure 72C) were taken 72 hours post-transduction, and transduced GFP-positive cells were quantified (Figure 72D). [Figure 72C]Screening of deletion mutants that ablate VSV-G tropism is shown. Lentiviral transduction on a panel of K562 cells is shown in Figures 72A-72B. K562 parental cells or K562-EGFR cells were transduced with the indicated retargeted lentiviruses presenting EGFm123 at an MOI of 20. Lentivirus pseudotyped with WT-G was used as a control. Celigo images (Figures 72A-72B) and Nikon images (Figure 72C) were taken 72 hours post-transduction, and transduced GFP-positive cells were quantified (Figure 72D). [Figure 72D] Screening of deletion mutants that ablate VSV-G tropism is shown. Lentiviral transduction on a panel of K562 cells is shown in Figures 72A-72B. K562 parental cells or K562-EGFR cells were transduced with the indicated retargeted lentiviruses presenting EGFm123 at an MOI of 20. Lentivirus pseudotyped with WT-G was used as a control. Celigo images (Figures 72A-72B) and Nikon images (Figure 72C) were taken 72 hours post-transduction, and transduced GFP-positive cells were quantified (Figure 72D). DETAILED DESCRIPTION OF THE INVENTION

[0197] Rhabdovirus glycoproteins are the only viral proteins found on the virion surface and mediate binding of viral particles to cellular receptors and subsequent viral entry into cells (infection). Vesicular stomatitis virus (VSV) glycoprotein (G) mediates infection of a wide variety of cell types from a wide range of species through interactions with the relatively ubiquitous low-density lipoprotein receptor (LDLR) and related receptor family members (Finkelshtein et al., 2013, Nikolic et al., 2018). VSV-G can be used to pseudotype lentiviruses for multiple therapeutic applications, including chimeric antigen receptor (CAR)-T cells, and several VSV-G-pseudotyped lentiviruses are in clinical trials (Munis et al., 2020).

[0198] CAR-T cells can be used to treat hematologic cancers. However, the logistical challenges associated with ex vivo production of CAR-T cells may be a limiting factor in the wider clinical adoption of this therapy. Currently, CAR-T cells are primarily engineered ex vivo using VSV-G pseudotyped lentiviral vectors to deliver the CAR to target T cells harvested from the patient. The modified CAR-T cells are then expanded ex vivo and infused back into the patient. In vivo CAR-T therapy involves delivering the CAR-encoding viral vector directly to the patient to achieve in-patient T cell modification and expansion, effectively overcoming many of the logistical challenges currently associated with CAR-T therapy. However, in vivo CAR-T therapy requires effective targeting of the CAR-encoding viral vector to T cells while limiting off-target effects. Because most candidate lentiviral vector therapies used with CAR-T cells are currently pseudotyped with VSV-G, solutions for effectively targeting VSV-G apply to both oncolytic VSV therapy and CAR-T cells.

[0199] In addition to proper targeting of VSV-G-based therapies, effective therapies must also circumvent natural barriers to infection long enough to reach target cells / tissues upon delivery into the body. Oncolytic VSV and VSV-G-pseudotyped lentiviral vector therapies can be inactivated by the complement system (DePolo et al., 2000; Mills and Cooper, 1978). Production of VSV or VSV-pseudotyped lentiviral vectors in cells expressing high levels of CD55 can significantly enhance virus / vector resistance to complement inactivation (Schauber-Plewa, C. et al., 2005; Johnson et al., 2012). However, as described in this disclosure, additional natural barriers that block oncolytic VSV or VSV-G-pseudotyped lentiviruses from binding to cells have been identified in human serum (blood). This barrier is ApoB-100-containing lipoproteins, which outcompete VSV-G for binding to the low-density lipoprotein receptor (LDLR), thereby preventing VSV-G-based therapies from being efficiently delivered to cells. Therefore, effective therapeutic delivery also requires overcoming or bypassing competition with ApoB-100-containing lipoproteins.

[0200] The disclosed recombinant fusion proteins have the advantage of significantly enhancing targeting to cellular receptors highly expressed in various cancers while also avoiding inhibition by LDL / VLDL during therapeutic delivery. Extensive data with different linker combinations highlight the importance of linker sequence and length in determining the function of retargeted G proteins and emphasize the need for appropriate linkers to deliver functional G-based therapeutics.

[0201] The present disclosure demonstrates exemplary VSV-G retargeting using LDLR blinding mutations (e.g., corresponding to positions H8, K47, Y209, and / or R354 in SEQ ID NO: 8) combined with scFv antibodies against EGFR or HER2, or nanobodies, as well as specific retargeting in the absence of blinding mutations using VSV-G with modified EGF (EGFm123) or stem cell factor (SCF). The distantly related rhabdovirus glycoprotein (FLAV-G) can be retargeted to EGFR and HER2, with HER2 retargeting requiring optimization of the linker sequence / length. The present disclosure can be expanded to include additional glycoproteins from the rhabdovirus family instead of VSV or FLAV-G, such as those detailed below. Receptors other than EGFR and HER2 can be targeted in a similar manner, for example, using scFv molecules or other ligands. While the data presented herein demonstrate the use of scFvs, nanobodies, and natural receptor ligands to target G, other targeting molecules, including, but not limited to, scFvs, affibodies, darpins, peptides, nanobodies, and natural or modified natural receptor ligands, can also be used. While the data presented herein primarily demonstrate VSV targeting using replication-competent viruses, a VSV platform in which the retargeted glycoprotein is not genome-encoded but rather provided in trans can also be used when a replicating vector is not desired. Furthermore, VSV glycoproteins can be used to pseudotype other viruses, including lentiviral vectors for gene therapy applications. In these applications, a retargeted rhabdovirus glycoprotein would be highly desirable to tailor therapeutic targeting to the cells of interest. To that end, the present disclosure demonstrates that the strategies used to specifically retarget VSV-G in the context of replicating VSV can be transferred to pseudotyped lentiviral vectors.

[0202] Importantly, the data described herein demonstrate that targeting the G protein not only achieved specific targeting to the cells of interest, but also circumvented a previously unrecognized natural barrier to infection: competition between VSV-G and LDL / VLDL for LDLR binding. Because LDL / VLDL exhibited a dose-dependent inhibitory response, an alternative approach to counteracting competition could be pretreatment of patients with LDL / VLDL-lowering medications to limit the amount of these competing molecules in the bloodstream at the time of treatment. However, such an alternative would require a delay in treatment long enough to allow blood LDL / VLDL levels to fall to a level that does not cause inhibition of the delivered therapy.

[0203] Further details of the compositions and methods of the present disclosure are described in various exemplary embodiments below.

[0204] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0205] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0206] The term "about" or "approximately" includes within a statistically significant range of a value. Such a range may be within an order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The permissible variations encompassed by the term "about" or "approximately" depend on the particular system under study and can be readily understood by one of ordinary skill in the art.

[0207] The term "antigen" refers to any agent (e.g., a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portion thereof, or combination thereof) that when introduced into a host, animal, or human (either directly or upon expression, e.g., a DNA vaccine) possesses the immune system, is recognized by the host's immune system, and can elicit or elicit an immune response.

[0208] The terms "viral element" and "viral component" are used herein to refer to viral genes (e.g., genes encoding polymerase or structural proteins) or other elements of the viral genome (e.g., packaging signals, regulatory elements, LTRs, ITRs, etc.).

[0209] The term " oncolytic virus " is used herein to refer to the virus that can infect and replicate in tumor cells, so that tumor cells can be killed.Oncolytic virus can also be capable of replication.By way of non-limiting example, oncolytic virus can comprise any of the virus groups that comprise rhabdovirus, that is, rhabdoviridae, such as vesicular stomatitis virus (VSV).

[0210] As used herein, the term "vesiculovirus" refers to any virus of the genus Vesiculovirus. Non-limiting examples of vesiculovirus include vesicular stomatitis virus (VSV) (e.g., VSV-New Jersey, VSV-Indiana), Alagoas vesiculovirus, Cocal vesiculovirus, Jurona vesiculovirus, Carajas vesiculovirus, Maraba vesiculovirus, Piry vesiculovirus, Calchaqui vesiculovirus, Yug Bogdanovac vesiculovirus, Isfahan vesiculovirus, Chandipura vesiculovirus, Perinct vesiculovirus, and Porton-S vesiculovirus. Vesiculovirus (VSV) of the genus Vesiculovirus is the prototype rhabdovirus. Although VSV is used as an example in this disclosure, the present disclosure may also be applied to other vesiculoviruses and other rhabdoviruses. There are two major serotypes of VSV, New Jersey and Indiana, both of which infect insects and mammals and can cause disease in cattle, horses, and pigs. The VSV genome consists of 11-12 kb of single-stranded negative-sense RNA that encodes five viral proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and viral polymerase (also known as large protein) (L). G monomers associate to form trimeric spikes anchored to the viral membrane.

[0211] The terms "vector," "expression vector," and "cloning vector" refer to any vehicle by which a nucleotide sequence, e.g., an RNA or DNA sequence, encoding, for example, a foreign gene, can be introduced into a cell (e.g., a host cell) to genetically modify the cell and promote expression (e.g., transcription and translation) of the introduced nucleotide sequence. Non-limiting examples of vectors include synthetic RNA and DNA molecules, plasmids, viruses, phages, and the like. In some embodiments, the vector may be a viral vector, including, but not limited to, a baculovirus vector, a herpesvirus vector, a lentivirus vector, a retrovirus vector, a vaccinia virus vector, an adeno-associated virus vector, an adenovirus vector, and an alphavirus vector.

[0212] The term "replication competent" is used herein to refer to viruses (including wild-type and recombinant viruses) that are capable of infecting and spreading within cells.

[0213] The term "pseudotyped" in reference to viral particles described herein refers to viral particles, including their lipid envelope or capsid molecules, e.g., proteins, glycoproteins, etc., that are mutated and / or heterologous compared to molecules typically found on the surface of the virus from which the particle is derived, and may influence, contribute to, direct, redirect, and / or completely alter the tropism of the viral particle compared to the reference wild-type virus from which the viral particle is derived. In some embodiments, the viral particle is pseudotyped such that it recognizes, binds, and / or infects a different target (ligand or cell) than the reference wild-type virus from which the viral particle is derived. In some embodiments, the viral particle is pseudotyped such that it does not recognize, bind, and / or infect the target (ligand or cell) of the reference wild-type virus from which the viral particle is derived.

[0214] The term "encoding" can refer to coding from either the (+) or (-) sense strand of a polynucleotide, for example, for expression in a viral particle.

[0215] The terms "antibody" and "antibody(s)" refer to monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFvs), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), intrabodies, minibodies, diabodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against antigen-specific TCRs), as well as epitope-binding fragments of any of the above. The terms "antibody" and "antibody(s)" also refer to covalently linked diabodies, such as those disclosed in U.S. Patent Application Publication No. 2007 / 0004909, and Ig-DARTS, such as those disclosed in U.S. Patent Application Publication No. 2009 / 0060910. Antibodies useful in the present disclosure include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0216] The terms "T cells" or "T lymphocytes" are used herein in the broadest sense to refer to all types of immune cells that express CD3, including, but not limited to, T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), tumor-infiltrating cytotoxic T cells (TILs; CD8+ T cells), CD4+CD8+ T cells, T regulatory cells (Tregs), and NK-T cells. T cells may include thymocytes, naive T cells, memory T cells, immature T cells, mature T cells, resting T cells, or activated T cells. T cells may also include "gamma-delta T cells (γδ T cells)," which refers to a small, specialized subset of T cells that possess a distinct TCR on their surface; unlike the majority of T cells, whose TCRs are composed of two glycoprotein chains, designated α- and β-TCR chains, the TCR in γδ T cells is composed of a γ chain and a δ chain.

[0217] The terms "major histocompatibility complex," "MHC," and "MHC molecule" encompass naturally occurring MHC molecules, as well as individual chains of MHC molecules (e.g., MHC class I α (heavy) chain, β2-microglobulin, MHC class II α chain, MHC class II β chain), individual subunits of such chains of MHC molecules (e.g., α1, α2, and / or α3 subunits of the MHC class I α chain, α1 and / or α2 subunits of the MHC class II α chain, β1 and / or β2 subunits of the MHC class II β chain), and fragments, variants, and various derivatives (including fusion proteins) thereof, which retain the ability to display antigenic peptides for recognition by TCRs, e.g., antigen-specific TCRs. MHC class I molecules contain a peptide-binding groove formed by the α1 and α2 domains of the heavy α chain that can accommodate peptides of approximately 8-10 amino acids. Despite the fact that both classes of MHC bind to a core of approximately nine amino acids within a peptide, the MHC class II peptide-binding groove (the α1 domain of a class II MHC polypeptide associated with the β1 domain of a class II MHC β polypeptide) allows for a wider range of peptide lengths. Peptides that bind to MHC class II typically vary between 13 and 17 amino acids in length, although shorter or longer lengths are not uncommon. As a result, peptides may shift within the MHC class II peptide-binding groove, altering which 9-mer is directly located within the groove at any given time. Conventional identification of specific MHC variants is used herein. For example, HLA-B17 refers to a human leukocyte antigen from the B gene cluster (hence, class I MHC) gene locus (known as locus) number 17, while the gene HLA-DR11 refers to a human leukocyte antigen encoded by a gene from the DR region (hence, class II MHC) locus number 11.

[0218] "Operably linked" or similar terms refer to a juxtaposition in which the described components are in a relationship permitting them to function in their intended manner. For example, a control sequence "operably linked" to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the control sequences. "Operably linked" sequences include both expression control sequences adjacent to a gene of interest and expression control sequences acting in trans or remotely to regulate the gene of interest (or sequence of interest). The term "expression control sequence" encompasses polynucleotide sequences necessary to affect the expression and processing of coding sequences to which they are ligated. "Expression control sequences" include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance polypeptide stability; and, if desired, sequences that enhance polypeptide secretion. The nature of such control sequences varies depending on the host organism. For example, in prokaryotes, such control sequences generally include promoters, ribosomal binding sites, and transcription termination sequences, while in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0219] The term "host cell" refers to any cell that contains a heterologous nucleic acid. As a non-limiting example, the heterologous nucleic acid may be a vector. A host cell may be, for example, but not limited to, a cell from any organism that is used, engineered, modified, selected, transformed, or grown for the cellular production of a substance, such as the cellular expression of an RNA or DNA sequence, a gene, a protein, or an enzyme.

[0220] "Individual" or "subject" or "animal" refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models of disease (e.g., mice, rats). In a preferred embodiment, the subject is a human.

[0221] The terms "nucleic acid," "polynucleotide," and "nucleotide," used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides (RNA), deoxyribonucleotides (DNA), or analogs or modified versions thereof. These include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases. A single-stranded nucleic acid can be a sense strand or an antisense strand.

[0222] Nucleic acids are said to have a "5' end" and a "3' end" because mononucleotides react to form oligonucleotides in such a way that the 5' phosphate of one mononucleotide pentose ring is unidirectionally linked to the 3' oxygen of its neighbor via a phosphodiester bond. An end of an oligonucleotide is called the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is called the "3' end" if its 3' oxygen is not linked to the 5' phosphate of another mononucleotide pentose ring. A nucleic acid sequence may also be said to have 5' and 3' ends, even if it is internal to a larger oligonucleotide. In either a linear or circular DNA molecule, distinct elements are referred to as "upstream" or "downstream" 5' or 3' elements.

[0223] The term "fragment," when referring to a protein, means a protein that is shorter or has fewer amino acids than the full-length protein. A fragment can be, for example, an N-terminal fragment (i.e., removal of a portion of the C-terminus of the protein), a C-terminal fragment (i.e., removal of a portion of the N-terminus of the protein), or an internal fragment. The term "fragment," when referring to a nucleic acid, means a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. A fragment can be, for example, a 5' fragment (i.e., removal of a portion of the 3' end of the nucleic acid), a 3' fragment (i.e., removal of a portion of the 5' end of the protein), or an internal fragment.

[0224] As used herein, the term "derivative" refers to a nucleic acid or protein, or a variant or analog thereof, that contains one or more mutations and / or chemical modifications compared to the corresponding full-length wild-type nucleic acid or protein. Non-limiting examples of chemical modifications that contain nucleic acids include, for example, modifications to the base moiety, sugar moiety, phosphate moiety, phosphate-sugar backbone, or combinations thereof.

[0225] "Sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to the residues of the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage sequence identity is used in the context of proteins, non-identical residue positions often differ by conservative amino acid substitutions (amino acid residues are replaced with other amino acid residues that have similar chemical properties (e.g., charge or hydrophobicity) and therefore do not alter the functional properties of the molecules). When sequences differ by conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thereby increasing the percentage sequence identity. Thus, for example, conservative substitutions are given a score of zero to one, with identical amino acids being given a score of one and non-conservative substitutions being given a score of zero. Scoring of conservative substitutions is calculated, for example, as carried out in the program PC / GENE.

[0226] "Percentage of sequence identity" includes a value determined by comparing two optimally aligned sequences (preferably the maximum number of identical residues) over a comparison window, and the portion of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of identical positions, dividing the number of identical positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Unless otherwise specified (for example, when the shorter sequence contains a linked heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.

[0227] The terms "treating" or "treatment" of a state, disorder, disease, or condition include (1) preventing, delaying, or reducing the likelihood of the onset and / or appearance of at least one clinical or subclinical symptom of a state, disorder, disease, or condition developing in a subject who may be affected by or prone to the state, disorder, disease, or condition, but who has not yet experienced or exhibited clinical or subclinical symptoms of the state, disorder, disease, or condition; or (2) inhibiting the state, disorder, disease, or condition, i.e., preventing, reducing, or delaying the onset or recurrence of the disease, or at least one clinical or subclinical symptom thereof; or (3) alleviating the state, disorder, disease, or condition, i.e., causing regression of the state, disorder, disease, or condition, or at least one clinical or subclinical symptom of the state, disorder, disease, or condition. The benefit to a subject being treated is either statistically significant or at least perceptible to the patient or to the physician.

[0228] When applied to a dose or amount, the term "effective" refers to the amount of a compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject in need thereof. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that was effective when administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general health of the subject, the severity of the condition being treated, the particular drug or drugs used, the mode of administration, etc.

[0229] The phrase "pharmaceutically acceptable," when used in connection with the compositions described herein, refers to molecular entities and other components of such compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to a mammal (e.g., a human). Preferably, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in mammals, more specifically, humans.

[0230] Terms such as "administration" refer to and include the administration of a composition to a subject or system (e.g., a cell, organ, tissue, organism, or associated component or set of components thereof). One of skill in the art will understand that the route of administration can vary depending, for example, on the subject or system to which the composition is being administered, the nature of the composition, the purpose of the administration, etc. For example, in certain embodiments, administration to an animal subject (e.g., a human or rodent) can be bronchial (including by bronchial infusion), buccal, intestinal, transcutaneous, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal infusion), transdermal, intravaginal, and / or intravitreal. In some embodiments, administration can include intermittent dosing. In some embodiments, administration can involve continuous administration (e.g., perfusion) for at least a selected period of time.

