Modified rhabdovirus glycoproteins and uses thereof

Abstract

The present disclosure provides a recombinant fusogenic protein comprising a rhabdovirus glycoprotein (G) and a targeting molecule linked to the N-terminus of the rhabdovirus glycoprotein. In addition, related recombinant polynucleotides, host cells and pharmaceutical compositions are also provided. Also provided are recombinant viruses (e.g., recombinant pseudotype viruses) and cell-derived nanovesicles comprising the recombinant polynucleotides. Furthermore, provided are methods of using the recombinant fusogenic proteins, polynucleotides, virus and cell-derived nanovesicles and / or pharmaceutical compositions thereof, including their use in the treatment of cancer.

Description

[0001] Cross-reference to related applications

[0002] 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 which are incorporated herein by reference in their entirety.

[0003] sequence list

[0004] This application includes a sequence list that has been electronically submitted in XML format, which is incorporated herein by reference in its entirety. The XML copy was created on January 19, 2024, and is named 250298_000603_SL.xml, with a size of 266,253 bytes. Invention Field

[0005] This disclosure provides a recombinant fusion-promoting protein comprising a rhabdoviral glycoprotein (G) and a targeting molecule attached to the N-terminus of the rhabdoviral glycoprotein. Additionally, related recombinant polynucleotides, host cells, and pharmaceutical compositions are provided. Recombinant viruses (e.g., recombinant pseudoviruses) and cell-derived nanovesicles comprising said recombinant polynucleotides are also provided. Furthermore, methods of using said recombinant fusion-promoting protein, polynucleotides, viruses, and cell-derived nanovesicles and / or pharmaceutical compositions thereof are provided, including their use in cancer treatment. Background of the Invention

[0007] A major challenge in developing targeted vector therapies is delivering therapeutic drugs to cells / tissues specific to the target disease. For success, the delivered vector therapy must be specific to the target cells / tissues and minimize off-target delivery that could lead to toxicity. Furthermore, the therapeutic vector must overcome natural barriers such as the patient's innate immune response and persist long enough to reach the target cells / tissues.

[0008] Vesicular stomatitis virus (VSV) possesses oncolytic properties and is currently undergoing clinical trials 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 to improve the targeted delivery of VSV to tumor tissues to maximize therapeutic efficacy and minimize potential toxicities associated with infection of healthy tissues. One of the most concerning off-target effects associated with VSV is neurotoxicity. Lost virus entering other tissues, including the liver and spleen, reduces the efficacy of oncolytic VSV. Strategies for tropistically directing VSV to tissues of interest often negatively impact viral adaptation, thereby reducing the effectiveness of VSV therapy.

[0009] Therefore, there is an unmet need in the field for improved VSV-based therapies. Invention Overview

[0011] As described in the background section above, there is a significant need in the art for improved VSV-based therapies. This application aims to address these and other needs.

[0012] In one aspect, this article provides a recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises:

[0013] (i) the rhabdoviral glycoprotein (G) or a functional fragment or derivative thereof; and

[0014] (ii) a targeting molecule, wherein the targeting molecule may be attached to the N-terminus of, for example, the rhabdoviral glycoprotein or a functional fragment or derivative thereof via a linker, the linker being sensitive to proteolytic cleavage via an endogenous protease or an exogenously added protease.

[0015] In some embodiments, the linker comprises arginine (R) and / or lysine (K) residues.

[0016] In some embodiments, the connector is included in a sequence selected from:

[0017] KRAAASGGS(G4S)2GPK(SEQ ID NO: 174);

[0018] KRAAASGGS(G4S)2(SEQ ID NO:2);

[0019] (EAAAK)3(SEQ ID NO: 3);

[0020] KR(EAAAK)3(SEQ ID NO: 4);

[0021] AAARGSPK(G4S)3(SEQ ID NO:5);

[0022] RAAARGSPK(G4S)3(SEQ ID NO:169);

[0023] AAARGSPK(G4S)3K(SEQ ID NO: 19);

[0024] K(G4S)3(SEQ ID NO: 20);

[0025] KR(G4S)3(SEQ ID NO: 21);

[0026] (G4S)3GPK(SEQ ID NO: 6); and

[0027] AAA(G4S)3K(SEQ ID NO: 7).

[0028] In some embodiments, the N-terminus of the rhabdoviral glycoprotein or its functional fragment or derivative to which the targeting molecule is attached via the linker does not contain one or more amino acids present at the N-terminus of the mature wild-type rhabdoviral glycoprotein.