[0231] In accordance with the disclosure herein there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art, such techniques being fully explained in the literature. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (herein referred to as “Sambrook et al., 1989”), DNA Cloning: A Practical Approach, Volumes I and II (DNGlover ed.1985), Oligonucleotide Synthesis (MJGait ed.1984), Nucleic Acid Hybridization[BDHames&S.J.Higgins eds.(1985)], Transcription And Translation[BDHames&S.J.Higgins,eds.(1984)], Animal Cell Culture[RIFreshney,ed.(1986)], Immobilized Cells And Enzymes[IRL Press, (1986)], B. Perbal, A Practical Guide To Molecular Cloning (1984), Ausubel, FMet See, e.g., J. Med., 1994. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994.These techniques are described in Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985), U.S. Patent No. 5,071,743, Fukuoka et al., Biochem. Biophys. Res. Commun. 263:357-360 (1999), Kim and Maas, BioTech. 28:196-198 (2000), Parikh and Guengerich, BioTech. 24:4 28-431 (1998), Ray and Nickoloff, BioTech. 13:342-346 (1992), Wang et al., BioTech. 19:556-559 (1995), Wang and Malcolm, BioTech. 26:680-682 (1999), Xu and Gong, BioTech. 26:639-641 (1999), U.S. Patent Nos. 5,789,166 and 5,932,419, Hogrefe, Strategies 14.3:74-75 (2001), U.S. Patent Nos. 5,702,931, 5,780,270, and 6,242,222, Angag and Schutz, Biotech. 30:486-488 (2001), Wang and Wilkinson, Biotech. 29:976-978 (2000), Kang et al., Biotech. 20:44-46 (1996), Ogel and McPherson, Protein Engineer. 5:467-468 (1992), Kirsch and This includes site-directed mutagenesis, such as that described in Joly, Nucl. Acids. Res. 26:1848-1850 (1998), Rhem and Hancock, J. Bacteriol. 178:3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28:197-198 (1995), Barrenttino et al., Nuc. Acids. Res. 22:541-542 (1993), Tessier and Thomas, Meths. Molec. Biol. 57:229-237, and Pons et al., Meth. Molec. Biol. 67:209-218.

[0232] fusion proteins In one aspect, the present disclosure provides recombinant fusion protein.Generally, fusion protein can comprise membrane-embedded polypeptide that can mediate the fusion of two lipid membranes, and at least one of them can incorporate polypeptide.In some embodiments, fusion protein described herein can comprise (i) rhabdovirus glycoprotein (G) or its functional fragment or derivative, and (ii) targeting molecule.Targeting molecule can be linked to the N-terminus of rhabdovirus glycoprotein or its functional fragment or derivative via linker.

[0233] Rhabdovirus is a member of the Rhabdoviridae family of the Mononegavirales order, and includes more than 150 viruses of vertebrates, invertebrates, and plants.Examples of rhabdovirus include rabies virus (RABV) from the Lyssavirus genus, vesiculovirus from the Vesiculovirus genus, viral hemorrhagic septicemia virus (VHSV), and infectious hematopoietic necrosis virus, all of which are from the Novirhabdovirus genus.Members of the Lyssavirus genus can cause fatal meningoencephalitis in humans and animals, while VSV (vesiculovirus genus) can cause symptoms clinically identical to those of foot-and-mouth disease in cattle, and can occasionally cause limited infection in humans.Dimarhabdoviruses is a supergroup of rhabdoviruses that infect mammals and mosquitoes.

[0234] Rhabdoviruses are bullet-shaped, enveloped viruses with negative-sense, single-stranded RNA genomes ranging from 11 to 15 kb in length. Rhabdovirus genomes can contain up to 10 genes, of which only five are common to all members of the family. These five common rhabdovirus genes encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and viral polymerase (also known as large protein) (L). The rhabdovirus genome associates with N, L, and P to form a nucleocapsid, which is condensed by the M protein into a tightly coiled helical structure. The condensed nucleocapsid is surrounded by a lipid bilayer containing the viral glycoprotein G, which constitutes the spikes that protrude from the viral surface. Rhabdoviruses enter host cells via the endocytic pathway and then fuse with the cellular membrane within the acidic environment of the endosome. Both receptor recognition and membrane fusion are mediated by a single transmembrane viral glycoprotein (G). Fusion between the viral envelope and the endosomal membrane is triggered via low pH-induced (intraendosomal) structural rearrangement of G, releasing the viral genome and associated proteins into the cytoplasm of the target cell.

[0235] In some embodiments, the rhabdovirus described herein can be a vesiculovirus, or its functional fragment or derivative. Examples of vesiculoviruses that can be used in the present disclosure are listed in Table 1. [Table 1]

[0236] In some embodiments, the rhabdovirus disclosed herein can comprise VSV.In some embodiments, VSV can be replication-competent VSV.In some embodiments, VSV can be replication-incompetent.

[0237] A "fusogen" (e.g., a fusogenic protein such as a recombinant fusogenic protein described herein) or "fusogenic molecule" can refer to any molecule capable of inducing membrane fusion when present on the surface of a virus. In some embodiments, fusogens can act on cellular membranes to prevent spontaneous membrane fusion and promote fusion, which can occur in a controlled and / or regulated manner. Upon activation, fusogens can extend trimers anchored at one end by their transmembrane domains, exposing amphipathic loops or hydrophobic fusion peptides that insert into the target membrane. At this time, the two interacting domains are positioned in different membranes. Regulated refolding of the fusogenic complex into a hairpin-like structure then brings the fusion peptide and transmembrane domain to the same end of the molecule, generating a pulling force that brings the two membranes into a closed (approximately 1 nm) position. The accumulated energy from this event can drive fusion through the formation of a hemifusion stalk, in which only the contacting proximal leaflets of the membranes are fused, while the inner leaflet remains intact. The expansion of the hemifusion stalk and subsequent fusion of the distal leaflets completes the reaction by opening a fusion pore, allowing the contents of the two compartments to mix. The expansion of the fusion pore is considered the final energy barrier before membrane fusion becomes permanent. Without wishing to be bound by theory, there is evidence that viral fusogens mediate fusion via hemifusion. Many different protein and non-protein fusogenic molecules can be used herein. In some embodiments, the fusogenic molecule is a fusogenic protein or polypeptide.

[0238] In some embodiments, the fusogens described herein may be capable of inducing the fusion of the viral envelope of a virus described herein, for example, but not limited to, a rhabdovirus such as VSV, with the plasma membrane of a target cell. In some embodiments, the fusogens may mediate the fusion of the plasma membrane of the target cell and / or one or more cells adjacent to the target cell (i.e., adjacent cell(s)), thereby resulting in the formation of a multinucleated cell, i.e., a syncytium. In some embodiments, the viruses disclosed herein can induce cell-cell fusion, for example, via a fusogen capable of forming a syncytium. In some embodiments, the formation of a syncytium can occur during in vitro and / or in vivo infection with the virus. In some embodiments, cell-cell fusion may allow for more efficient spread of the virus to adjacent cell(s) than in the absence of such cell-cell fusion. The spread of the virus to adjacent cells associated with cell-cell fusion may allow for more efficient spread of the virus, for example, due to the absence of exposure of the virus to neutralizing antibodies and / or other host immune responses and / or molecules. In various embodiments, syncytium formation may allow the virus to evade or partially evade host defense mechanisms, such as, but not limited to, the humoral immune response, hi various embodiments, syncytium formation may allow the virus to evade or partially evade restriction factors that target viral particle assembly and / or release and / or viral entry into target cells.

[0239] In some embodiments, the fusogenic molecule is a viral fusogenic molecule. Non-limiting examples of viral fusogenic molecules include, for example, vesiculovirus fusogens (e.g., vesicular stomatitis virus G glycoprotein, alphavirus fusogens (e.g., Sindbis virus glycoprotein), orthomyxovirus fusogens (e.g., influenza HA protein), paramyxovirus fusogens (e.g., Nipah virus F protein or measles virus F protein), and fusogens from dengue virus (DV), Lassa fever virus, tick-borne encephalitis virus, dengue virus, hepatitis B virus, rabies virus, Semliki Forest virus, Ross River virus, Aura virus, Borna disease virus, Hantaan virus, SARS-CoV virus, and various fragments, variants, and derivatives thereof.

[0240] In some embodiments, the fusogenic molecule is heterologous to the virus from which the virus is derived, hi some embodiments, the fusogenic molecule is a mutant protein that does not bind to the fusogenic molecule's natural ligand(s).

[0241] There are two classes of viral fusogenic molecules, either of which can be used as a targeting molecule. Class I fusogens use a helical coiled-coil structure to induce membrane fusion, while Class II fusogens induce fusion with a 13-barrel structure. In some embodiments, Class I fusogens are used. In other embodiments, Class II fusogens are used. In still other embodiments, both Class I and Class II fusogens are used.

[0242] In some embodiments, recombinant fusion protein described herein can comprise rhabdovirus glycoprotein (G) or its functional fragment or derivative.In some embodiments, fusion molecule is vesicular stomatitis virus (VSV) envelope protein.In certain embodiments, recombinant fusion protein comprises VSV G protein (VSV-G or its fragment, variant, derivative or homolog).VSV-G can interact with the phospholipid component of cell membrane to mediate viral entry by membrane fusion.

[0243] In some embodiments, rhabdovirus G proteins described herein include, but are not limited to, vesicular stomatitis virus glycoprotein (VSV-G), Flanders virus glycoprotein (FLAV-G), Chandipura virus glycoprotein (CHPV-G), Perinetovirus glycoprotein (PERV-G), Piryvirus glycoprotein (PIRYV-G), Fukuoka virus glycoprotein (FUKV-G), Joint-Jakaka virus glycoprotein (JOIV-G), Kumasi virus glycoprotein (KRV-G), Isfahan glycoprotein (ISFV-G), Jurona glycoprotein (JURV-G), Mediterranean bat glycoprotein (MBV-G), Malpais Spring glycoprotein (MSPV-G), Radi glycoprotein (RADV-G), Rhinolophus affinis-G, Yugbugdanabok virus glycoprotein (Yugbugdanabok virus glycoprotein), and the like. Bugdanavoc glycoprotein (YBV-G), Yinshui bat glycoprotein (YSBV-G), Keurariva glycoprotein (KEUV-G), Kimberley glycoprotein (KIMV-G), Kanyawara glycoprotein (KYAV-G), La Joya glycoprotein (LJV-G), Mosquiero glycoprotein (MQOV-G), Parry Creek glycoprotein (PCV-G), Bas Congo glycoprotein (BASV-G), Bovine Ephemeral Fever virus glycoprotein (BEFV-G), Curionopolis glycoprotein (CURV-G), Drosophila melanogaster sigma virus glycoprotein (DMelSV-G), Niakha glycoprotein (NIAV-G), Puerto Almanduras The glycoprotein of the present invention may include the glycoprotein of the rhabdovirus G of the almandras (PTAMV-G), or the glycoprotein of the rhabdovirus G of the Tupaia (TUPTV-G), or functional fragments or derivatives thereof. Non-limiting examples of the amino acid sequences of the rhabdovirus G described herein are shown in SEQ ID NOs: 8-15, 17, 60-108, and 157.

[0244] In some embodiments, fusion protein can comprise targeting molecule as described herein.In some embodiments, targeting molecule is linked to the N-terminus of rhabdovirus glycoprotein.In some embodiments, the targeting molecule that is linked to N-terminus can be linked to rhabdovirus glycoprotein via linker as described herein.

[0245] In some embodiments, the N-terminus of rhabdovirus G, or a functional fragment or derivative thereof, to which a targeting molecule can be attached, for example, via a linker, does not contain a rhabdovirus glycoprotein signal sequence. In some embodiments, the rhabdovirus G, or a functional fragment or derivative thereof, to which a targeting molecule can be attached, is, for example, a mature rhabdovirus G that does not contain a rhabdovirus glycoprotein signal sequence. In some embodiments, the rhabdovirus glycoprotein signal sequence may be necessary to ensure that rhabdovirus G containing a signal sequence, such as nascent rhabdovirus G, can enter the endoplasmic reticulum (ER). In some embodiments, the rhabdovirus glycoprotein signal sequence is cleaved from nascent rhabdovirus G. Non-limiting examples of the amino acid sequence of a rhabdovirus G signal sequence are set forth in SEQ ID NOs: 109-137. In some embodiments, rhabdovirus G contains a signal sequence. Non-limiting examples of the amino acid sequence of a rhabdovirus glycoprotein containing a signal sequence are set forth in SEQ ID NOs: 80-108.

[0246] In some embodiments, the N-terminus of a rhabdovirus glycoprotein, or a functional fragment or derivative thereof, to which a targeting molecule can be attached, for example, via a linker, does not include one or more amino acids present at the N-terminus of a mature wild-type (WT) rhabdovirus glycoprotein described herein. For example, in certain embodiments, the N-terminus of a mature WT rhabdovirus glycoprotein, or a functional fragment or derivative thereof, to which a targeting molecule can be attached, does not include 1 to 2, 1 to 4, 1 to 6, 1 to 8, 1 to 10, 1 to 12, 1 to 14, 1 to 16, 1 to 18, 1 to 20, 1 to 22, 1 to 24, 1 to 26, 1 to 28, 1 to 30, 1 to 32, 1 to 34, 1 to 36, 1 to 38, 1 to 40, 1 to 42, 1 to 44, 1 to 46, 1 to 48, or 1 to 50 or more amino acids present at the N-terminus of a mature WT rhabdovirus glycoprotein described herein. In some embodiments, the N-terminus of the mature WT rhabdovirus glycoprotein, or its functional fragment or derivative, to which the targeting molecule can be bound, does not include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more amino acids present at the N-terminus of the mature WT rhabdovirus glycoprotein described herein. In some embodiments, the mature WT rhabdovirus glycoprotein, or its functional fragment or derivative, can be the mature WT vesicular stomatitis virus glycoprotein (VSV-G) described herein, or its functional fragment or derivative.

[0247] In some embodiments, the N-terminus of rhabdovirus glycoprotein or its functional fragment or derivative, and targeting molecule can be linked, for example, via a linker, does not include one or more amino acids that can be coded by the 5 ' end of wild-type (WT) glycoprotein gene, for example, the signal sequence (for example, rhabdovirus glycoprotein signal sequence) described herein that can be coded by the 5 ' end of wild-type (WT) glycoprotein gene, or its fragment or derivative.In some embodiments, when the N-terminus of rhabdovirus glycoprotein or its functional fragment or derivative does not include one or more amino acids (for example, signal sequence) that can be coded by the 5 ' end of WT glycoprotein gene, rhabdovirus glycoprotein can include mature rhabdovirus glycoprotein, i.e., the rhabdovirus glycoprotein that does not include signal peptide described herein. In certain embodiments, the N-terminus of a mature WT rhabdovirus glycoprotein, or functional fragment or derivative thereof, to which a targeting molecule can be attached, does not include 1 to 2, 1 to 4, 1 to 6, 1 to 8, 1 to 10, 1 to 12, 1 to 14, 1 to 16, 1 to 18, 1 to 20, 1 to 22, 1 to 24, 1 to 26, 1 to 28, 1 to 30, 1 to 32, 1 to 34, 1 to 36, 1 to 38, 1 to 40, 1 to 42, 1 to 44, 1 to 46, 1 to 48, or 1 to 50 or more amino acids encoded by the 5' end of the WT glycoprotein gene. In some embodiments, the N-terminus of a mature WT rhabdovirus glycoprotein, or functional fragment or derivative thereof, to which a targeting molecule can be attached, does not include about 10 to 32 amino acids encoded by the 5' end of the WT glycoprotein gene. In some embodiments, the N-terminus of the mature WT rhabdovirus glycoprotein, or functional fragment or derivative thereof, to which a targeting molecule can be attached, does not include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more amino acids encoded by the 5' end of the WT glycoprotein gene.In some embodiments, when the N-terminus of a rhabdovirus glycoprotein, or a functional fragment or derivative thereof, does not contain one or more amino acids encoded by the 5' end of the WT glycoprotein gene, the rhabdovirus glycoprotein can comprise a mature rhabdovirus glycoprotein without a signal peptide sequence, such as the signal sequences set forth in SEQ ID NOS: 109-137 described herein. Non-limiting examples of amino acid sequences of mature rhabdovirus glycoproteins without a signal sequence are set forth in SEQ ID NOS: 8-15, 17, 60-79. In some embodiments, a mature WT rhabdovirus glycoprotein, or a functional fragment or derivative thereof, can be a mature WT vesicular stomatitis virus glycoprotein (VSV-G) described herein, or a functional fragment or derivative thereof.

[0248] In some embodiments, the fusion proteins described herein may comprise VSV-G. In some embodiments, VSV-G comprises the amino acid sequence of SEQ ID NO: 8 or 80, or a functional fragment or derivative thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 8 or 80. In certain embodiments, the nucleotide sequence encoding VSV-G comprises the amino acid sequence of SEQ ID NO:8 or 80, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:8 or 80. In certain embodiments, the VSV-G comprises the amino acid sequence of SEQ ID NO:8 or 80.

[0249] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from VSV-G. In some embodiments, VSV-G comprises the sequence of SEQ ID NO: 8. In some embodiments, VSV-G consists of the sequence of SEQ ID NO: 8.

[0250] In some embodiments, the VSV-G described herein can be derived from, for example, the VSV-G described in U.S. Patent Publication No. 2020 / 0216502, the contents of which are incorporated by reference in their entirety for all purposes. By way of example, and not limitation, the VSV-G, or a functional fragment or derivative thereof, can include the amino acid sequence of SEQ ID NO: 157, or a functional fragment or derivative thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 157.

[0251] In some embodiments, the fusogenic proteins described herein, or functional fragments or derivatives thereof, may comprise a rhabdovirus glycoprotein (G) comprising one or more mutations compared to the corresponding WT sequence of the rhabdovirus glycoprotein.

[0252] In some embodiments, the rhabdovirus glycoprotein described herein, or its functional fragment or derivative, can comprise amino acid mutation(s) at one or more positions.Non-limiting examples of amino acid mutation include amino acid substitution, insertion, and / or deletion.Amino acid substitution can mean that an amino acid residue is replaced with a substituted amino acid residue at the same position.The inserted amino acid residue can be inserted at any position, and some or all of the inserted amino acid residues can be inserted so that they are directly adjacent to each other, or none of the inserted amino acid residues can be inserted so that they are directly adjacent to another inserted amino acid residue.

[0253] In some embodiments, a rhabdovirus glycoprotein, or a functional fragment or derivative thereof, may contain an amino acid mutation(s) at one or more positions. As a non-limiting example, a rhabdovirus glycoprotein, or a functional fragment or derivative thereof, may contain one or more amino acid mutation(s) at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 0, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 1, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410 , 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, or 700 or more amino acid positions.

[0254] In some embodiments, rhabdovirus protein or its fragment or derivative can comprise one or more mutations, that is, amino acid substitution, insertion or deletion, or combinations thereof, at one or more positions of its amino acid sequence compared with the amino acid sequence of WT rhabdovirus protein.For example, the VSV-G protein described herein can comprise one or more amino acids in the amino acid sequence of parent VSV-G protein with similar or homologous amino acid(s) or different amino acid(s).

[0255] In some embodiments, the rhabdovirus G protein described herein has an amino acid sequence that is at least about 50% or more, about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more of its WT sequence. , 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or 100% identity and may include any amino acid sequence that has the activity of the WT sequence.

[0256] In some embodiments, a VSV-G polypeptide described herein, or a functional fragment or derivative thereof, can contain one or more mutations compared to its corresponding WT VSV-G sequence (see, e.g., SEQ ID NO: 8). In some embodiments, the one or more mutations in VSV-G can, for example, reduce or eliminate binding of the VSV-G protein, or a functional fragment or derivative thereof, to the low-density lipoprotein receptor (LDLR).

[0257] In some embodiments, the one or more mutations in the VSV-G polypeptide, or functional fragments or derivatives thereof, include one or more amino acid substitutions and / or deletions at positions corresponding to H8, K47, Y209, and / or R354 of SEQ ID NO:8. In some embodiments, the one or more mutations in the VSV-G polypeptide or functional fragment or derivative thereof may include, for example, one or more amino acid substitutions and / or deletions at amino acid positions corresponding to positions 8, 47, 209, or 354, or both 8 and 47, or both 8 and 209, or both 8 and 354, or both 47 and 209, or both 47 and 354, or both 209 and 354, or a combination of 8, 47, and 209, or a combination of 8, 47, and 354, or a combination of 8, 209, and 354, or a combination of 47, 209, and 354, or a combination of 8, 47, 209, and 354, or a combination of 8, 47, 209, and 354,

[0258] In various embodiments, the mutant VSV-G polypeptide may include, for example, a mutation in SEQ ID NO: 8, in which the amino acid at position 8 may be substituted with any amino acid other than H, preferably other than Y; a mutation in which the amino acid at position 47 may be substituted with any amino acid other than K; a mutation in which the amino acid at position 209 may be substituted with any amino acid other than Y, preferably other than H; and / or a mutation in which the amino acid at position 354 may be substituted with any amino acid other than R.

[0259] In some embodiments, the mutant VSV-G polypeptides described herein may comprise a fusogen comprising the sequence of SEQ ID NO: 8, with amino acid substitutions at (i) positions K47, (ii) R354, and (iii) H8 or Y209.

[0260] In some embodiments, the mutant VSV-G polypeptide described herein may consist of the sequence of SEQ ID NO: 8, with amino acid substitutions at (i) positions K47, (ii) R354, and (iii) H8 or Y209.

[0261] In some embodiments, the mutant VSV-G polypeptides described herein may comprise a fusogen comprising the sequence of SEQ ID NO: 8, with amino acid substitutions at positions K47, R354, H8, and Y209.

[0262] In some embodiments, the mutant VSV-G polypeptide described herein can consist of the sequence of SEQ ID NO: 8, with amino acid substitutions at positions K47, R354, H8, and Y209.

[0263] In some embodiments, the one or more mutations in the VSV-G polypeptide, or functional fragment or derivative thereof, comprise an amino acid deletion at one or more positions corresponding to positions H8, K47, Y209, and / or R354 in SEQ ID NO:8.

[0264] In some embodiments, the mutant VSV-G polypeptides described herein may comprise a functional fragment of SEQ ID NO: 8, or a derivative thereof, having amino acid deletions at (i) positions K47, (ii) R354, and (iii) H8 or Y209.

[0265] In some embodiments, the mutant VSV-G polypeptide described herein may consist of the sequence of SEQ ID NO: 8, with amino acid deletions at (i) position K47, (ii) position PR54, and (iii) position H8 or Y209.

[0266] In some embodiments, a mutant VSV-G polypeptide described herein can comprise the sequence of SEQ ID NO: 8, which has an amino acid deletion at position K47.

[0267] In some embodiments, the mutant VSV-G polypeptide described herein can consist of the sequence of SEQ ID NO: 8, with an amino acid deletion at position K47.

[0268] Other exemplary mutations in VSV-G that can reduce or eliminate binding of the VSV-G protein, or a functional fragment or derivative thereof, to the low-density lipoprotein receptor (LDLR) are described in US2020 / 0216502, which is incorporated herein by reference in its entirety.

[0269] In some embodiments, VSV-G, or a functional fragment or derivative thereof, may comprise one or more mutations that increase viral titer. Non-limiting examples of mutations that increase viral titer are M184T and F250L, as specified relative to their positions in SEQ ID NO: 8. In some embodiments, VSV-G, or a functional fragment or derivative thereof, may comprise one or more mutations in VSV-G that increase viral titer, such as, but not limited to, M184T and / or F250L, as specified relative to their positions in SEQ ID NO: 8.

[0270] Other examples of mutations that increase viral titer include those described in US2022 / 0162266, the entire contents of which are incorporated herein by reference, such as H22N and S422I in the ectodomain of the VSV Indiana glycoprotein G (or similar substitutions, e.g., S422F, S422M, S422L, or S422V, or equivalent substitutions at equivalent positions in the G ectodomain of other VSV strains).

[0271] In various embodiments, a recombinant fusion protein of the present disclosure comprises a fusogen, or a functional fragment or derivative thereof, having a minimum of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% amino acid sequence identity to a vesicular stomatitis virus glycoprotein (VSV-G) comprising the sequence of SEQ ID NO: 8. In various embodiments, a recombinant fusion protein of the present disclosure comprises a fusogen comprising a vesicular stomatitis virus glycoprotein (VSV-G) consisting of the sequence of SEQ ID NO: 8. In various embodiments, a recombinant fusion protein of the present disclosure comprises a fusogen, or a functional fragment or derivative thereof, having a minimum of at least 60% amino acid sequence identity to a vesicular stomatitis virus glycoprotein (VSV-G) comprising the sequence of SEQ ID NO: 8. In such embodiments, the fusogen, or a functional fragment or derivative thereof, may comprise one or more amino acid deletions at one or more positions corresponding to positions H8, K47, Y209, and / or R354 in SEQ ID NO: 8. In some embodiments, the fusogen comprises SEQ ID NO: 8, or a functional fragment or derivative thereof, having one or more amino acid deletions at one or more positions selected from H8, K47, Y209, and / or R354. In some embodiments, the fusogen, or functional fragment or derivative thereof, may further comprise one or more viral titer-increasing mutations, such as, but not limited to, viral titer-increasing mutations, at one or more positions corresponding to positions M184 and F250 of SEQ ID NO: 8. In some embodiments, the one or more viral titer-increasing mutations in VSV-G, or a functional fragment or derivative thereof, may be, for example, M184T and / or F250L, as specified relative to the positions in SEQ ID NO: 8.

[0272] In some embodiments, the recombinant fusion proteins described herein comprise a fusogen comprising an N-terminus that can be attached to a targeting molecule, for example, via a linker. In some embodiments, the recombinant fusogen protein further comprises a targeting molecule located at the N-terminus of the fusogen. In some embodiments, the targeting molecule is attached to the N-terminus of the fusogen, or a functional or derivative thereof, via a linker.

[0273] In some embodiments, the N-terminus of the fusogen does not include a signal sequence described herein and / or does not include one or more amino acids present at the N-terminus of a mature WT fusogen. As a non-limiting example, the N-terminus of a mature WT fusogen, or functional fragment or derivative thereof, to which a targeting molecule can be attached does not include 1 to 2, 1 to 4, 1 to 6, 1 to 8, 1 to 10, 1 to 12, 1 to 14, 1 to 16, 1 to 18, 1 to 20, 1 to 22, 1 to 24, 1 to 26, 1 to 28, 1 to 30, 1 to 32, 1 to 34, 1 to 36, 1 to 38, 1 to 40, 1 to 42, 1 to 44, 1 to 46, 1 to 48, or 1 to 50 or more amino acids present at the N-terminus of a mature WT fusogen described herein. In some embodiments, the N-terminus of a mature WT fusogen, or functional fragment or derivative thereof, to which a targeting molecule can be attached does not include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more amino acids present at the N-terminus of a mature WT fusogen described herein.

[0274] In some embodiments, recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (FLAV-G) from Flanders virus.In some embodiments, FLAV-G comprises the amino acid sequence of SEQ ID NO:9 or 81, or its functional fragment or derivative has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with SEQ ID NO:9 or 81. In certain embodiments, the nucleotide sequence encoding FLAV-G comprises the amino acid sequence of SEQ ID NO:9 or 81, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:9 or 81. In certain embodiments, FLAV-G comprises the amino acid sequence of SEQ ID NO:9 or 81.

[0275] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from FLAV-G. In some embodiments, FLAV-G comprises the sequence of SEQ ID NO: 9. In some embodiments, FLAV-G consists of the sequence of SEQ ID NO: 9.

[0276] In some embodiments, the recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (CHPV-G) derived from Chandipura virus. In some embodiments, CHPV-G comprises the amino acid sequence of SEQ ID NO: 10 or 82, or its functional fragment or derivative that has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 10 or 82. In certain embodiments, the nucleotide sequence encoding CHPV-G comprises the amino acid sequence of SEQ ID NO: 10 or 82, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 10 or 82. In certain embodiments, the CHPV-G comprises the amino acid sequence of SEQ ID NO: 10 or 82.

[0277] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from CHPV-G. In some embodiments, CHPV-G comprises the sequence of SEQ ID NO: 10. In some embodiments, CHPV-G consists of the sequence of SEQ ID NO: 10.

[0278] In some embodiments, the recombinant fusion protein described herein can comprise a rhabdovirus glycoprotein (PERV-G) derived from a Perinetovirus. In some embodiments, the PERV-G comprises the amino acid sequence of SEQ ID NO: 11 or 87, or a functional fragment or derivative thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 11 or 87. In certain embodiments, the nucleotide sequence encoding PERV-G comprises the amino acid sequence of SEQ ID NO: 11 or 87, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 11 or 87. In certain embodiments, the PERV-G comprises the amino acid sequence of SEQ ID NO: 11 or 87.

[0279] In some embodiments, the recombinant fusion proteins described herein comprise a rhabdovirus glycoprotein derived from PERV-G. In some embodiments, the PERV-G comprises the sequence of SEQ ID NO: 11. In some embodiments, the PERV-G consists of the sequence of SEQ ID NO: 11.

[0280] In some embodiments, the recombinant fusion protein described herein can comprise a rhabdovirus glycoprotein (PIRYV-G) derived from a Piryvirus. In some embodiments, PIRYV-G comprises the amino acid sequence of SEQ ID NO: 12 or 88, or a functional fragment or derivative thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 12 or 88. In certain embodiments, the nucleotide sequence encoding PIRYV-G comprises the amino acid sequence of SEQ ID NO: 12 or 88, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 12 or 88. In certain embodiments, PIRYV-G comprises the amino acid sequence of SEQ ID NO: 12 or 88.

[0281] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from PIRYV-G. In some embodiments, PIRYV-G comprises the sequence of SEQ ID NO: 12. In some embodiments, PIRYV-G consists of the sequence of SEQ ID NO: 12.

[0282] In some embodiments, the recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (FUKV-G) derived from Fukuoka virus. In some embodiments, FUKV-G comprises the amino acid sequence of SEQ ID NO: 13 or 93, or a functional fragment or derivative thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 13 or 93. In certain embodiments, the nucleotide sequence encoding FUKV-G comprises the amino acid sequence of SEQ ID NO: 13 or 93, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 13 or 93. In certain embodiments, the FUKV-G comprises the amino acid sequence of SEQ ID NO: 13 or 93.

[0283] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from FUKV-G. In some embodiments, FUKV-G comprises the sequence of SEQ ID NO: 13. In some embodiments, FUKV-G consists of the sequence of SEQ ID NO: 13.

[0284] In some embodiments, the recombinant fusion protein described herein can comprise a rhabdovirus glycoprotein (JOIV-G) derived from the Joint Jacaka virus. In some embodiments, JOIV-G comprises the amino acid sequence of SEQ ID NO: 14 or 94, or a functional fragment or derivative thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 14 or 94. In certain embodiments, the nucleotide sequence encoding JOIV-G comprises the amino acid sequence of SEQ ID NO: 14 or 94, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 14 or 94. In certain embodiments, JOIV-G comprises the amino acid sequence of SEQ ID NO: 14 or 94.

[0285] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from JOIV-G. In some embodiments, JOIV-G comprises the sequence of SEQ ID NO: 14. In some embodiments, JOIV-G consists of the sequence of SEQ ID NO: 14.

[0286] In some embodiments, the recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (KRV-G) derived from Kumasi virus. In some embodiments, KRV-G comprises the amino acid sequence of SEQ ID NO: 15 or 97, or its functional fragment or derivative has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 15 or 97. In certain embodiments, the nucleotide sequence encoding KRV-G comprises the amino acid sequence of SEQ ID NO: 15 or 97, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 15 or 97. In certain embodiments, KRV-G comprises the amino acid sequence of SEQ ID NO: 15 or 97.

[0287] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from KRV-G. In some embodiments, KRV-G comprises the sequence of SEQ ID NO: 15. In some embodiments, KRV-G consists of the sequence of SEQ ID NO: 15.

[0288] In some embodiments, the recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (KEUV-G) derived from Keurariba virus. In some embodiments, KEUV-G comprises the amino acid sequence of SEQ ID NO: 17 or 95, or a functional fragment or derivative thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 17 or 95. In certain embodiments, the nucleotide sequence encoding KEUV-G comprises the amino acid sequence of SEQ ID NO: 17 or 95, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 17 or 95. In certain embodiments, KEUV-G comprises the amino acid sequence of SEQ ID NO: 17 or 95.

[0289] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from KEUV-G. In some embodiments, KEUV-G comprises the sequence of SEQ ID NO: 17. In some embodiments, KEUV-G consists of the sequence of SEQ ID NO: 17.

[0290] In some embodiments, the recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (ISFV-G) derived from Isfahan virus. In some embodiments, ISFV-G comprises the amino acid sequence of SEQ ID NO: 60 or 83, or its functional fragment or derivative that has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 60 or 83. In certain embodiments, the nucleotide sequence encoding ISFV-G comprises the amino acid sequence of SEQ ID NO: 60 or 83, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 60 or 83. In certain embodiments, the ISFV-G comprises the amino acid sequence of SEQ ID NO: 60 or 83.

[0291] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from ISFV-G. In some embodiments, ISFV-G comprises the sequence of SEQ ID NO: 60. In some embodiments, ISFV-G consists of the sequence of SEQ ID NO: 60.

[0292] In some embodiments, the recombinant fusion protein described herein can comprise a rhabdovirus glycoprotein (JURV-G) derived from Duronavirus. In some embodiments, JURV-G comprises the amino acid sequence of SEQ ID NO: 61 or 84, or a functional fragment or derivative thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 61 or 84. In certain embodiments, the nucleotide sequence encoding JURV-G comprises the amino acid sequence of SEQ ID NO: 61 or 84, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 61 or 84. In certain embodiments, JURV-G comprises the amino acid sequence of SEQ ID NO: 61 or 84.

[0293] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from JURV-G. In some embodiments, JURV-G comprises the sequence of SEQ ID NO: 61. In some embodiments, JURV-G consists of the sequence of SEQ ID NO: 61.

[0294] In some embodiments, the recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (MBV-G) derived from Mediterranean bat virus. In some embodiments, MBV-G comprises the amino acid sequence of SEQ ID NO: 62 or 85, or a functional fragment or derivative thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 62 or 85. In certain embodiments, the nucleotide sequence encoding MBV-G comprises the amino acid sequence of SEQ ID NO: 62 or 85, or a nucleotide sequence encoding a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 62 or 85. In certain embodiments, the MBV-G comprises the amino acid sequence of SEQ ID NO: 62 or 85.

[0295] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from MBV-G. In some embodiments, MBV-G comprises the sequence of SEQ ID NO: 62. In some embodiments, MBV-G consists of the sequence of SEQ ID NO: 62.

[0296] In some embodiments, the recombinant fusion protein described herein can comprise a rhabdovirus glycoprotein (MSPV-G) derived from Malpais Springs virus. In some embodiments, MSPV-G comprises the amino acid sequence of SEQ ID NO: 63 or 86, or a functional fragment or derivative thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 63 or 86. In certain embodiments, the nucleotide sequence encoding MSPV-G comprises the amino acid sequence of SEQ ID NO: 63 or 86, or a nucleotide sequence encoding a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 63 or 86. In certain embodiments, the MSPV-G comprises the amino acid sequence of SEQ ID NO: 63 or 86.

[0297] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from MSPV-G. In some embodiments, MSPV-G comprises the sequence of SEQ ID NO: 63. In some embodiments, MSPV-G consists of the sequence of SEQ ID NO: 63.

[0298] In some embodiments, recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (RADV-G) derived from Radivirus.In some embodiments, RADV-G comprises the amino acid sequence of SEQ ID NO: 64 or 89, or its functional fragment or derivative has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with SEQ ID NO: 64 or 89. In certain embodiments, the nucleotide sequence encoding RADV-G comprises the amino acid sequence of SEQ ID NO: 64 or 89, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 64 or 89. In certain embodiments, RADV-G comprises the amino acid sequence of SEQ ID NO: 64 or 89.

[0299] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from RADV-G. In some embodiments, RADV-G comprises the sequence of SEQ ID NO: 64. In some embodiments, RADV-G consists of the sequence of SEQ ID NO: 64.

[0300] In some embodiments, the recombinant fusion proteins described herein may comprise a rhabdovirus glycoprotein derived from the Rhinolophus affinis virus (Rhinolophus affinis G). In some embodiments, Rhinolophus affinis G comprises the amino acid sequence of SEQ ID NO: 65 or 90, or a functional fragment or derivative thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 65 or 90. In certain embodiments, a nucleotide sequence encoding Rhinolophus affinis G comprises the amino acid sequence of SEQ ID NO: 65 or 90, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 65 or 90. In certain embodiments, Rhinolophus affinis G comprises the amino acid sequence of SEQ ID NO: 65 or 90.

[0301] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from Rhinolophus affinis G. In some embodiments, the Rhinolophus affinis G comprises the sequence of SEQ ID NO: 65. In some embodiments, the Rhinolophus affinis G consists of the sequence of SEQ ID NO: 65.

[0302] In some embodiments, the recombinant fusion protein described herein can comprise a rhabdovirus glycoprotein (YBV-G) derived from the YBV virus. In some embodiments, the YBV-G comprises the amino acid sequence of SEQ ID NO: 66 or 91, or a functional fragment or derivative thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 66 or 91. In certain embodiments, the nucleotide sequence encoding YBV-G comprises the amino acid sequence of SEQ ID NO: 66 or 91, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 66 or 91. In certain embodiments, YBV-G comprises the amino acid sequence of SEQ ID NO: 66 or 91.

[0303] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from YBV-G. In some embodiments, YBV-G comprises the sequence of SEQ ID NO: 66. In some embodiments, YBV-G consists of the sequence of SEQ ID NO: 66.

[0304] In some embodiments, the recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (YSBV-G) derived from the Japanese bat virus. In some embodiments, YSBV-G comprises the amino acid sequence of SEQ ID NO: 67 or 92, or its functional fragment or derivative has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 67 or 92. In certain embodiments, the nucleotide sequence encoding YSBV-G comprises the amino acid sequence of SEQ ID NO: 67 or 92, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 67 or 92. In certain embodiments, YSBV-G comprises the amino acid sequence of SEQ ID NO: 67 or 92.

[0305] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from YSBV-G. In some embodiments, YSBV-G comprises the sequence of SEQ ID NO: 67. In some embodiments, YSBV-G consists of the sequence of SEQ ID NO: 67.

[0306] In some embodiments, the recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (KIMV-G) derived from Kimberly virus.In some embodiments, KIMV-G comprises the amino acid sequence of SEQ ID NO: 68 or 96, or its functional fragment or derivative has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 68 or 96. In certain embodiments, the nucleotide sequence encoding KIMV-G comprises the amino acid sequence of SEQ ID NO: 68 or 96, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 68 or 96. In certain embodiments, the KIMV-G comprises the amino acid sequence of SEQ ID NO: 68 or 96.

[0307] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from KIMV-G. In some embodiments, KIMV-G comprises the sequence of SEQ ID NO: 68. In some embodiments, KIMV-G consists of the sequence of SEQ ID NO: 68.

[0308] In some embodiments, the recombinant fusion protein described herein can comprise a rhabdovirus glycoprotein (KYAV-G) derived from Kanyawara virus. In some embodiments, the KYAV-G comprises the amino acid sequence of SEQ ID NO: 69 or 98, or a functional fragment or derivative thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 69 or 98. In certain embodiments, the nucleotide sequence encoding KYAV-G comprises the amino acid sequence of SEQ ID NO:69 or 98, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:69 or 98. In certain embodiments, the KYAV-G comprises the amino acid sequence of SEQ ID NO:69 or 98.

[0309] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from KYAV-G. In some embodiments, KYAV-G comprises the sequence of SEQ ID NO: 69. In some embodiments, KYAV-G consists of the sequence of SEQ ID NO: 69.