[0029] In some embodiments, the rhabdoviral glycoprotein is vesicular stomatitis virus glycoprotein (VSV-G) or a functional fragment or derivative thereof.

[0030] In some implementations, the VSV-G contains the sequence SEQ ID NO: 8.

[0031] In some implementations, the VSV-G consists of the sequence SEQ ID NO: 8.

[0032] In some embodiments, the targeting molecule is capable of interfering with the ability of the VSV-G or its functional fragments or derivatives to interact with the low-density lipoprotein receptor (LDLR).

[0033] In some embodiments, the VSV-G or a functional fragment or derivative thereof contains one or more mutations, wherein the one or more mutations reduce or eliminate the binding of the VSV-G peptide or a functional fragment or derivative thereof to LDLR.

[0034] In some embodiments, the one or more mutations in the VSV-G or its functional fragments or derivatives include one or more amino acid substitutions and / or deletions at positions corresponding to H8, K47, Y209 or R354 in SEQ ID NO: 8.

[0035] 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.

[0036] 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.

[0037] In some embodiments, the VSV-G comprises or consists of SEQ ID NO: 8, and the one or more mutations are substitutions at the H8 position.

[0038] In some embodiments, the one or more mutations in the VSV-G or its functional fragments or derivatives include the deletion of one or more amino acids corresponding to the positions H8, K47, Y209 or R354 in SEQ ID NO: 8.

[0039] In some implementations, the VSV-G comprises or consists of SEQ ID NO: 8.

[0040] In some implementations, the one or more deletions are deletions at position K47.

[0041] In some implementations, the one or more missing values ​​are missing values ​​at the H8 position.

[0042] In some implementations, the one or more deletions are deletions at positions H8 and K47.

[0043] In some implementations, the VSV-G or its functional fragments or derivatives further include one or more mutations that increase viral titers.

[0044] In some embodiments, the one or more viral titer-enhancing mutations in the VSV-G or its functional fragments or derivatives are M184T and / or F250L as specified at the position in SEQ ID NO: 8.

[0045] In some embodiments, the rhabdoviral glycoprotein is a glycoprotein derived from Flanders virus (FLAV-G).

[0046] In some implementations, the FLAV-G contains the sequence SEQ ID NO: 9.

[0047] In some implementations, the FLAV-G consists of the sequence SEQ ID NO: 9.

[0048] In some embodiments, the rhabdoviral glycoprotein is a glycoprotein derived from Chandipura virus (CHPV-G).

[0049] In some implementations, the CHPV-G contains the sequence SEQ ID NO: 10.

[0050] In some implementations, the CHPV-G consists of the sequence SEQ ID NO: 10.

[0051] In some embodiments, the rhabdoviral glycoprotein is a glycoprotein derived from the Perinet virus (PERV-G).

[0052] In some implementations, the PERV-G contains the sequence SEQ ID NO: 11.

[0053] In some implementations, the PERV-G consists of the sequence SEQ ID NO: 11.

[0054] In some embodiments, the rhabdoviral glycoprotein is a glycoprotein derived from Piry virus (PIRYV-G).

[0055] In some implementations, the PIRYV-G contains the sequence SEQ ID NO: 12.

[0056] In some implementations, the PIRYV-G consists of the sequence SEQ ID NO: 12.

[0057] In some embodiments, the rhabdovirus glycoprotein is a glycoprotein derived from the Fukuoka virus (FUKV-G).

[0058] In some implementations, the FUKV-G contains the sequence SEQ ID NO: 13.

[0059] In some implementations, the FUKV-G consists of the sequence SEQ ID NO: 13.

[0060] In some embodiments, the rhabdoviral glycoprotein is a glycoprotein derived from the Joinjakaka virus (JOIV-G).

[0061] In some implementations, the JOIV-G contains the sequence SEQ ID NO: 14.

[0062] In some implementations, the JOIV-G consists of the sequence SEQ ID NO: 14.

[0063] In some embodiments, the rhabdoviral glycoprotein is a glycoprotein derived from the Kumasi virus (KRV-G).

[0064] In some implementations, the KRV-G contains the sequence SEQ ID NO: 15.

[0065] In some implementations, the KRV-G consists of the sequence SEQ ID NO: 15.

[0066] In some embodiments, the rhabdoviral glycoprotein is a glycoprotein derived from the Keuraliba virus (KEUV-G).