[0310] In some embodiments, the recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (LJV-G) derived from La Jolla virus. In some embodiments, LJV-G comprises the amino acid sequence of SEQ ID NO: 70 or 99, or a functional fragment or derivative thereof that has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 70 or 99. In certain embodiments, the nucleotide sequence encoding LJV-G comprises the amino acid sequence of SEQ ID NO: 70 or 99, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 70 or 99. In certain embodiments, LJV-G comprises the amino acid sequence of SEQ ID NO: 70 or 99.

[0311] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from LJV-G. In some embodiments, LJV-G comprises the sequence of SEQ ID NO: 70. In some embodiments, LJV-G consists of the sequence of SEQ ID NO: 70.

[0312] In some embodiments, the recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (MQOV-G) derived from Mosquito virus. In some embodiments, MQOV-G comprises the amino acid sequence of SEQ ID NO: 71 or 100, or a functional fragment or derivative thereof that has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 71 or 100. In certain embodiments, the nucleotide sequence encoding MQOV-G comprises the amino acid sequence of SEQ ID NO: 71 or 100, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 71 or 100. In certain embodiments, MQOV-G comprises the amino acid sequence of SEQ ID NO: 71 or 100.

[0313] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from MQOV-G. In some embodiments, MQOV-G comprises the sequence of SEQ ID NO: 71. In some embodiments, MQOV-G consists of the sequence of SEQ ID NO: 71.

[0314] In some embodiments, the recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (PCV-G) derived from Parry Creek virus. In some embodiments, PCV-G comprises the amino acid sequence of SEQ ID NO: 72 or 101, or a functional fragment or derivative thereof that has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 72 or 101. In certain embodiments, the nucleotide sequence encoding PCV-G comprises the amino acid sequence of SEQ ID NO: 72 or 101, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 72 or 101. In certain embodiments, the PCV-G comprises the amino acid sequence of SEQ ID NO: 72 or 101.

[0315] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from PCV-G. In some embodiments, PCV-G comprises the sequence of SEQ ID NO: 72. In some embodiments, PCV-G consists of the sequence of SEQ ID NO: 72.

[0316] In some embodiments, the recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (BASV-G) derived from Bascongo virus. In some embodiments, BASV-G comprises the amino acid sequence of SEQ ID NO: 73 or 102, or a functional fragment or derivative thereof that has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 73 or 102. In certain embodiments, the nucleotide sequence encoding BASV-G comprises the amino acid sequence of SEQ ID NO: 73 or 102, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 73 or 102. In certain embodiments, BASV-G comprises the amino acid sequence of SEQ ID NO: 73 or 102.

[0317] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from BASV-G. In some embodiments, BASV-G comprises the sequence of SEQ ID NO: 73. In some embodiments, BASV-G consists of the sequence of SEQ ID NO: 73.

[0318] In some embodiments, the recombinant fusion protein described herein can comprise a rhabdovirus glycoprotein (BEFV-G) derived from bovine ephemeral fever glycovirus. In some embodiments, the BEFV-G comprises the amino acid sequence of SEQ ID NO: 74 or 103, or a functional fragment or derivative thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 74 or 103. In certain embodiments, the nucleotide sequence encoding BEFV-G comprises the amino acid sequence of SEQ ID NO: 74 or 103, or a nucleotide sequence encoding a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 74 or 103. In certain embodiments, the BEFV-G comprises the amino acid sequence of SEQ ID NO: 74 or 103.

[0319] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from BEFV-G. In some embodiments, BEFV-G comprises the sequence of SEQ ID NO: 74. In some embodiments, BEFV-G consists of the sequence of SEQ ID NO: 74.

[0320] In some embodiments, the recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (CURV-G) derived from Cryonopolis virus.In some embodiments, CURV-G comprises the amino acid sequence of SEQ ID NO: 75 or 104, or its functional fragment or derivative has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 75 or 104. In certain embodiments, the nucleotide sequence encoding CURV-G comprises the amino acid sequence of SEQ ID NO: 75 or 104, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 75 or 104. In certain embodiments, CURV-G comprises the amino acid sequence of SEQ ID NO: 75 or 104.

[0321] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from CURV-G. In some embodiments, CURV-G comprises the sequence of SEQ ID NO: 75. In some embodiments, CURV-G consists of the sequence of SEQ ID NO: 75.

[0322] In some embodiments, the recombinant fusion protein described herein can comprise a rhabdovirus glycoprotein (DMelSV-G) derived from Drosophila melanogaster sigma virus. In some embodiments, DMelSV-G comprises the amino acid sequence of SEQ ID NO: 76 or 105, or a functional fragment or derivative thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 76 or 105. In certain embodiments, the nucleotide sequence encoding DMelSV-G comprises the amino acid sequence of SEQ ID NO: 76 or 105, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 76 or 105. In certain embodiments, DMelSV-G comprises the amino acid sequence of SEQ ID NO: 76 or 105.

[0323] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from DMelSV-G. In some embodiments, DMelSV-G comprises the sequence of SEQ ID NO: 76. In some embodiments, DMelSV-G consists of the sequence of SEQ ID NO: 76.

[0324] In some embodiments, the recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (NIAV-G) derived from Niaka virus.In some embodiments, NIAV-G comprises the amino acid sequence of SEQ ID NO: 77 or 106, or its functional fragment or derivative has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 77 or 106. In certain embodiments, the nucleotide sequence encoding NIAV-G comprises the amino acid sequence of SEQ ID NO: 77 or 106, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 77 or 106. In certain embodiments, the NIAV-G comprises the amino acid sequence of SEQ ID NO: 77 or 106.

[0325] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from NIAV-G. In some embodiments, NIAV-G comprises the sequence of SEQ ID NO: 77. In some embodiments, NIAV-G consists of the sequence of SEQ ID NO: 77.

[0326] In some embodiments, the recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (PTAMV-G) derived from Puerto Armandras virus. In some embodiments, PTAMV-G comprises the amino acid sequence of SEQ ID NO: 78 or 107, or a functional fragment or derivative thereof that has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 78 or 107. In certain embodiments, the nucleotide sequence encoding PTAMV-G comprises the amino acid sequence of SEQ ID NO: 78 or 107, or a nucleotide sequence encoding a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 78 or 107. In certain embodiments, PTAMV-G comprises the amino acid sequence of SEQ ID NO: 78 or 107.

[0327] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from PTAMV-G. In some embodiments, PTAMV-G comprises the sequence of SEQ ID NO: 78. In some embodiments, PTAMV-G consists of the sequence of SEQ ID NO: 78.

[0328] In some embodiments, the recombinant fusion protein described herein can comprise the rhabdovirus glycoprotein (TUPTV-G) derived from Tupaiavirus. In some embodiments, TUPTV-G comprises the amino acid sequence of SEQ ID NO: 79 or 108, or a functional fragment or derivative thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 79 or 108. In certain embodiments, the nucleotide sequence encoding TUPTV-G comprises the amino acid sequence of SEQ ID NO: 79 or 108, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 79 or 108. In certain embodiments, TUPTV-G comprises the amino acid sequence of SEQ ID NO: 79 or 108.

[0329] In some embodiments, the recombinant fusion protein described herein comprises a rhabdovirus glycoprotein derived from TUPTV-G. In some embodiments, TUPTV-G comprises the sequence of SEQ ID NO: 79. In some embodiments, TUPTV-G consists of the sequence of SEQ ID NO: 79.

[0330] In some embodiments, the recombinant fusion proteins described herein may comprise a rhabdovirus glycoprotein described herein, which may be truncated, e.g., at the N-terminus and / or C-terminus(s), thereby resulting in the production of a truncated rhabdovirus glycoprotein, e.g., a truncated functional fragment, that can retain the ability to confer at least the activity of the rhabdovirus glycoprotein.

[0331] In some embodiments, the recombinant fusion proteins described herein may be fragments of the rhabdovirus glycoproteins described herein, wherein the cytoplasmic tail of the rhabdovirus glycoprotein has been removed or truncated and / or optionally replaced with another sequence.

[0332] In some embodiments, the recombinant fusion protein described herein may be a fragment of a rhabdovirus glycoprotein described herein, and the fragment of the rhabdovirus glycoprotein may include, but is not limited to, a truncated cytoplasmic tail. As a non-limiting example, the cytoplasmic tail of the rhabdovirus glycoprotein may be truncated at 2 to 80, 3 to 75, 4 to 70, 5 to 65, 6 to 60, 7 to 55, 8 to 50, 9 to 45, 10 to 40, 11 to 35, 12 to 30, 13 to 25, 14 to 20, or 15 to 35 amino acids from the C-terminus. In certain embodiments, the truncated cytoplasmic tail of the rhabdovirus glycoprotein may be truncated at 10 to 40 amino acids from the C-terminus. In some embodiments, the cytoplasmic tail of a rhabdovirus glycoprotein is comprised of from the C-terminus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, In some embodiments, the cytoplasmic tail of a rhabdovirus glycoprotein can be truncated 30 amino acids from the C-terminus.

[0333] In some embodiments, the cytoplasmic tail of a rhabdovirus glycoprotein described herein can be truncated up to 40 amino acids from the C-terminus. In some embodiments, the cytoplasmic tail of a rhabdovirus glycoprotein can be truncated 10 to 40 amino acids from the C-terminus. In some embodiments, the cytoplasmic tail of a rhabdovirus glycoprotein can be truncated 30 amino acids from the C-terminus.

[0334] In some embodiments, the recombinant fusion protein described herein can further comprise a cytoplasmic tail from VSV-G, or a functional fragment or derivative thereof. A non-limiting example of a cytoplasmic tail is the amino acid sequence of CIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 16). In some embodiments, the cytoplasmic tail of the rhabdovirus glycoprotein described herein comprises the amino acid sequence of SEQ ID NO: 16, or a functional fragment or derivative thereof that has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 16. In certain embodiments, the nucleotide sequence encoding the cytoplasmic tail of rhabdovirus glycoprotein comprises the amino acid sequence of SEQ ID NO: 16, or the nucleotide sequence encoding its variant that has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 16. In certain embodiments, the cytoplasmic tail of rhabdovirus glycoprotein comprises the amino acid sequence of SEQ ID NO: 16.

[0335] In some embodiments, the fusogenic proteins included in the recombinant viruses of the present disclosure may also include other fusogens. For example, a form of hemagglutinin (HA) from influenza A / fowl plague virus / Rostock / 34 (FPV), a class I fusogen, may be used. In some embodiments, a form of FPV HA may be used. HA-mediated fusion is generally independent of receptor binding. As another example, the Sindbis virus glycoprotein (a class II fusogen) from the alphavirus family may be used.

[0336] In some embodiments, the fusogenic molecule is Sindbis virus envelope protein (SIN). SINdbis virus transfers SINdbis viral RNA into cells through low-pH-mediated membrane fusion. SIN contains five structural proteins: E1, E2, E3, 6K, and capsid. E2 contains a receptor-binding sequence that allows wild-type SIN to bind, while E1 is known to contain the properties necessary for membrane fusion. E1, E2, and E3 are encoded by a polyprotein, the amino acid sequences of which are provided, for example, by accession numbers VHWVB, VHWVB2, and P03316, and the nucleic acid sequences of which are provided, for example, by accession numbers SVU90536 and V01403.

[0337] In some embodiments, the Sindbis virus envelope protein is mutated (SINmu). In certain embodiments, the mutation reduces the natural tropism of the Sindbis virus. In certain embodiments, in SINmu, which comprises SIN proteins E1, E2, and E3, at least one of E1, E2, or E3 is mutated compared to the wild-type sequence. For example, one or more of the E1, E2, or E3 proteins can be mutated at one or more amino acid positions. In addition, combinations of mutations in E1, E2, and E3 are encompassed by the fusogens described herein, such as mutations in E1 and E2, or E2 and E3, or E3 and E1, or E1, E2, and E3. In certain embodiments, at least E2 is mutated.

[0338] In some embodiments, SINmu comprises the following envelope protein mutations compared to wild-type Sindbis virus envelope protein: (i) a deletion of E3 amino acids 61-64, (ii) E2 KE159-160AA, and (iii) E2 SLKQ68-71AAAA. In some embodiments, SINmu comprises the envelope protein mutation E1 AK226-227SG.

[0339] Other Togaviridae family envelopes from the alphavirus genus, such as Semliki Forest virus, Ross River virus, and equine encephalitis virus, can also be used to pseudotype the vectors described herein. Envelope protein sequences for such alphaviruses are known in the art.

[0340] Linker In certain aspects, the present disclosure provides a recombinant fusion protein, comprising rhabdovirus glycoprotein (G) or its functional fragment or derivative and a targeting molecule that can be linked to the N-terminus of rhabdovirus glycoprotein or its functional fragment or derivative via a linker.In some embodiments, the linker can be sensitive to proteolytic cleavage, for example, through naturally occurring cell-associated proteases.In some embodiments, the linker can be sensitive to proteolytic cleavage, for example, through endogenous proteases.In some embodiments, the linker can be sensitive to proteolytic cleavage, for example, through exogenously added proteases.In some embodiments, the linker can be, for example, but not limited to, arginine (R) and / or lysine (K) residue.

[0341] In some embodiments, linkers of the present disclosure may not be susceptible to proteolytic cleavage by endogenous proteases. In some embodiments, linkers of the present disclosure may not be susceptible to proteolytic cleavage by exogenously added proteases.

[0342] In some embodiments, the linker can be 1 to 50 amino acids in length. By way of non-limiting example, the linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more amino acids in length. In some embodiments, the linker can be 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 35, 1 to 40, or 1 to 50 or more amino acids in length.

[0343] In some embodiments, the linker may be optimized so that the linker does not impose any constraints on the conformation and / or interactions of the linked partners.

[0344] In some embodiments, the linker may be a flexible linker. Suitable linkers can be easily selected and may be of any of a variety of suitable lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids. Exemplary flexible linkers include glycine polymers (Gly) and glycine polymers (Gly). n , glycine-serine polymer (GS) n (where n is an integer of at least 1 (eg, 1-20)), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.

[0345] In some embodiments, the linker has the sequence (GGGS) n(SEQ ID NO: 42), where n=1-10, or n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0346] In some embodiments, the linker has the sequence (GGGGS) n (SEQ ID NO: 43), where n=1-10, or n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0347] In some embodiments, the linker can be a rigid linker.

[0348] In some embodiments, a linker of the present disclosure can comprise any linker shown in Table 2, or a variant thereof having at least 50, at least 55, at least 60, at least 65, 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, or at least 99% sequence identity to a linker sequence shown in Table 2. Linker sequences are shown in underlined text. [Table 2-1] [Table 2-2] [Table 2-3]

[0349] Further non-limiting examples of linkers that may be used include any of the various linker sequences shown in Table 5, or variants thereof having at least 50, at least 55, at least 60, at least 65, 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, or at least 99% sequence identity to a linker sequence shown in Table 5.

[0350] In some embodiments, a linker described herein may comprise a linker sequence set forth in the amino acid sequence of SEQ ID NO:1-7, 18-57, or 164-174, or a variant thereof having at least at least 50, at least 55, at least 60, at least 65, 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, or at least 99% sequence identity to the linker sequence set forth in the amino acid sequence of SEQ ID NO:1-7, 18-57, or 164-174.

[0351] In some embodiments, the linker sequences described herein may be included within an amino acid sequence that includes one or more amino acids of an antibody sequence or antigen-binding fragment thereof described herein. As a non-limiting example, the antigen-binding fragment may be any of the various antigen-binding fragments described herein, such as, but not limited to, a single-chain fragment variable (scFv) described herein.

[0352] In some embodiments, the linker sequences and / or amino acid sequences comprising a linker sequence described herein may comprise one or more amino acids comprising the amino acid sequence EIKR (SEQ ID NO: 58). In some embodiments, the linker sequences and / or amino acid sequences comprising a linker sequence described herein may comprise the amino acid sequence EIK of an antibody or antigen-binding fragment thereof described herein. In some embodiments, the linker sequences and / or amino acid sequences comprising a linker sequence described herein may comprise the amino acid sequence EI of an antibody or antigen-binding fragment thereof described herein. In some embodiments, the linker sequences and / or amino acid sequences comprising a linker sequence described herein may comprise any one of the amino acids E, I, K, R, or any combination thereof of an antibody or antigen-binding fragment thereof described herein.

[0353] In some embodiments, the linker sequences and / or amino acid sequences comprising a linker sequence described herein may comprise one or more amino acids comprising the amino acid sequence LGAK (SEQ ID NO: 59). In some embodiments, the linker sequences and / or amino acid sequences comprising a linker sequence described herein may comprise the sequence LGAK (SEQ ID NO: 59) of an antibody or antigen-binding fragment thereof described herein. In some embodiments, the linker sequences and / or amino acid sequences comprising a linker sequence described herein may comprise the amino acid sequence LG of an antibody or antigen-binding fragment thereof described herein. In some embodiments, the linker sequences and / or amino acid sequences comprising a linker sequence described herein may comprise amino acid L or G, or any combination thereof, of an antibody or antigen-binding fragment thereof described herein.

[0354] In some embodiments, the linker sequences described herein may be included within an amino acid sequence that includes one or more amino acids of the starting amino acid sequence of the VSV-G protein, or fragment or derivative thereof, described herein. In some embodiments, the linker sequences and / or amino acid sequences that include a linker sequence described herein may include one or more amino acids that include the amino acid sequence KFT of the VSV-G protein. In some embodiments, the linker sequences and / or amino acid sequences that include a linker sequence described herein may include one or more amino acids that include the amino acid sequence FT of the VSV-G protein. In some embodiments, the linker sequences and / or amino acid sequences that include a linker sequence described herein may include any one of the amino acids K, F, or T of the VSV-G protein, or any combination thereof.

[0355] In some embodiments, the linkers described herein can be cleavable or non-cleavable. In some embodiments, the linker is a cleavable linker. In other embodiments, the linker is a non-cleavable linker. The cleavable linker can be a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker. These linkers are generally only cleavable intracellularly and are preferably stable in the extracellular environment.

[0356] Protease-sensitive linkers are cleavable by protease enzyme activity. These linkers typically comprise peptide sequences and can be 2-10 amino acids, about 2-5 amino acids, about 5-10 amino acids, about 10 amino acids, about 5 amino acids, about 3 amino acids, or about 2 amino acids in length. In some embodiments, the peptide sequence may comprise natural amino acids, such as cysteine ​​or alanine, or unnatural or modified amino acids. Unnatural amino acids include 3-amino acids, homoamino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and others known in the art. In some embodiments, the protease-sensitive linker comprises a valine-citrulline or alanine-citrulline dipeptide sequence. In some embodiments, the protease-sensitive linker can be cleaved by lysosomal proteases, such as cathepsin B, and / or endosomal proteases.

[0357] A pH-sensitive linker is a covalent bond that readily degrades in high or low pH environments. In some embodiments, the pH-sensitive linker can be cleaved at a pH in the range of 4 to 6. In some embodiments, the pH-sensitive linker comprises a hydrazone or a cyclic acetal. In some embodiments, the pH-sensitive linker is cleaved in an endosome or lysosome.

[0358] In some embodiments, the glutathione-sensitive linker comprises a disulfide moiety. In some embodiments, the glutathione-sensitive linker is cleaved by a disulfide exchange reaction with intracellular glutathione species. In some embodiments, the disulfide moiety further comprises at least one amino acid, e.g., a cysteine ​​residue.

[0359] Non-limiting examples of cleavable linkers are set forth in the amino acid sequences of SEQ ID NOs: 1-7, 18-26, 30, 169, and 174. In some embodiments, the cleavable linker may comprise SEQ ID NOs: 1-7, 18-26, and 30, or a variant thereof having at least 50, at least 55, at least 60, at least 65, 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, or at least 99% sequence identity to SEQ ID NOs: 1-7, 18-26, and 30, or to SEQ ID NOs: 1-7, 18-26, and 30, 169, and 174. In some embodiments, the cleavable linker may be susceptible to cleavage via proteolytic cleavage. In some embodiments, proteolytic cleavage may occur by naturally occurring cell-associated proteases (e.g., endogenous proteases) and / or by exogenously added proteases. In some embodiments, proteolytic cleavage may occur by endogenous proteases. In some embodiments, proteolytic cleavage can occur by an exogenous protease.

[0360] In some embodiments, the protease capable of proteolytic cleavage, e.g., proteolytic cleavage of the linker described herein, can belong to a class of proteases, such as, but not limited to, serine proteases, cystine proteases, or metalloproteinases. In some embodiments, the endogenous protease can be found, for example, but not limited to, in the ER, Golgi, the cell surface of cells from which viruses can be released, the supernatant (or body fluids), the surface of cells that can be targeted by viruses, or the endocytic compartment of a target cell. Protease cleavage signals for various proteases have been extensively studied, for example, using high-throughput proteomics approaches known in the art. Those skilled in the art will understand that there are numerous cleavage signal options for a given protease, which can vary greatly depending on the context. In some embodiments, protease cleavage signal pairings can have different cleavage kinetics, and many membrane proteins can be only partially shed from the cell surface, for example, due to slower kinetics and / or regulated activity of their cleaving proteases (e.g., sheddases). In some embodiments, the proteases described herein may include any of the various proteases, or variants thereof, described in, for example, Encyclopedia of Cell Biology. 2016:650-60; Int. J. Mol. Sci. 2020, 21(18), 6805 and / or Molecular Neurobiology volume 56, pages 3090-3112, 2019 (both of which are incorporated by reference in their entirety for all purposes).

[0361] In some embodiments, the linker can be a non-cleavable linker (also known as a non-cleavable linker). Generally, a non-cleavable linker cannot be readily degraded in a cell or physiological environment. Non-limiting examples of non-cleavable linkers are set forth in the amino acid sequences of SEQ ID NOs: 27-29, 31, 32, 36, and 37. In some embodiments, the cleavable linker can include SEQ ID NOs: 27-29, 31, 32, 36, and 37, or variants thereof having at least 50, at least 55, at least 60, at least 65, 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, or at least 99% sequence identity to SEQ ID NOs: 27-29, 31, and 32.

[0362] In various embodiments, the linkers described herein may be contained within a sequence including, for example, but not limited to, AAASGGS(G4S)2GPK (SEQ ID NO: 1); KRAAASGGS(G4S)2GPK (SEQ ID NO: 174), KRAAASGGS(G4S)2 (SEQ ID NO: 2); (EAAAK)3 (SEQ ID NO: 3); KR(EAAAK)3 (SEQ ID NO: 4); AAARGSPK(G4S)3 (SEQ ID NO: 5); KRAAARGSPK(G4S)3 (SEQ ID NO: 18); RAAARGSPK(G4S)3 (SEQ ID NO: 169); AAARGSPK(G4S)3K (SEQ ID NO: 19); K(G4S)3 (SEQ ID NO: 20); KR(G4S)3 (SEQ ID NO: 21); (G4S)3GPK (SEQ ID NO: 6); or AAA(G4S)3K (SEQ ID NO: 7).

[0363] In some embodiments, the linkers described herein may be contained within a sequence set forth in SEQ ID NOs: 1-7, 18-57, or 164-174, or a variant thereof, or a sequence having at least 50, at least 55, at least 60, at least 65, 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, or at least 99% sequence identity to a sequence set forth in SEQ ID NOs: 1-7, 18-57, or 164-174.

[0364] In some embodiments, the linkers described herein may be contained within a sequence shown in Table 5, or a variant thereof, or a sequence having at least 50, at least 55, at least 60, at least 65, 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, or at least 99% sequence identity to a sequence shown in Table 5.

[0365] In some embodiments, the linkers described herein may be contained within a sequence shown in Table 2, or a variant thereof, or a sequence having at least 50, at least 55, at least 60, at least 65, 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, or at least 99% sequence identity to a sequence shown in Table 2.

[0366] In some embodiments, the linker sequences described herein and / or amino acid sequences comprising the linker sequences described herein may include the amino acid sequence alanine-alanine-alanine (AAA), which may be replaced with the amino acid sequence glycine-glycine-glycine (GGG) (i.e., the linker and / or amino acid sequence comprising the linker may include an AAA to GGG mutation), which may allow for more freedom of movement of the domains displayed on the linkers described herein (e.g., targeting molecules described herein).

[0367] Both alanine (A) and glycine (G) are the simplest nonpolar neutral amino acids, but glycine (G) is hydrophilic and alanine (A) is hydrophobic. In some embodiments, the linker described herein can be positioned between the N-terminus of VSV-G (e.g., blinded VSV-G) and a displayed domain (e.g., a targeting molecule described herein) that can bind to a receptor on a desired cell, for example. Therefore, because the linker will be exposed to the external environment, placing a hydrophilic amino acid (e.g., glycine (G)) in the linker sequence can allow for greater flexibility and movement of the displayed domain, which can improve the targeting of the displayed domain. Hydrophobic amino acids (e.g., alanine (A)) can affect the tertiary conformation of the protein in the external hydrophilic environment and can drive the linker itself in addition to the displayed domain. This can reduce the displayed domain's ability to move to access its desired receptor. In some embodiments, hydrophilic amino acids (glycine (G)) can facilitate manipulation of the viral particles described herein, for example, when folding is impaired by the presence of hydrophobic amino acids (e.g., alanine (A)).

[0368] As a non-limiting example, a linker sequence described herein may comprise the amino acid sequence RAAASGGS(G4S)2 (SEQ ID NO: 172), which, when mutated to include the AAA to GGG mutation described herein, may comprise the amino acid sequence RGGGSGGS(G4S)2 (SEQ ID NO: 176). As another non-limiting example, an amino acid sequence comprising a linker sequence may comprise the amino acid sequence KRAAASGGS(G4S)2 (SEQ ID NO: 2), which, when mutated to include the AAA to GGG mutation described herein, may comprise the amino acid sequence KRGGGSGGS(G4S)2 (SEQ ID NO: 175). In some embodiments, a linker sequence described herein may comprise the amino acid sequence RGGGSGGS(G4S)2 (SEQ ID NO: 176). In some embodiments, an amino acid sequence comprising a linker sequence described herein may comprise the amino acid sequence KRGGGSGGS(G4S)2 (SEQ ID NO: 175).

[0369] As yet another non-limiting example, a linker sequence described herein can comprise the amino acid sequence RAAASGGS(G4S)2GP (SEQ ID NO: 171), which, when mutated to include the AAA to GGG mutation described herein, can comprise the amino acid sequence RGGGSGGS(G4S)2GP (SEQ ID NO: 178). As yet another non-limiting example, an amino acid sequence comprising a linker sequence can comprise the amino acid sequence KRAAASGGS(G4S)2GPK (SEQ ID NO: 174), which, when mutated to include the AAA to GGG mutation described herein, can comprise the amino acid sequence KRGGGSGGS(G4S)2GPK (SEQ ID NO: 177). In some embodiments, a linker sequence described herein can comprise the amino acid sequence RGGGSGGS(G4S)2GP (SEQ ID NO: 178). In some embodiments, an amino acid sequence comprising a linker sequence described herein can comprise the amino acid sequence KRGGGSGGS(G4S)2GPK (SEQ ID NO: 177).

[0370] In some embodiments, a linker sequence described herein may comprise the amino acid sequence (EAAAK)3 (SEQ ID NO: 3), which when mutated to include the AAA to GGG mutation described herein, may comprise the amino acid sequence (EGGGK)3 (SEQ ID NO: 205). In some embodiments, a linker sequence described herein may comprise the amino acid sequence (EGGGK)3 (SEQ ID NO: 205).

[0371] In some embodiments, a linker sequence described herein may comprise the amino acid sequence R(EAAAK)3 (SEQ ID NO: 173), which, when mutated to include an AAA to GGG mutation described herein, may comprise the amino acid sequence R(EGGGK)3 (SEQ ID NO: 207). In some embodiments, an amino acid sequence comprising a linker sequence may comprise the amino acid sequence KR(EAAAK)3 (SEQ ID NO: 4), which, when mutated to include an AAA to GGG mutation described herein, may comprise the amino acid sequence KR(EGGGK)3 (SEQ ID NO: 206). In some embodiments, a linker sequence described herein may comprise the amino acid sequence R(EGGGK)3 (SEQ ID NO: 207). In some embodiments, an amino acid sequence comprising a linker sequence described herein may comprise the amino acid sequence KR(EGGGK)3 (SEQ ID NO: 206).

[0372] In some embodiments, a linker sequence described herein may comprise the amino acid sequence AAARGSPK(G4S)3 (SEQ ID NO:5), which, when mutated to include the AAA to GGG mutation described herein, may comprise the amino acid sequence GGGRGSPK(G4S)3 (SEQ ID NO:208). In some embodiments, a linker sequence described herein may comprise the amino acid sequence GGGRGSPK(G4S)3 (SEQ ID NO:208).

[0373] In some embodiments, a linker sequence described herein may comprise the amino acid sequence RAAARGSPK(G4S)3 (SEQ ID NO: 169), which, when mutated to include the AAA to GGG mutation described herein, may comprise the amino acid sequence RGGGRGSPK(G4S)3 (SEQ ID NO: 209). In some embodiments, a linker sequence described herein may comprise the amino acid sequence RGGGRGSPK(G4S)3 (SEQ ID NO: 209).

[0374] In some embodiments, an amino acid sequence comprising a linker sequence may comprise the amino acid sequence AAARGSPK(G4S)3K (SEQ ID NO: 19), which, when mutated to comprise an AAA to GGG mutation as described herein, may comprise the amino acid sequence GGGRGSPK(G4S)3K (SEQ ID NO: 210). In some embodiments, an amino acid sequence comprising a linker sequence as described herein may comprise the amino acid sequence GGGRGSPK(G4S)3K (SEQ ID NO: 210).

[0375] In some embodiments, a linker sequence described herein may comprise the amino acid sequence AAA(G4S)3 (SEQ ID NO: 34), which, when mutated to include an AAA to GGG mutation described herein, may comprise the amino acid sequence GGG(G4S)3 (SEQ ID NO: 212). In some embodiments, an amino acid sequence comprising a linker sequence may comprise the amino acid sequence AAA(G4S)3K (SEQ ID NO: 7), which, when mutated to include an AAA to GGG mutation described herein, may comprise the amino acid sequence GGG(G4S)3K (SEQ ID NO: 211). In some embodiments, a linker sequence described herein may comprise the amino acid sequence GGG(G4S)3K (SEQ ID NO: 212). In some embodiments, an amino acid sequence comprising a linker sequence described herein may comprise the amino acid sequence GGG(G4S)3K (SEQ ID NO: 211).

[0376] targeting molecule In various embodiments, the recombinant fusion protein described herein can comprise rhabdovirus glycoprotein (G), or its functional fragment or derivative, and targeting molecule.Targeting molecule can be located at the N-terminus of rhabdovirus glycoprotein.In certain aspects, targeting molecule can be linked to the N-terminus of rhabdovirus glycoprotein, or its functional fragment or derivative, for example, by any of the various linkers described herein or their combinations.In some embodiments, the N-terminus of the rhabdovirus glycoprotein, or its functional fragment or derivative, to which targeting molecule is linked via a linker, does not contain one or more amino acids present at the N-terminus of mature wild-type rhabdovirus glycoprotein.In some embodiments, the rhabdovirus glycoprotein (G) that is linked to targeting molecule can comprise any of the various rhabdovirus glycoproteins described herein, or its functional fragment or derivative.For example, as described herein, rhabdovirus glycoproteins include vesicular stomatitis virus glycoprotein (VSV-G), Flanders virus glycoprotein (FLAV-G), Chandipura virus glycoprotein (CHPV-G), Perinetovirus glycoprotein (PERV-G), Piryvirus glycoprotein (PIRYV-G), Fukuoka virus glycoprotein (FUKV-G), Joint-Jakaka virus glycoprotein (JOIV-G), Kumasi virus glycoprotein (KRV-G), Isfahan glycoprotein (ISFV-G), Jurona glycoprotein (JURV-G), Mediterranean bat glycoprotein (MBV-G), Malpais spring glycoprotein (MSPV-G), Radi glycoprotein (RADV-G), Rhinolophus affinis-G, Yugbugdanabok glycoprotein (YBV-G), Inshui bat glycoprotein (YSBV-G), Keuraliba glycoprotein (KEUV-G), Kimberly glycoprotein (KIMV-G), Kanyawara glycoprotein (KYAV-G), La Jolla glycoprotein (LJV-G), Mosquiero glycoprotein (MQOV-G), Parry Creek glycoprotein (PCV-G), Bascongo glycoprotein (BASV-G), Bovine ephemeral fever glycoprotein (BEFV-G), Clionopolis glycoprotein (CURV-G), Drosophila melanogaster sigma virus glycoprotein (DMelSV-G), Niaka glycoprotein (NIAV-G), Puerto Armandros glycoprotein (PTAMV-G), or Tupaia rubdo virus (TUPTV-G), or a functional fragment or derivative thereof.

[0377] In some embodiments, when rhabdovirus G protein comprises VSV-G or its functional fragment or derivative, targeting molecule can prevent VSV-G or its functional fragment or derivative from interacting with low-density lipoprotein receptor (LDLR).In some embodiments, targeting molecule can prevent VSV-G or its functional fragment or derivative from interacting with LDLR, for example, by steric hindrance.

[0378] Non-limiting examples of targeting molecules include antibodies or antigen-binding fragments thereof, affibodies, darpins, peptides, natural or modified natural receptor ligands, T cell receptors (TCRs) or fragments or derivatives thereof, or MHC-peptide complexes or fragments or derivatives thereof.

[0379] In some embodiments, the targeting molecule targets, for example, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), mucin 16 (MUC16), cKit, alpha-v beta-3 (αVβ3) integrin, insulin-like growth factor 1 receptor (IGF1R), B-cell maturation antigen (BCMA), Nectin-4, mitogen-activated protein kinase kinase (MEK), cluster of differentiation 44 (CD44), CD3, CD4, CD28, stem cell factor (SCF), thrombopoietin, c-Met, CXCR4, IL2R, or interleukin 3 (IL-3).

[0380] In some embodiments, the targeting molecules described herein target cells, such as, but not limited to, cancer cells, including tumor cells, including cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testicle, tongue, or uterus. In addition, the cancer may be of the following histological types, specifically, but not limited to: neoplasia, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; calcifying carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma, familial polyposis coli; solid tumor; carcinoma tumor, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulating sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma carcinoma); skin adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; cerumen gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease; breast; acinic cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor, malignant; and loblastoma, malignant; Sertoli cell carcinoma; Leiden Zich cell tumor, malignant; Lipid cell tumor, malignant; Paraganglioma, malignant; Extramammary paraganglioma, malignant; Pheochromocytoma; Hemangioangiosarcoma; Malignant melanoma; Amelanotic melanoma; Superficial spreading melanoma; Malignant melanoma in giant pigmented nevus; Epithelioid cell melanoma; Blue nevus, malignant; Sarcoma; Fibrosarcoma; Fibrohistiocytoma, malignant; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor, malignant; Mixed Müllerian tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymoma, malignant;Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; ovarial goiter, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; hemangioendothelial sarcoma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; adjacent cortical sarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastoma; ameloblastoma, malignant; ameloblastoma; ameloblastoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal Tumors; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory nerve tumor; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranulomatous; malignant lymphoma, small lymphocytic; malignant lymphoma, giant cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. ;

[0381] In some embodiments, the targeting molecule is selected from a humanized antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single-chain fragment variable (scFv), a bis-scFv, an (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a single heavy chain antibody, a bispecific antibody or binding fragment thereof, a bispecific T-cell engager (BiTE), a triabody, or a chemically modified derivative thereof.

[0382] The antibodies described herein may comprise immunoglobulin molecules comprising four polypeptide chains, two heavy chains (HC) and two light chains (LC), interconnected by disulfide bonds (e.g., IgG). In various embodiments, each antibody heavy chain (HC) comprises a heavy chain variable region ("HCVR" or "V"). H ") and a heavy chain constant region, and each antibody light chain (LC) comprises a light chain variable region ("LCVR" or "V L ") and the light chain constant region (CL). H and V L The regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs).

[0383] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant domain, e.g., of the IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, and IgG4 (e.g., containing S228P and / or S108P mutations)), or IgM type. In some embodiments, the antibody or antigen-binding fragment thereof may comprise a light chain constant domain, e.g., of the kappa or lambda type. In one embodiment of the present disclosure, the V H can be linked to a human heavy chain constant domain (e.g., IgG), and V L can be linked to a human light chain constant domain (e.g., kappa).

[0384] In some embodiments, the assignment of amino acids to each framework or CDR domain follows the definitions in Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991), Kabat (1978) Adv. Prot. Chem. 32:1-75, Kabat et al., (1977) J. Biol. Chem. 252:6609-6616, Chothia et al., (1987) J Mol. Biol. 196:901-917, or Chothia et al., (1989) Nature 342:878-883. Thus, the present disclosure includes antibodies and antigen-binding fragments comprising a VH CDR and a VL CDR, wherein the VH and VL comprise the amino acid sequences set forth herein (or variants thereof), and the CDRs are as defined, e.g., according to Kabat and / or Chothia.

[0385] In some embodiments, the antibody or antigen-binding fragment thereof may comprise a heavy chain constant domain of, e.g., an IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, and IgG4 (e.g., containing an S228P and / or S108P mutation)), or IgM type. In embodiments of the present disclosure, the antigen-binding protein, e.g., an antibody or antigen-binding fragment, comprises a light chain constant domain of, e.g., a kappa or lambda type.

[0386] The term "human" antibody or antigen-binding fragment, as used herein, includes antibodies and fragments having variable and constant regions derived from human amino acid sequences, e.g., human germline immunoglobulin sequences, whether in a human cell or grafted into a non-human cell, e.g., a mouse cell. The human antibodies and antigen-binding fragments of the present disclosure, in one embodiment of the present disclosure, may include, for example, amino acid residues in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., with mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) are grafted onto human FR sequences. The term includes antibodies recombinantly produced in a non-human mammal or in the cells of a non-human mammal. The term is not intended to include antibodies isolated from or generated in a human subject.

[0387] The present disclosure includes chimeric antibodies and antigen-binding fragments thereof, as well as methods for using them. As used herein, a "chimeric antibody" is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, wherein the first and second antibodies are derived from different species. (See, e.g., US4816567, and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855). The present disclosure includes chimeric antibodies comprising a variable domain and a non-human constant domain.

[0388] Antigen-binding fragments of antibodies, in various embodiments, include antibodies that are less than the complete antibody, but still specifically bind to an antigen, including, for example, at least one variable domain. The variable domain can be of any size or amino acid composition and generally includes at least one (e.g., three) CDR(s) adjacent to or in frame with one or more framework sequences. LV associated with the domain H For antigen-binding fragments containing domains, V H The V domain and VL domain can be positioned relative to each other in any suitable configuration. For example, the variable region is a dimer, H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of non-covalently linked monomeric V H and / or V L It may include a domain.

[0389] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present disclosure include: (i) a V H -CH1, (ii) V H -CH2, (iii) V H -CH3, (iv)V H -CH1-CH2, (v) V H -CH1-CH2-CH3, (vi) V H -CH2-CH3, (vii)V H -CL, (viii)V L -CH1, (ix)V L -CH2, (x)V L -CH3, (xi)V L -CH1-CH2, (xii) V L -CH1-CH2-CH3, (xiii)V L -CH2-CH3, and (xiv) V L-CL. In any arrangement of variable and constant domains, including any of the exemplary arrangements listed above, the variable and constant domains can be either directly linked to each other or linked by a complete or partial hinge or linker region. The hinge region can consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure can be linked to each other and / or to one or more monomeric V H Domain or V L It may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain arrangements listed above in non-covalent association with the domains (e.g., by disulfide bond(s)).