[0067] In some implementations, the KEUV-G contains the sequence SEQ ID NO: 17.

[0068] In some implementations, the KEUV-G contains the sequence SEQ ID NO: 17.

[0069] In some embodiments, the cytoplasmic tail of the rhabdoviral glycoprotein has been removed or truncated, and optionally replaced by another sequence.

[0070] In some embodiments, the cytoplasmic tail of the glycoprotein is truncated from the C-terminus by up to 40 amino acids.

[0071] In some embodiments, the cytoplasmic tail of the rhabdovirus glycoprotein is truncated by 10 to 40 amino acids from the C-terminus.

[0072] In some embodiments, the cytoplasmic tail of the rhabdovirus glycoprotein is truncated by 30 amino acids from the C-terminus.

[0073] In some embodiments of any of the above-described recombinant fusion-promoting proteins, the recombinant fusion-promoting protein may also include a cytoplasmic tail from VSV-G or a functional fragment or derivative thereof.

[0074] In some implementations, the cytoplasmic tail of VSV-G contains the sequence CIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 16).

[0075] On the other hand, this article provides a recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises a fusogen having at least 60% amino acid sequence identity with the vesicular stomatitis virus glycoprotein (VSV-G) or a functional fragment or derivative thereof comprising SEQ ID NO: 8, wherein the fusogen or a 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.

[0076] In some embodiments, the fusion element comprises sequence SEQ ID NO: 8 or a functional fragment or derivative thereof, and has one or more amino acid deletions at positions H8, K47, Y209 or R354.

[0077] In some embodiments, the fusion element comprises or consists of the sequence SEQ ID NO: 8, and has an amino acid deletion at the H8 position.

[0078] In some embodiments, the fusion element comprises or consists of the sequence SEQ ID NO: 8, and contains an amino acid deletion at positions H8 and K47.

[0079] In some embodiments, the fusion element comprises the sequence SEQ ID NO: 8 and contains an amino acid deletion at (i) K47, (ii) R354 and (iii) H8 or Y209 positions.

[0080] In some embodiments, the fusion element consists of the sequence SEQ ID NO: 8 and contains amino acid deletions at positions (i) K47, (ii) R354 and (iii) H8 or Y209.

[0081] In some embodiments, the fusion element comprises the sequence SEQ ID NO: 8 and contains an amino acid deletion at position K47.

[0082] In some embodiments, the fusion element consists of the sequence SEQ ID NO: 8 and contains an amino acid deletion at position K47.

[0083] In another aspect, this article provides a recombinant fusion-promoting protein comprising a fusionin having the sequence SEQ ID NO: 8 and containing amino acid substitutions at (i) K47, (ii) R354 and (iii) H8 or Y209 positions.

[0084] In some embodiments, the fusion element consists of the sequence SEQ ID NO: 8 and contains amino acid substitutions at positions (i) K47, (ii) R354 and (iii) H8 or Y209.

[0085] In another aspect, this article provides a recombinant fusion-promoting protein comprising a fusionin having the sequence SEQ ID NO: 8 and containing amino acid substitutions at positions K47, R354, H8, and Y209.

[0086] In some embodiments, the fusion element consists of the sequence SEQ ID NO: 8 and contains amino acid substitutions at positions K47, R354, H8, and Y209.

[0087] In some embodiments, the fusionin or its functional fragments or derivatives further comprise one or more mutations that increase viral titers.

[0088] In some embodiments, the one or more mutations that increase viral titer are located at one or more positions corresponding to the M184 and / or F250 positions in SEQ ID NO: 8.

[0089] In some embodiments of any of the above-described recombinant fusion-promoting proteins, the fusion-promoting protein may further comprise a targeting molecule located at the N-terminus of the fusionin or a functional fragment or derivative thereof.

[0090] In some embodiments, the targeting molecule is attached to the N-terminus of the fusionin or its functional fragment or derivative via a connector.

[0091] In some embodiments, the adapter is sensitive to proteolytic cleavage via endogenous or exogenously added proteases.

[0092] In some embodiments, the linker comprises arginine (R) and / or lysine (K) residues.