[0390] Antibodies and antigen-binding fragments thereof can be monospecific or multispecific (eg, bispecific).

[0391] The antibodies and antigen-binding fragments described herein may be fused to other polypeptide molecules, such as, but not limited to, an epitope (e.g., FLAG) or tag sequence (e.g., His tag sequence, etc.), to allow for detection and / or isolation of the antibody or antigen-binding fragment, a ligand or portion thereof that binds to a transmembrane receptor protein, an enzyme or portion thereof that is catalytically active, a polypeptide or peptide that promotes oligomerization such as a leucine zipper domain, a polypeptide or peptide that increases stability such as an immunoglobulin constant region (e.g., an Fc domain), a half-life extending polypeptide (e.g., albumin or an albumin-binding peptide / protein), a functional or non-functional antibody, or heavy or light chain thereof, and / or a polypeptide having an activity distinct from the antibody or antigen-binding fragment of the present disclosure.

[0392] In some embodiments, the antibodies or antigen-binding fragments thereof described herein may be post-translationally modified, including, for example, Glu or Gln cyclization at the N-terminus, loss of the N-terminal positive charge, Lys variants at the C-terminus, deamidation (Asn to Asp), isomerization (Asp to isoAsp), deamidation (Gln to Glu), oxidation (Cys, His, Met, Tyr, Trp), and / or disulfide bond heterogeneity (shuffling, thioether, and trisulfide formation).

[0393] In some embodiments, the antigen binding protein comprises a fragment antigen binding region (Fab). In some embodiments, the antigen binding protein comprises a single-chain fragment variable (scFv). In some embodiments, the scFv comprises domains arranged in the following orientation from N-terminus to C-terminus: HCVR-LCVR. In some embodiments, the scFv comprises domains arranged in the following orientation from N-terminus to C-terminus: LCVR-HCVR, where the scFv variable regions are connected by a linker, such as, but not limited to, any of the various linkers described herein.

[0394] The terms "specifically binds" or "binds specifically" refer to a targeting molecule (e.g., an antibody or antigen-binding fragment thereof) that has binding affinity for an antigen. The present disclosure includes targeting molecules that specifically bind to, for example, but are not limited to, EGFR, HER2, MUC16, cKit, αVβ3 integrin, IGF1R, BCMA, Nectin-4, MEK, CD44, CD3, CD4, CD28, stem cell factor, thrombopoietin, c-Met, CXCR4, IL2R, or IL-3. In some embodiments, the targeting molecules disclosed herein may comprise an scFv that targets the tumor antigen HER2 (also referred to as human epidermal growth factor receptor 2, HER-2, c-erbB-2, C-ErbB-2, C-ERB-2, c-ERB2, etc.). In some embodiments, the anti-HER2 scFv comprises domains arranged in the following orientation from N-terminus to C-terminus: HCVR-LCVR. In some embodiments, the anti-HER2 scFv comprises domains arranged in the following orientation from C-terminus to N-terminus: LCVR-HCVR. In some embodiments, the scFv variable regions of the anti-HER2 scFv are connected by a linker, such as, but not limited to, those described herein. In some embodiments, the anti-HER2 scFv specifically binds to human HER2. By way of example, an anti-HER2 scFv that can be used in the practice of the present disclosure can include, for example, any anti-HER2 scFv described by Shier and colleagues (Shier et al., "Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site." J Mol Biol. 1996 Nov 8;263(4):551-67, the contents of which are incorporated herein by reference in their entireties), or a variant thereof.In some embodiments, the anti-HER2 scFv can include clone C6-B1D2, such as that described in Shier et al. (1996), in some cases at about, e.g., 0.15 x 10. -10 K of M d It can bind to human HER2.

[0395] In some embodiments, the anti-HER2 scFv described herein is derived from the antibody C6B1D2. In some embodiments, the anti-HER2 scFv derived from the antibody C6B1D2 comprises the amino acid sequence of SEQ ID NO: 158, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the anti-HER2 scFv derived from the antibody C6B1D2 comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 158, or an amino acid sequence having at least 80% identity thereto.

[0396] In some embodiments, an anti-HER2 scFv derived from antibody C6B1D2 comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, a nucleotide sequence encoding the HCVR of an anti-HER2 scFv derived from antibody C6B1D2 comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence having at least 80% identity thereof. In some embodiments, an anti-HER2 scFv derived from antibody C6B1D2 comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 160, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, a nucleotide sequence encoding the LCVR of an anti-HER2 scFv derived from antibody C6B1D2 comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 160, or an amino acid sequence having at least 80% identity thereof. In some embodiments, an anti-HER2 scFv derived from antibody C6B1D2 comprises a linker sequence between the HCVR and LCVR, wherein the linker sequence comprises the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence with at least 80% sequence identity. In some embodiments, a nucleotide sequence encoding the linker between the HCVR and LCVR of an anti-HER2 scFv derived from antibody C6B1D2 comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence with at least 80% identity thereto. In some embodiments, an anti-HER2 scFv derived from antibody C6B1D2 comprises domains arranged in the following orientation from N-terminus to C-terminus: HCVR-LCVR. In some embodiments, an anti-HER2 scFv derived from antibody C6B1D2 comprises domains arranged in the following orientation from N-terminus to C-terminus: LCVR-HCVR, wherein the anti-HER2 scFv variable regions are connected by a linker, such as, but not limited to, any of the various linkers described herein.

[0397] In some embodiments, the anti-HER2 scFv described herein is derived from the antibody C6.5. In some embodiments, the anti-HER2 scFv derived from the antibody C6.5 comprises the amino acid sequence of SEQ ID NO: 192, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the anti-HER2 scFv derived from the antibody C6.5 comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 192, or an amino acid sequence having at least 80% identity thereto.

[0398] In some embodiments, an anti-HER2 scFv derived from antibody C6.5 comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 193, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the HCVR of an anti-HER2 scFv derived from antibody C6.5 comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 193, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, an anti-HER2 scFv derived from antibody C6.5 comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 194, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the LCVR of an anti-HER2 scFv derived from antibody C6.5 comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 194, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, an anti-HER2 scFv derived from antibody C6.5 comprises a linker sequence between the HCVR and LCVR, wherein the linker sequence comprises the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the linker between the HCVR and LCVR of the anti-HER2 scFv derived from antibody C6.5 comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence having at least 80% identity thereto. In some embodiments, the anti-HER2 scFv derived from antibody C6.5 comprises domains arranged in the following orientation from N-terminus to C-terminus: HCVR-LCVR. In some embodiments, the anti-HER2 scFv derived from antibody C6.5 comprises domains arranged in the following orientation from N-terminus to C-terminus: LCVR-HCVR, wherein the anti-HER2 scFv variable regions are connected by a linker, such as, but not limited to, any of the various linkers described herein.

[0399] In some embodiments, the targeting molecules disclosed herein can comprise an scFv that targets the tumor antigen EGFR (which may also be called epidermal growth factor receptor, ERBB1, receptor tyrosine-protein kinase ErbB-1, etc.). In some embodiments, the anti-EGFR scFv comprises domains arranged in the following orientation from N-terminus to C-terminus: HCVR-LCVR. In some embodiments, the anti-EGFR scFv comprises domains arranged in the following orientation from C-terminus to N-terminus: LCVR-HCVR. In some embodiments, the scFv variable regions of the anti-EGFR scFv are connected by a linker, such as, but not limited to, the linkers described herein. In some embodiments, the anti-EGFR scFv specifically binds to human EGFR. By way of example, anti-EGFR scFvs that can be used in the practice of the present disclosure can include, for example, any of the anti-EGFR scFvs described in Nakamura and colleagues (Nakamura et al., Antibody-targeted cell fusion, Nature Biotechnology volume 22, pages 331-336 (2004), the contents of which are incorporated herein by reference in their entirety), or variants thereof.

[0400] In some embodiments, the anti-EGFR scFv described herein comprises the amino acid sequence of SEQ ID NO: 161, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the anti-EGFR scFv comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 161, or an amino acid sequence having at least 80% identity thereof.

[0401] In some embodiments, the anti-EGFR scFv described herein comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 162, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the HCVR of the anti-EGFR scFv comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 162, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the anti-EGFR scFv comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 163, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the LCVR of the anti-EGFR scFv comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 163, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the anti-EGFR scFv comprises a linker sequence between the HCVR and LCVR, wherein the linker sequence comprises the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the linker between the HCVR and LCVR of the anti-EGFR scFv comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence having at least 80% identity thereto. In some embodiments, the anti-EGFR scFv comprises domains arranged in the following orientation from N-terminus to C-terminus: HCVR-LCVR. In some embodiments, the anti-EGFR scFv comprises domains arranged in the following orientation from N-terminus to C-terminus: LCVR-HCVR, wherein the anti-EGFR scFv variable regions are connected by a linker, such as, but not limited to, any of the various linkers described herein.

[0402] In some embodiments, the targeting molecules disclosed herein may comprise an scFv targeting the tumor antigen cKit (which may also be referred to as c-kit, KIT Proto-Oncogene, Receptor Tyrosine Kinase, SCFR, V-Kit Hardy-Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog, Obesity / Stem Cell Growth Factor Receptor Kit, CD117, PBT, Tyrosine-Protein Kinase Kit, Piebald Trait Protein, Proto-Oncogene C-Kit, P145 C-Kit, C-Kit, V-Kit Hardy-Zuckerman 4 Feline Sarcoma Viral Oncogene-Like Protein, Proto-Oncogene Tyrosine-Protein Kinase Kit, C-Kit Protooncogene, Piebald Trait, CD117 antigen, MASTC, C-KIT, etc.). In some embodiments, the anti-cKit scFv comprises domains arranged in the following orientation from N-terminus to C-terminus: HCVR-LCVR. In some embodiments, the anti-cKit scFv comprises domains arranged in the following orientation from C-terminus to N-terminus: LCVR-HCVR. In some embodiments, the scFv variable regions of the anti-cKit scFv are connected by a linker, such as, but not limited to, the linkers described herein. In some embodiments, the anti-cKit scFv specifically binds to human cKit.

[0403] In some embodiments, the anti-cKit scFv described herein comprises the amino acid sequence of SEQ ID NO: 195, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the anti-cKit scFv comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 195, or an amino acid sequence having at least 80% identity thereof.

[0404] In some embodiments, the anti-cKit scFv described herein comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 196, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the HCVR of the anti-cKit scFv comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 196, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the anti-cKit scFv comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 197, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the LCVR of the anti-cKit scFv comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 197, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the anti-cKit scFv comprises a linker sequence between the HCVR and LCVR, wherein the linker sequence comprises the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the linker between the HCVR and LCVR of the anti-cKit scFv comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence having at least 80% identity thereto. In some embodiments, the anti-cKit scFv comprises domains arranged in the following orientation from N-terminus to C-terminus: HCVR-LCVR. In some embodiments, the anti-cKit scFv comprises domains arranged in the following orientation from N-terminus to C-terminus: LCVR-HCVR, wherein the anti-cKit scFv variable regions are connected by a linker, such as, but not limited to, any of the various linkers described herein.

[0405] In some embodiments, the targeting molecule disclosed herein may comprise a nanobody that targets the tumor antigen EGFR. A non-limiting example of a nanobody that targets EGFR is nanobody Nb 7D12 (anti-EGFR) (also referred to as EGFRNb). In some embodiments, nanobody Nb 7D12 (anti-EGFR) may comprise the amino acid sequence QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSALE (SEQ ID NO: 187), or an amino acid sequence having at least 80% identity thereto. Another non-limiting example of a nanobody that targets EGFR is nanobody Nb 9G8 (anti-EGFR). In some embodiments, nanobody Nb 9G8 (anti-EGFR) may comprise the amino acid sequence EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSALE (SEQ ID NO: 188), or an amino acid sequence with at least 80% identity thereof.

[0406] In some embodiments, the targeting molecule can be a ligand (e.g., a natural receptor ligand) or a fragment or derivative thereof, such as, but not limited to, EGFm123 (modified EGF), human stem cell factor (hSCF), and human insulin-like growth factor 1 (hIGF1). In some embodiments, EGFm123 of the present disclosure can comprise the amino acid sequence NSYSECPPSYDGYCLHDGVCRYIEALDSYACNCVVGYAGERCQYRDLRWWGRR (SEQ ID NO: 186), or an amino acid sequence having at least 80% identity thereto. In some embodiments, a human stem cell factor (hSCF) of the present disclosure can comprise the amino acid sequence EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAA (SEQ ID NO: 189), or an amino acid sequence with at least 80% identity thereof. In some embodiments, a human insulin-like growth factor 1 (hIGF1) of the present disclosure can comprise the amino acid sequence GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA (SEQ ID NO: 190), or an amino acid sequence with at least 80% identity thereof.

[0407] In some embodiments, the targeting molecule disclosed herein may comprise a T cell receptor (TCR) or a fragment or derivative thereof. TCR can recognize peptides presented in the context of major histocompatibility complex (MHC) molecules. Peptide-MHC (pMHC) complexes can be recognized by TCRs, with the peptide (antigenic determinant) and TCR idiotype providing the specificity of the interaction. Thus, the antigens described herein may encompass peptides presented in the context of MHC molecules. Peptides that can be presented on MHC molecules may also comprise epitopes described herein. In some embodiments, epitopes may not only be those naturally presented by antigen-presenting cells (APCs), but may also be any desired peptide, as long as it is recognized by immune cells when properly presented to cells of the immune system. For example, peptides with artificially prepared amino acid sequences may also be used as epitopes. In some embodiments, in addition to a fusogen, such as, but not limited to, VSV-G or a functional fragment or derivative thereof, described herein, the viral particles described herein may further exhibit a TCR binding molecule. In some embodiments, the TCR binding molecule and fusogen may be comprised within a recombinant fusion protein described herein. In some embodiments, the TCR binding molecule may comprise a TCR-specific antibody or portion thereof.

[0408] In various embodiments, the TCR binding molecules described herein can comprise a peptide presented in the context of an MHC molecule, e.g., an antigenic determinant associated with (or within) the peptide-binding groove of an MHC. In some embodiments, the viral particles described herein, e.g., viral particles comprising a recombinant fusogenic protein described herein, can bind to an antigen-specific T cell receptor (TCR), wherein the recombinant viral particle comprises (i) a peptide (p) presented in the context of a major histocompatibility complex (MHC) molecule, i.e., a pMHC complex, and (ii) a lipid envelope comprising a fusogen. In some embodiments, the antigen-specific TCR specifically binds to the pMHC complex.

[0409] MHC molecules are generally classified into two categories: class I and class II MHC. MHC class I molecules are integral membrane proteins containing a glycoprotein heavy chain, also referred to herein as the α chain, which has three extracellular domains (i.e., α1, α2, and α3) and two extracellular domains (i.e., a transmembrane domain (TM) and a cytoplasmic domain (CYT)). The heavy chain is noncovalently associated with a soluble subunit called β2-microglobulin (β2m or B2M). MHC class II proteins are heterodimeric integral membrane proteins containing one α chain and one β chain in noncovalent association. The α chain has two extracellular domains (α1 and α2) and two extracellular domains (a TM domain and a CYT domain). The β chain contains two extracellular domains (β1 and β2) and two extracellular domains (a TM domain and a CYT domain).

[0410] The domain organization of class I and class II MHC forms the antigenic determinant binding site, or peptide-binding groove. The peptide-binding groove refers to the portion of the MHC protein that forms a cavity to which a peptide, e.g., an antigenic determinant, can bind. The conformation of the peptide-binding groove changes upon peptide binding, allowing for proper alignment of amino acid residues important for TCR binding to the peptide-MHC (pMHC) complex.

[0411] The MHCs described herein include fragments of the MHC chains sufficient to form a peptide-binding groove. For example, the peptide-binding groove of a class I protein can include portions of the α1 and α2 domains of the heavy chain, which can form two β-pleated sheets and two α-helices. The inclusion of a portion of the β2-microglobulin chain stabilizes the complex. In most versions of MHC class II molecules, the interaction between the α and β chains can occur in the absence of peptide, but the two-chain complex of an MHC class I is unstable until the binding groove is filled with peptide. The peptide-binding groove of a class II protein can include portions of the α1 and β1 domains, which can form two β-pleated sheets and two α-helices. The first portion of the α1 domain forms the first β-pleated sheet, and the second portion of the α1 domain forms the first α-helix. The first portion of the β1 domain forms the second β-pleated sheet, and the second portion of the β1 domain forms the second α-helix. X-ray crystal structures of class II proteins with peptides engaged in the protein's binding groove indicate that one or both ends of the engaged peptide may protrude beyond the MHC protein. Thus, the ends of the class II α1 and β1 α-helices form an open cavity, preventing the end of the peptide bound in the binding groove from becoming buried. Furthermore, X-ray crystal structures of class II proteins indicate that the N-terminus of the MHC β-chain apparently protrudes unstructured from the side of the MHC protein, as the first four amino acid residues of the β-chain could not be assigned by X-ray crystallography.

[0412] Many human and other mammalian MHC molecules are known in the art, and any MHC class I or class II molecule can be part of the TCR binding molecules described herein.

[0413] MHC molecules useful in the viral particles described herein include naturally occurring full-length MHC molecules, as well as individual chains of MHC molecules (e.g., MHC class I α (heavy) chain, β2-microglobulin, MHC class II α chain, and MHC class II β chain), individual subunits of such chains of MHC molecules (e.g., α1, α2, and / or α3 subunits of the MHC class I α chain, α1 and / or α2 subunits of the MHC class II α chain, β1 and / or β2 subunits of the MHC class II β chain), and fragments, mutants, and various derivatives thereof, which retain the ability to present antigenic determinants for recognition by antigen-specific TCRs. In a specific embodiment, the MHC comprises a transmembrane domain embedded in the lipid envelope of the viral particle.

[0414] Naturally occurring MHC molecules are encoded by a cluster of genes on human chromosome 6 or mouse chromosome 17. These MHC molecules, designated H-2 in mice and HLA (human leukocyte antigens) in humans, are classified as either class I or class II molecules. MHC class I molecules specifically bind to CD8 molecules expressed on cytotoxic T lymphocytes (CD8+ T cells), while MHC class II molecules specifically bind to CD4 molecules expressed on helper T lymphocytes (CD4+ T cells). MHC specificities include, but are not limited to, HLA specificities such as A (e.g., A1-A74), B (e.g., B1-B77), C (e.g., C1-C11), D (e.g., D1-D26), E, ​​G, DR (e.g., DR1-DR8), DQ (e.g., DQ1-DQ9), and DP (e.g., DP1-DP6). More preferably, the HLA specificities include A1, A2, A3, A11, A23, A24, A28, A30, A33, B7, B8, B35, B44, B53, B60, B62, DR1, DR2, DR3, DR4, DR7, DR8, and DR-11.

[0415] The MHCs described herein can be derived from any mammalian or avian species, for example, primates (eg, humans), rodents, rabbits, horses, cows, dogs, cats, pigs, etc.

[0416] Naturally occurring MHC class I molecules bind peptides derived from proteolytically degraded proteins, particularly endogenously synthesized proteins, by cells. The resulting small peptides are transported to the endoplasmic reticulum, where they are routed through the Golgi apparatus and associated with nascent MHC class I molecules before being presented on the cell surface for recognition by cytotoxic T lymphocytes.

[0417] Naturally occurring MHC class I molecules consist of an α (heavy) chain associated with β2-microglobulin. The heavy chain consists of subunits α1 to α3. The β2-microglobulin protein and the α3 subunit of the heavy chain are associated. In certain embodiments, the β2-microglobulin and the α3 subunit are covalently linked. In certain embodiments, the β2-microglobulin and the α3 subunit are non-covalently linked. The α1 and α2 subunits of the heavy chain fold to form a groove for peptides, e.g., antigenic determinants, to be presented and recognized by TCRs.

[0418] Class I molecules bind to peptides about 8-9 amino acids in length. Every human has three to six different Class I molecules, each of which can bind many different types of peptides.

[0419] In some embodiments, the MHC comprises (i) a Class I MHC polypeptide, or a fragment, variant, or derivative thereof, and optionally (ii) a β2 microglobulin polypeptide, or a fragment, variant, or derivative thereof. In one particular embodiment, the Class I MHC polypeptide is linked to the β2 microglobulin polypeptide by a peptide linker.

[0420] In some embodiments, the class I MHC polypeptide is a human class I MHC polypeptide selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In another specific embodiment, the class I MHC polypeptide is a mouse class I MHC polypeptide selected from the group consisting of H-2K, H-2D, H-2L, H2-IA, H2-IB, H2-IJ, H2-IE, and H2-IC.

[0421] In some embodiments, the viral particle comprises one or more MHC class I α heavy chains. In some embodiments, the MHC class I α heavy chains are fully human. In some embodiments, the MHC class I α heavy chains are humanized. Humanized MHC class I α heavy chains are described, for example, in U.S. Patent Publication Nos. 2013 / 0111617, 2013 / 0185819, and 2014 / 0245467, both of which are incorporated by reference in their entireties. In some embodiments, the MHC class I α heavy chain comprises a human extracellular domain (human α1, α2, and / or α3 domains) and a cytoplasmic domain of another species. In some embodiments, the class I α heavy chain polypeptide is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, or HLA-L. In some embodiments, the HLA-A sequence can be an HLA-A*0201 sequence. In various embodiments, the peptide-MHC may include all domains of an MHC class I heavy chain.

[0422] In some embodiments, the viral particle comprises β2-microglobulin. In some embodiments, the β2-microglobulin is fully human. In some embodiments, the β2-microglobulin is humanized. Humanized β2-microglobulin polypeptides are described, for example, in U.S. Patent Publication Nos. 2013 / 0111617 and 2013 / 0185819, both of which are incorporated by reference in their entireties.

[0423] In some embodiments, the MHC class I molecule comprises mutations in the β2-microglobulin (β2m or B2M) polypeptide and heavy chain sequence that affect the disulfide bond between B2M and the heavy chain. In some cases, the heavy chain is an HLA, and the disulfide bond links one of the following residue pairs: B2M residue 12, HLA residue 236; B2M residue 12, HLA residue 237; B2M residue 8, HLA residue 234; B2M residue 10, HLA residue 235; B2M residue 24, HLA residue 236; B2M residue 28, HLA residue 232; B2M residue 98, HLA residue 192; B2M residue 99, HLA residue 234; B2M residue 3, HLA residue 120; B2M residue 31, HLA residue 96; B2M residue 53, HLA residue 35; B2M residue 60, HLA Residue 96; B2M residue 60, HLA residue 122; B2M residue 63, HLA residue 27; B2M residue Arg3, HLA residue Glyl20; B2M residue His31, HLA residue Gln96 ;B2M residue Asp53, HLA residue Arg35;B2M residue Trp60, HLA residue Gln96;B2M residue Trp60, HLA residue Aspl22;B2M residue Tyr63, H LA residue Tyr27; B2M residue Lys6, HLA residue Glu232; B2M residue Gln8, HLA residue Arg234; B2M residue TyrlO, HLA residue Pro235; B2M residue Group Ser1, HLA residue Gln242; B2M residue Asn24, HLA residue Ala236; B2M residue Ser28, HLA residue Glu232; B2M residue Asp98, HLA residue His192; and B2M residue Met99, HLA residue Arg234, first linker position Gly2, heavy chain (HLA) position Tyr84; light chain (B2M) position Arg12, HLA Ala236; and / or B2M residue Arg12, HLA residue Gly237. See, e.g., International Patent Application Publication No. WO 2015 / 195531.

[0424] In some embodiments, the antigenic determinant amino acid sequence can be that of a peptide capable of being presented by an MHC class I molecule. In certain embodiments, the sequence can comprise about 8 to about 15 contiguous amino acids. In certain embodiments, the peptide sequence can be that of a protein fragment, e.g., derived from a portion of an infectious agent or a cellular protein, e.g., a protein expressed by a cancer cell, and the peptide can bind to an MHC class I heavy chain.

[0425] In some embodiments, at least one chain of the MHC and peptide is included within the fusion protein described herein. In one specific embodiment, the MHC and peptide are separated by a linker sequence. For example, the single-chain molecule can comprise, from the amino to the carboxy terminus, an antigenic determinant, a β2-microglobulin sequence, and a class I α (heavy) chain sequence. Alternatively, the single-chain molecule can comprise, from the amino to the carboxy terminus, an antigenic determinant, a class I α (heavy) chain sequence, and a β2-microglobulin sequence. The single-chain molecule can further comprise a signal peptide sequence at the amino terminus. In certain embodiments, there can be a linker sequence between the peptide sequence and the β2-microglobulin sequence. In certain embodiments, there can be a linker sequence between the β2-microglobulin sequence and the class I α (heavy) chain sequence. The single-chain molecule can further comprise a signal peptide sequence at the amino terminus, and a first linker sequence extending between the peptide sequence and the β2-microglobulin sequence and / or a second linker sequence extending between the β2-microglobulin sequence and the class I heavy chain sequence. In certain embodiments, the β2-microglobulin and class I α (heavy) chain sequences can be human, murine, or porcine.

[0426] In some embodiments, the single-chain molecule can include a first flexible linker between the peptide ligand segment and the β2-microglobulin segment. For example, the linker can extend from the carboxy terminus of the peptide ligand segment and connect to the amino te...

Claims

1. A recombinant fusion protein, the fusion protein comprising: (i) a rhabdovirus glycoprotein (G), or a functional fragment or derivative thereof; (ii) a targeting molecule, wherein the targeting molecule is attached to the N-terminus of the rhabdovirus glycoprotein, or the functional fragment or derivative thereof, via a linker, wherein the linker is susceptible to proteolytic cleavage by an endogenous protease or an exogenously added protease.

2. The recombinant fusion protein of claim 1 , wherein the linker comprises arginine (R) and / or lysine (K) residues.

3. The linker is KRAAASGGS(G ４ S) ２ GPK (SEQ ID NO: 174), KRAAASGGS(G ４ S) ２ (SEQ ID NO: 2), (EAAAK) ３ (SEQ ID NO: 3), KR (EAAAK) ３ (SEQ ID NO: 4), AAARGSPK(G ４ S) ３ (SEQ ID NO: 5), RAAAARGSPK(G ４ S) ３ (SEQ ID NO: 169), AAARGSPK(G ４ S) ３ K (SEQ ID NO: 19), K (G ４ S) ３ (SEQ ID NO: 20), KR (G ４ S) ３ (SEQ ID NO: 21), (G ４ S) ３ GPK (SEQ ID NO: 6), and AAA (G ４ S) ３ 2. The recombinant fusion protein of claim 1, wherein the fusion protein is comprised within a sequence selected from the group consisting of:

4. 4. The recombinant fusion protein of any one of claims 1 to 3, wherein the N-terminus of the rhabdovirus glycoprotein, or the functional fragment or derivative thereof, to which the targeting molecule is attached via a linker, does not include one or more amino acids present at the N-terminus of a mature wild-type rhabdovirus glycoprotein.

5. The recombinant fusion protein according to any one of claims 1 to 4, wherein the rhabdovirus glycoprotein is vesicular stomatitis virus glycoprotein (VSV-G), or a functional fragment or derivative thereof.

6. The recombinant fusion protein of claim 5, wherein the VSV-G comprises the sequence of SEQ ID NO:

8.

7. The recombinant fusion protein of claim 6, wherein the VSV-G consists of the sequence of SEQ ID NO:

8.

8. 8. The recombinant fusion protein of any one of claims 5 to 7, wherein the targeting molecule is capable of interfering with the ability of the VSV-G, or the functional fragment or derivative thereof, to interact with the low density lipoprotein receptor (LDLR).

9. 9. The recombinant fusion protein of claim 5, wherein the VSV-G, or the functional fragment or derivative thereof, comprises one or more mutations that reduce or eliminate binding of the VSV-G polypeptide, or the functional fragment or derivative thereof, to LDLR.

10. 10. The recombinant fusion protein of claim 9, wherein the one or more mutations in the VSV-G, or the functional fragment or derivative thereof, comprise one or more amino acid substitutions and / or deletions at positions corresponding to H8, K47, Y209, or R354 in SEQ ID NO:

8.

11. 11. The recombinant fusion protein of claim 10, wherein the VSV-G comprises or consists of SEQ ID NO: 8, and the one or more mutations are substitutions at positions K47 and R354.

12. 11. The recombinant fusion protein of claim 10, wherein the VSV-G comprises or consists of SEQ ID NO: 8, and the one or more mutations are substitutions at positions K47, R354, and Y209.

13. 11. The recombinant fusion protein of claim 10, wherein the VSV-G comprises or consists of SEQ ID NO: 8, and the one or more mutations are substitutions at position H8.

14. 11. The recombinant fusion protein of claim 10, wherein the one or more mutations in the VSV-G, or the functional fragment or derivative thereof, comprise one or more amino acid deletions at positions corresponding to H8, K47, Y209, or R354 in SEQ ID NO:

8.

15. 15. The recombinant fusion protein of claim 14, wherein the VSV-G comprises or consists of SEQ ID NO:

8.

16. 16. The recombinant fusion protein of claim 15, wherein the one or more deletions is a deletion at position K47.

17. 16. The recombinant fusion protein of claim 15, wherein the one or more deletions are deletions at position H8.

18. 16. The recombinant fusion protein of claim 15, wherein the one or more deletions are deletions at positions H8 and K47.

19. The recombinant fusion protein of any one of claims 5 to 18, wherein the VSV-G, or the functional fragment or derivative thereof, further comprises one or more mutations that increase viral titer.

20. 20. The recombinant fusion protein of claim 19, wherein the one or more viral titer-increasing mutations in the VSV-G, or the functional fragment or derivative thereof, are M184T and / or F250L, as specified relative to their positions in SEQ ID NO:

8.

21. The recombinant fusion protein according to any one of claims 1 to 4, wherein the rhabdovirus glycoprotein is a glycoprotein derived from Flanders virus (FLAV-G).

22. 22. The recombinant fusion protein of claim 21, wherein the FLAV-G comprises the sequence of SEQ ID NO:

9.

23. 23. The recombinant fusion protein of claim 22, wherein said FLAV-G consists of the sequence of SEQ ID NO:

9.

24. The recombinant fusion protein according to any one of claims 1 to 4, wherein the rhabdovirus glycoprotein is a glycoprotein derived from Chandipura virus (CHPV-G).

25. 25. The recombinant fusion protein of claim 24, wherein the CHPV-G comprises the sequence of SEQ ID NO:

10.

26. 26. The recombinant fusion protein of claim 25, wherein said CHPV-G consists of the sequence of SEQ ID NO:

10.

27. The recombinant fusion protein according to any one of claims 1 to 4, wherein the rhabdovirus glycoprotein is a glycoprotein derived from Perinet virus (PERV-G).

28. 28. The recombinant fusion protein of claim 27, wherein the PERV-G comprises the sequence of SEQ ID NO:

11.

29. 29. The recombinant fusion protein of claim 28, wherein the PERV-G consists of the sequence of SEQ ID NO:

11.

30. The recombinant fusion protein according to any one of claims 1 to 4, wherein the rhabdovirus glycoprotein is a glycoprotein (PIRYV-G) derived from Piry virus.

31. 31. The recombinant fusion protein of claim 30, wherein said PIRYV-G comprises the sequence of SEQ ID NO:

12.

32. 32. The recombinant fusion protein of claim 31, wherein said PIRYV-G consists of the sequence of SEQ ID NO:

12.

33. The recombinant fusion protein according to any one of claims 1 to 4, wherein the rhabdovirus glycoprotein is a glycoprotein derived from Fukuoka virus (FUKV-G).

34. The recombinant fusion protein of claim 33, wherein the FUKV-G comprises the sequence of SEQ ID NO:

13.

35. The recombinant fusion protein of claim 34, wherein the FUKV-G consists of the sequence of SEQ ID NO:

13.

36. The recombinant fusion protein according to any one of claims 1 to 4, wherein the rhabdovirus glycoprotein is a glycoprotein derived from Joinjakaka virus (JOIV-G).

37. 37. The recombinant fusion protein of claim 36, wherein the JOIV-G comprises the sequence of SEQ ID NO:

14.

38. 38. The recombinant fusion protein of claim 37, wherein the JOIV-G consists of the sequence of SEQ ID NO:

14.

39. The recombinant fusion protein according to any one of claims 1 to 4, wherein the rhabdovirus glycoprotein is a glycoprotein derived from Kumasi virus (KRV-G).

40. 40. The recombinant fusion protein of claim 39, wherein the KRV-G comprises the sequence of SEQ ID NO:

15.

41. The recombinant fusion protein of claim 40, wherein the KRV-G consists of the sequence of SEQ ID NO:

15.

42. The recombinant fusion protein according to any one of claims 1 to 4, wherein the rhabdovirus glycoprotein is a glycoprotein derived from Keuraliva virus (KEUV-G).

43. 43. The recombinant fusion protein of claim 42, wherein the KEUV-G comprises the sequence of SEQ ID NO:

17.

44. 44. The recombinant fusion protein of claim 43, wherein the KEUV-G comprises the sequence of SEQ ID NO:

17.

45. 45. The recombinant fusion protein of any one of claims 21 to 44, wherein the cytoplasmic tail of the rhabdovirus glycoprotein has been removed or truncated and, optionally, replaced with another sequence.

46. 46. ​​The recombinant fusion protein of claim 45, wherein the cytoplasmic tail of the glycoprotein is truncated at most 40 amino acids from the C-terminus.

47. 47. The recombinant fusion protein of claim 46, wherein the cytoplasmic tail of the rhabdovirus glycoprotein is truncated 10 to 40 amino acids from the C-terminus.

48. 48. The recombinant fusion protein of claim 47, wherein the cytoplasmic tail of the rhabdovirus glycoprotein is truncated 30 amino acids from the C-terminus.

49. The recombinant fusion protein of any one of claims 45 to 48, further comprising a cytoplasmic tail from VSV-G, or a functional fragment or derivative thereof.

50. 50. The recombinant fusion protein of claim 49, wherein the cytoplasmic tail of VSV-G comprises the sequence CIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 16).

51. A recombinant fusion protein, comprising a fusogen having at least 60% amino acid sequence identity to vesicular stomatitis virus glycoprotein (VSV-G) comprising SEQ ID NO: 8, or a functional fragment or derivative thereof, wherein the fusogen, or the functional fragment or derivative thereof, comprises one or more amino acid deletions at positions corresponding to H8, K47, Y209, or R354 in SEQ ID NO:

8.

52. 52. The recombinant fusion protein of claim 51, wherein the fusogen comprises the sequence of SEQ ID NO: 8, or a functional fragment or derivative thereof, having one or more amino acid deletions at positions H8, K47, Y209, or R354.

53. 53. The recombinant fusion protein of claim 52, wherein the fusogen comprises or consists of the sequence of SEQ ID NO: 8, having an amino acid deletion at position H8.

54. 53. The recombinant fusion protein of claim 52, wherein the fusogen comprises or consists of the sequence of SEQ ID NO: 8, having amino acid deletions at positions H8 and K47.

55. The recombinant fusion protein of claim 52, wherein the fusogen comprises the sequence of SEQ ID NO: 8, having amino acid deletions at (i) position K47, (ii) position R354, and (iii) position H8 or Y209.

56. The recombinant fusion protein of claim 55, wherein the fusogen consists of the sequence of SEQ ID NO: 8, having amino acid deletions at (i) position K47, (ii) position R354, and (iii) position H8 or Y209.

57. 53. The recombinant fusion protein of claim 52, wherein the fusogen comprises the sequence of SEQ ID NO: 8, having an amino acid deletion at position K47.

58. 58. The recombinant fusion protein of claim 57, wherein the fusogen consists of the sequence of SEQ ID NO: 8, having an amino acid deletion at position K47.

59. A recombinant fusion protein comprising a fusogen having the sequence of SEQ ID NO: 8, with amino acid substitutions at (i) position K47, (ii) position R354, and (iii) position H8 or Y209.

60. The recombinant fusion protein of claim 59, wherein the fusogen consists of the sequence of SEQ ID NO: 8, having amino acid substitutions at (i) position K47, (ii) position R354, and (iii) position H8 or Y209.

61. A recombinant fusion protein comprising a fusogen having the sequence of SEQ ID NO: 8, with amino acid substitutions at positions K47, R354, H8, and Y209.

62. 62. The recombinant fusion protein of claim 61, wherein the fusogen consists of the sequence of SEQ ID NO: 8, with amino acid substitutions at positions K47, R354, H8, and Y209.

63. 63. The recombinant fusion protein of any one of claims 51 to 62, wherein the fusogen, or the functional fragment or derivative thereof, further comprises one or more viral titer-increasing mutations.

64. 64. The recombinant fusion protein of claim 63, wherein the one or more viral titer-increasing mutations are at one or more positions corresponding to positions M184 and / or F250 in SEQ ID NO:

8.

65. 65. The recombinant fusion protein of any one of claims 51 to 64, further comprising a targeting molecule positioned at the N-terminus of said fusogen, or said functional fragment or derivative thereof.

66. 66. The recombinant fusion protein of claim 65, wherein the targeting molecule is attached to the N-terminus of the fusogen, or the functional fragment or derivative thereof, via a linker.

67. 67. The recombinant fusion protein of claim 66, wherein the linker is susceptible to proteolytic cleavage by an endogenous protease or by an exogenously added protease.

68. 68. The recombinant fusion protein of claim 67, wherein the linker comprises arginine (R) and / or lysine (K) residues.

69. The linker is KRAAASGGS(G ４ S) ２ GPK (SEQ ID NO: 174), KRAAASGGS(G ４ S) ２ (SEQ ID NO: 2), (EAAAK) ３ (SEQ ID NO: 3), KR (EAAAK) ３ (SEQ ID NO: 4), AAARGSPK(G ４ S) ３ (SEQ ID NO: 5), RAAAARGSPK(G ４ S) ３ (SEQ ID NO: 169), AAARGSPK(G ４ S) ３ K (SEQ ID NO: 19), K (G ４ S) ３ (SEQ ID NO: 20), KR (G ４ S) ３ (SEQ ID NO: 21), (G ４ S) ３ GPK (SEQ ID NO: 6), or AAA (G ４ S) ３ 68. The recombinant fusion protein of claim 67, wherein the fusion protein is comprised within a sequence selected from:

70. 67. The recombinant fusion protein of claim 66, wherein the linker is not susceptible to proteolytic cleavage by endogenous proteases or by exogenously added proteases.

71. A recombinant fusion protein described in any one of claims 65 to 70, wherein the N-terminus of the fusogen, or its functional fragment or derivative, to which the targeting molecule is attached does not include one or more amino acids present at the N-terminus of a mature wild-type fusogen.

72. A recombinant fusion protein, the fusion protein comprising: (i) a glycoprotein from Flanders virus (FLAV-G), or a functional fragment or derivative thereof; (ii) a targeting molecule.

73. 73. The recombinant fusion protein of claim 72, wherein the FLAV-G comprises the sequence of SEQ ID NO:

9.

74. 74. The recombinant fusion protein of claim 73, wherein said FLAV-G consists of the sequence of SEQ ID NO:

9.

75. A recombinant fusion protein, the fusion protein comprising: (i) a glycoprotein from Chandipura virus (CHPV-G), or a functional fragment or derivative thereof; (ii) a targeting molecule.

76. 76. The recombinant fusion protein of claim 75, wherein the CHPV-G comprises the sequence of SEQ ID NO:

10.

77. 77. The recombinant fusion protein of claim 76, wherein said CHPV-G consists of the sequence of SEQ ID NO:

10.

78. A recombinant fusion protein, the fusion protein comprising: (i) a glycoprotein from Perinetovirus (PERV-G), or a functional fragment or derivative thereof; (ii) a targeting molecule.

79. 79. The recombinant fusion protein of claim 78, wherein the PERV-G comprises the sequence of SEQ ID NO:

11.

80. 80. The recombinant fusion protein of claim 79, wherein the PERV-G consists of the sequence of SEQ ID NO:

11.

81. A recombinant fusion protein, the fusion protein comprising: (i) a glycoprotein derived from a Piryvirus (PIRYV-G), or a functional fragment or derivative thereof; (ii) a targeting molecule.

82. 82. The recombinant fusion protein of claim 81, wherein said PIRYV-G comprises the sequence of SEQ ID NO:

12.

83. 83. The recombinant fusion protein of claim 82, wherein said PIRYV-G consists of the sequence of SEQ ID NO:

12.

84. A recombinant fusion protein, the fusion protein comprising: (i) a glycoprotein derived from Fukuoka virus (FUKV-G), or a functional fragment or derivative thereof; (ii) a targeting molecule.

85. 85. The recombinant fusion protein of claim 84, wherein the FUKV-G comprises the sequence of SEQ ID NO:

13.

86. 86. The recombinant fusion protein of claim 85, wherein the FUKV-G consists of the sequence of SEQ ID NO:

13.

87. A recombinant fusion protein, the fusion protein comprising: (i) a glycoprotein from Joint Jacaka virus (JOIV-G), or a functional fragment or derivative thereof; (ii) a targeting molecule.

88. 88. The recombinant fusion protein of claim 87, wherein the JOIV-G comprises the sequence of SEQ ID NO:

14.

89. 89. The recombinant fusion protein of claim 88, wherein the JOIV-G consists of the sequence of SEQ ID NO:

14.

90. A recombinant fusion protein, the fusion protein comprising: (i) a glycoprotein from Kumasi virus (KRV-G), or a functional fragment or derivative thereof; (ii) a targeting molecule.

91. 91. The recombinant fusion protein of claim 90, wherein the KRV-G comprises the sequence of SEQ ID NO:

15.

92. 92. The recombinant fusion protein of claim 91, wherein the KRV-G consists of the sequence of SEQ ID NO:

15.

93. A recombinant fusion protein, the fusion protein comprising: (i) a glycoprotein from Keurariba virus (KEUV-G), or a functional fragment or derivative thereof; (ii) a targeting molecule.

94. 94. The recombinant fusion protein of claim 93, wherein the KEUV-G comprises the sequence of SEQ ID NO:

17.

95. 95. The recombinant fusion protein of claim 94, wherein the KEUV-G consists of the sequence of SEQ ID NO:

17.

96. 96. The recombinant fusion protein of any one of claims 72 to 95, wherein the glycoprotein is a fragment, and the cytoplasmic tail of the glycoprotein has been removed or truncated and, optionally, replaced with another sequence.

97. 97. The recombinant fusion protein of claim 96, wherein the cytoplasmic tail of the glycoprotein is truncated by up to 40 amino acids from the C-terminus.

98. 98. The recombinant fusion protein of claim 97, wherein the cytoplasmic tail of the glycoprotein is truncated 10 to 40 amino acids from the C-terminus.

99. 99. The recombinant fusion protein of claim 98, wherein the cytoplasmic tail of the glycoprotein is truncated 30 amino acids from the C-terminus.

100. 100. The recombinant fusion protein of any one of claims 96 to 99, further comprising a cytoplasmic tail from VSV-G, or a functional fragment or derivative thereof.

101. 101. The recombinant fusion protein of claim 100, wherein the cytoplasmic tail of VSV-G comprises the sequence CIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 16).

102. The recombinant fusion protein of any one of claims 72 to 101, wherein said targeting molecule is located at the N-terminus of said glycoprotein, or said functional fragment or derivative thereof.

103. 103. The recombinant fusion protein of claim 102, wherein the targeting molecule is attached to the N-terminus of the glycoprotein, or the functional fragment or derivative thereof, via a linker.

104. 104. The recombinant fusion protein of claim 103, wherein the linker is susceptible to proteolytic cleavage by an endogenous protease or by an exogenously added protease.

105. 105. The recombinant fusion protein of claim 104, wherein the linker comprises arginine (R) and / or lysine (K) residues.

106. The linker is KRAAASGGS(G ４ S) ２ GPK (SEQ ID NO: 174), KRAAASGGS(G ４ S) ２ (SEQ ID NO: 2), (EAAAK) ３ (SEQ ID NO: 3), KR (EAAAK) ３ (SEQ ID NO: 4), AAARGSPK(G ４ S) ３ (SEQ ID NO: 5), RAAAARGSPK(G ４ S) ３ (SEQ ID NO: 169), AAARGSPK(G ４ S) ３ K (SEQ ID NO: 19), K (G ４ S) ３ (SEQ ID NO: 20), KR (G ４ S) ３ (SEQ ID NO: 21), (G ４ S) ３ GPK (SEQ ID NO: 6), and AAA (G ４ S) ３ 105. The recombinant fusion protein of claim 104, wherein the fusion protein is comprised within a sequence selected from:

107. 104. The recombinant fusion protein of claim 103, wherein the linker is not susceptible to proteolytic cleavage by endogenous proteases or by exogenously added proteases.

108. A recombinant fusion protein according to any one of claims 102 to 107, wherein the N-terminus of the glycoprotein, or the functional fragment or derivative thereof, to which the targeting molecule is attached, does not include one or more amino acids present at the N-terminus of a mature wild-type fusogen.

109. 109. The recombinant fusion protein of any one of claims 1-50 and 65-108, wherein the targeting molecule is an antibody or antigen-binding fragment thereof, an affibody, a darpin, a peptide, a natural or modified natural receptor ligand, a T-cell receptor or a fragment or derivative thereof, or an MHC-peptide complex or a fragment or derivative thereof.

110. 110. The recombinant fusion protein of claim 109, wherein the antibody or antigen-binding fragment thereof is a single-chain fragment variable (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a single heavy chain antibody.

111. 111. The recombinant fusion protein of any one of claims 1-50 and 65-110, wherein the targeting molecule targets EGFR, HER2, MUC16, cKit, αVβ3 integrin, IGF1R, BCMA, Nectin-4, MEK, CD44, CD3, CD4, CD28, stem cell factor, thrombopoietin, c-Met, CXCR4, IL2R, or IL-3.

112. A recombinant polynucleotide encoding the recombinant fusion protein of any one of claims 1 to 111.

113. 113. The recombinant polynucleotide of Claim 112, wherein said polynucleotide comprises a sequence encoding a signal peptide sequence, such signal sequence being positioned at the extreme N-terminus of the encoded recombinant fusion protein.

114. 114. The recombinant polynucleotide of claim 112 or 113, wherein the polynucleotide is DNA.

115. 114. The recombinant polynucleotide of claim 112 or 113, wherein the polynucleotide is RNA.

116. 112. A recombinant polynucleotide, wherein the recombinant polynucleotide is an RNA molecule comprising a nucleotide sequence that is a template for a positive-sense transcription product encoding a recombinant fusion protein of any one of claims 1 to 111.

117. 117. The recombinant polynucleotide of Claim 116, wherein the positive-sense transcript comprises a sequence encoding a signal peptide sequence, such signal sequence being positioned at the extreme N-terminus of the encoded recombinant fusion protein.

118. 118. The recombinant polynucleotide of claim 116 or 117, wherein the recombinant polynucleotide is an RNA molecule comprising: a nucleotide sequence that is a template for a positive-sense transcript encoding a vesicular stomatitis virus (VSV) nucleoprotein (N) polypeptide, or a functional fragment or derivative thereof; a nucleotide sequence that is a template for a positive-sense transcript encoding a VSV phosphoprotein (P) polypeptide, or a functional fragment or derivative thereof; a nucleotide sequence that is a template for a positive-sense transcript encoding a VSV matrix (M) polypeptide, or a functional fragment or derivative thereof; a nucleotide sequence that is a template for a positive-sense transcript encoding a fusion protein of any one of claims 1 to 111; and a nucleotide sequence that is a template for a positive-sense transcript encoding a VSV large protein (L) polypeptide, or a functional fragment or derivative thereof.

119. 119. The recombinant polynucleotide of claim 118, wherein the VSV M polypeptide is a mutant VSV M polypeptide.

120. 120. The recombinant polynucleotide of claim 119, wherein the mutant VSV M polypeptide comprises a mutation at methionine (M) 51.

121. 121. The recombinant polynucleotide of claim 120, wherein the mutation at methionine (M) 51 is a substitution of methionine (M) with arginine (R).

122. 122. The recombinant polynucleotide of any one of claims 112 to 121, wherein the polynucleotide is optimized for expression in a human cell.

123. A composition comprising a recombinant polynucleotide according to any one of claims 112 to 122 and a carrier and / or excipient.

124. A host cell comprising a recombinant polynucleotide according to any one of claims 112 to 122.

125. A recombinant pseudotyped virus or cell-derived nanovesicle comprising the recombinant polynucleotide of any one of claims 112 to 122.

126. A recombinant pseudotyped virus or cell-derived nanovesicle comprising one or more recombinant fusogenic proteins according to any one of claims 1 to 111.

127. 127. A recombinant pseudotyped virus or cell-derived nanovesicle according to claim 126, comprising two or more different recombinant fusogenic proteins according to any one of claims 1 to 111.

128. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 126 or 127, wherein the recombinant fusion protein forms a chimeric trimer with one or two different fusion proteins on the surface of the recombinant pseudotyped virus or cell-derived nanovesicle.

129. 129. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 128, wherein the chimeric trimer comprises: (i) at least one fusogenic protein of any one of claims 1 to 111; and (ii) a fusogenic protein comprising a rhabdovirus glycoprotein, or a functional fragment or derivative thereof, without a targeting molecule.

130. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 129, wherein the fusogenic protein (ii) comprises a fusogen having the sequence of SEQ ID NO: 8, with amino acid substitutions and / or deletions at one or more positions selected from K47, R354, H8, and Y209.

131. A recombinant pseudotyped virus or cell-derived nanovesicle comprising a chimeric trimer comprising: (i) one or two monomers of a first fusion protein, wherein the first fusion protein comprises a rhabdovirus glycoprotein, or a functional fragment or derivative thereof, and a targeting molecule, or a functional fragment or derivative thereof; and (ii) one or two monomers of a second fusion protein, wherein the second fusion protein comprises a rhabdovirus glycoprotein, or a functional fragment or derivative thereof, without a targeting molecule.

132. 132. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 131, wherein in the first fusogenic protein, the targeting molecule is attached to the rhabdovirus glycoprotein via a linker.

133. 133. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 132, wherein the linker is not susceptible to proteolytic cleavage by endogenous proteases or by exogenously added proteases.

134. 133. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 132, wherein the linker is susceptible to proteolytic cleavage by an endogenous protease or by an exogenously added protease.

135. 135. The recombinant pseudotyped virus or cell-derived nanovesicle of any one of claims 131 to 134, wherein the first fusogenic protein and / or the second fusogenic protein comprises a rhabdovirus glycoprotein comprising the sequence of SEQ ID NO: 8, with amino acid substitutions and / or deletions at one or more positions selected from K47, R354, H8, and Y209.

136. 136. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 135, wherein the first fusogenic protein and / or the second fusogenic protein comprises a rhabdovirus glycoprotein comprising a sequence according to any one of claims 10 to 18 or 52 to 54.

137. Recombinant pseudotyped virus or cell-derived nanovesicles comprising the glycoprotein from Flanders virus (FLAV-G), or a functional fragment or derivative thereof.

138. 138. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 137, wherein said FLAV-G comprises the sequence of SEQ ID NO:

9.

139. 139. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 138, wherein said FLAV-G consists of the sequence of SEQ ID NO:

9.

140. Recombinant pseudotyped virus or cell-derived nanovesicles comprising the glycoprotein from Chandipura virus (CHPV-G), or a functional fragment or derivative thereof.

141. 141. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 140, wherein said CHPV-G comprises the sequence of SEQ ID NO:

10.

142. 142. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 141, wherein said CHPV-G consists of the sequence of SEQ ID NO:

10.

143. Recombinant pseudotyped virus or cell-derived nanovesicles comprising a glycoprotein from a Perinetovirus (PERV-G), or a functional fragment or derivative thereof.

144. 144. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 143, wherein said PERV-G comprises the sequence of SEQ ID NO:

11.

145. 145. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 144, wherein said PERV-G consists of the sequence of SEQ ID NO:

11.

146. Recombinant pseudotyped virus or cell-derived nanovesicles comprising a glycoprotein from a Piryvirus (PIRYV-G), or a functional fragment or derivative thereof.

147. 147. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 146, wherein said PIRYV-G comprises the sequence of SEQ ID NO:

12.

148. 148. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 147, wherein said PIRYV-G consists of the sequence of SEQ ID NO:

12.

149. Recombinant pseudotyped virus or cell-derived nanovesicles comprising glycoprotein from Fukuoka virus (FUKV-G), or a functional fragment or derivative thereof.

150. 150. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 149, wherein said FUKV-G comprises the sequence of SEQ ID NO:

13.

151. 151. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 150, wherein said FUKV-G consists of the sequence of SEQ ID NO:

13.

152. Recombinant pseudotyped virus or cell-derived nanovesicles comprising glycoprotein from Joint-Jacaka virus (JOIV-G), or a functional fragment or derivative thereof.

153. 153. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 152, wherein said JOIV-G comprises the sequence of SEQ ID NO:

14.

154. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 153, wherein said JOIV-G consists of the sequence of SEQ ID NO:

14.

155. Recombinant pseudotyped virus or cell-derived nanovesicles comprising the glycoprotein from Kumasi virus (KRV-G), or a functional fragment or derivative thereof.

156. 156. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 155, wherein said KRV-G comprises the sequence of SEQ ID NO:

15.

157. 157. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 156, wherein said KRV-G consists of the sequence of SEQ ID NO:

15.

158. Recombinant pseudotyped virus or cell-derived nanovesicles comprising the glycoprotein from Keurariba virus (KEUV-G), or a functional fragment or derivative thereof.

159. 159. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 158, wherein said KEUV-G comprises the sequence of SEQ ID NO:

17.

160. 160. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 159, wherein said KEUV-G consists of the sequence of SEQ ID NO:

17.

161. 161. The recombinant pseudotyped virus or cell-derived nanovesicle of any one of claims 137 to 160, wherein the cytoplasmic tail of the glycoprotein has been removed or truncated and, optionally, replaced with another sequence.

162. 162. The recombinant fusion protein of claim 161, wherein the cytoplasmic tail of the glycoprotein is truncated at most 40 amino acids from the C-terminus.

163. 163. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 162, wherein the cytoplasmic tail of the glycoprotein is truncated 10 to 40 amino acids from the C-terminus.

164. 164. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 163, wherein the cytoplasmic tail of the glycoprotein is truncated 30 amino acids from the C-terminus.

165. 165. The recombinant pseudotyped virus or cell-derived nanovesicle of any one of claims 161 to 164, wherein said glycoprotein further comprises a cytoplasmic tail from VSV-G, or a functional fragment or derivative thereof.

166. 166. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 165, wherein the cytoplasmic tail of VSV-G comprises the sequence CIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 16).

167. 167. The recombinant pseudotyped virus of any one of claims 125 to 166, wherein the virus is a rhabdovirus.

168. 168. The recombinant rhabdovirus of claim 167, wherein the virus is a recombinant vesicular stomatitis virus (VSV).

169. 167. The recombinant pseudotyped virus of any one of claims 125 to 166, wherein the virus is a retrovirus.

170. 170. The recombinant pseudotyped virus of claim 169, wherein the retrovirus is a lentivirus (LV).

171. 171. The recombinant pseudotyped virus of any one of claims 125 to 170, wherein the virus is replication competent.

172. 171. The recombinant pseudotyped virus of any one of claims 125 to 170, wherein the virus is non-replicating.

173. 173. The recombinant pseudotyped virus or cell-derived nanovesicle of any one of claims 125 to 172, wherein the virus further comprises a molecular cargo.

174. 174. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 173, wherein the molecular cargo is a transgene encoding a therapeutic protein, a suicide gene, a toxic protein or peptide, an antibody or fragment thereof, a chimeric antigen receptor (CAR), a T-cell receptor (TCR), a gene editing system or component(s) thereof, an antisense oligonucleotide, a ribozyme, or an RNAi molecule.

175. 174. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 173, wherein the molecular cargo is a therapeutic protein, a toxic protein or peptide, an antibody or fragment thereof, a chimeric antigen receptor (CAR), a T-cell receptor (TCR), a gene editing system or component(s) thereof, an antisense oligonucleotide, a ribozyme, or an RNAi molecule.

176. 174. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 173, wherein the molecular cargo is a gene-editing ribonucleoprotein complex or component(s) thereof.

177. 177. The recombinant pseudotyped virus or cell-derived nanovesicle of claim 176, wherein the molecular cargo is a Cas9 protein complexed with a guide RNA (gRNA) specific for a gene of interest.

178. 178. A composition comprising a recombinant pseudotyped virus or cell-derived nanovesicle according to any one of claims 125 to 177 and a carrier and / or excipient.

179. 178. A method of reducing the susceptibility of a recombinant virus or nanovesicle to serum neutralization in a subject in need thereof, comprising administering to said subject a recombinant pseudotyped virus or cell-derived nanovesicle of any one of claims 125-177, or a composition of claim 178.

180. 178. A method of enhancing resistance to low density lipoprotein (LDL) and / or very low density lipoprotein (VLDL) mediated neutralization in a subject in need thereof, comprising administering to said subject a recombinant pseudotyped virus or cell-derived nanovesicle of any one of claims 125-177, or a composition of claim 178.

181. 178. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant pseudotyped virus or cell-derived nanovesicle of any one of claims 125-177, or a composition of claim 178.

182. 182. The method of claim 181, wherein the method does not include prior treatment with an LDL / VLDL-lowering medication.

183. 182. The method of claim 181, wherein the method further comprises pretreatment with an LDL / VLDL-lowering medication.

184. 178. A method of inducing an immune response in a subject in need thereof, comprising administering to the subject an effective amount of a recombinant pseudotyped virus or cell-derived nanovesicle of any one of claims 125-177, or a composition of claim 178.

185. 178. A method for delivering a molecular cargo to a cell in a subject in need thereof, comprising administering to the subject an effective amount of a recombinant pseudotyped virus or cell-derived nanovesicle of any one of claims 125 to 177, or a composition comprising said pseudotyped virus or cell-derived nanovesicle and a carrier and / or excipient, wherein said recombinant fusogenic protein within said recombinant pseudotyped virus or cell-derived nanovesicle comprises a targeting molecule that targets said cell.

186. The method of any one of claims 179 to 185, wherein the subject is a human.

187. 178. A method for ex vivo delivery of molecular cargo to a cell, comprising administering to said cell an effective amount of a recombinant pseudotyped virus or cell-derived nanovesicle of any one of claims 125 to 177, or a composition comprising said pseudotyped virus or cell-derived nanovesicle and a carrier and / or excipient, wherein said recombinant fusogenic protein within said recombinant pseudotyped virus or cell-derived nanovesicle comprises a targeting molecule that targets said cell.

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