[0093] In some embodiments, the connector is included in a sequence selected from:

[0094] KRAAASGGS(G4S)2GPK(SEQ ID NO: 174);

[0095] KRAAASGGS(G4S)2(SEQ ID NO:2);

[0096] (EAAAK)3(SEQ ID NO: 3);

[0097] KR(EAAAK)3(SEQ ID NO: 4);

[0098] AAARGSPK(G4S)3(SEQ ID NO:5);

[0099] RAAARGSPK(G4S)3(SEQ ID NO:169);

[0100] AAARGSPK(G4S)3K(SEQ ID NO: 19);

[0101] K(G4S)3(SEQ ID NO: 20);

[0102] KR(G4S)3(SEQ ID NO: 21);

[0103] (G4S)3GPK(SEQ ID NO: 6); or

[0104] AAA(G4S)3K(SEQ ID NO: 7).

[0105] In some embodiments, the adapter is insensitive to proteolytic cleavage by endogenous or exogenously added proteases.

[0106] In some embodiments, the N-terminus of the fusionin to which the targeting molecule is attached, or a functional fragment or derivative thereof, does not contain one or more amino acids present at the N-terminus of the mature wild-type fusionin.

[0107] On the other hand, this article provides a recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises:

[0108] (i) glycoprotein (FLAV-G) from Flanders virus or its functional fragments or derivatives, and

[0109] (ii) Targeting molecules.

[0110] In some implementations, the FLAV-G contains the sequence SEQ ID NO: 9.

[0111] In some implementations, the FLAV-G consists of the sequence SEQ ID NO: 9.

[0112] In another aspect, this article provides a recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises:

[0113] (i) glycoprotein (CHPV-G) from Chandipura virus or its functional fragments or derivatives, and

[0114] (ii) Targeting molecules.

[0115] In some implementations, the CHPV-G contains the sequence SEQ ID NO: 10.

[0116] In some implementations, the CHPV-G consists of the sequence SEQ ID NO: 10.

[0117] In another aspect, this article provides a recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises:

[0118] (i) glycoprotein (PERV-G) from the Perinet virus or its functional fragments or derivatives, and

[0119] (ii) Targeting molecules.

[0120] In some implementations, the PERV-G contains the sequence SEQ ID NO: 11.

[0121] In some implementations, the PERV-G consists of the sequence SEQ ID NO: 11.

[0122] In another aspect, this article provides a recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises:

[0123] (i) a glycoprotein (PIRYV-G) derived from Piry virus or a functional fragment or derivative thereof, and

[0124] (ii) Targeting molecules.

[0125] In some implementations, the PIRYV-G contains the sequence SEQ ID NO: 12.

[0126] In some implementations, the PIRYV-G consists of the sequence SEQ ID NO: 12.

[0127] In another aspect, this article provides a recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises:

[0128] (i) A glycoprotein (FUKV-G) or a functional fragment or derivative thereof derived from Fukuoka virus, and

[0129] (ii) Targeting molecules.

[0130] In some implementations, the FUKV-G contains the sequence SEQ ID NO: 13.

[0131] In some implementations, the FUKV-G consists of the sequence SEQ ID NO: 13.

[0132] In another aspect, this article provides a recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises:

[0133] (i) glycoprotein (JOIV-G) or its functional fragments or derivatives derived from Joinjakaka virus, and

[0134] (ii) Targeting molecules.

[0135] In some implementations, the JOIV-G contains the sequence SEQ ID NO: 14.

[0136] In some implementations, the JOIV-G consists of the sequence SEQ ID NO: 14.

[0137] In another aspect, this article provides a recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises:

[0138] (i) Glycoprotein (KRV-G) from Kumasi virus or its functional fragments or derivatives, and

[0139] (ii) Targeting molecules.

[0140] In some implementations, the KRV-G contains the sequence SEQ ID NO: 15.

[0141] In some implementations, the KRV-G consists of the sequence SEQ ID NO: 15.

[0142] In another aspect, this article provides a recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises:

[0143] (i) A glycoprotein (KEUV-G) or a functional fragment or derivative thereof derived from the Keuraliba virus, and

[0144] (ii) Targeting molecules.

[0145] In some implementations, the KEUV-G contains the sequence SEQ ID NO: 17.

[0146] In some implementations, the KEUV-G consists of the sequence SEQ ID NO: 17.

[0147] In some embodiments, the glycoprotein is a fragment in which the cytoplasmic tail of the glycoprotein has been removed or truncated and optionally replaced by another sequence.

[0148] In some embodiments, the cytoplasmic tail of the glycoprotein is truncated from the C-terminus by up to 40 amino acids.

[0149] In some embodiments, the cytoplasmic tail of the glycoprotein is truncated by 10 to 40 amino acids from the C-terminus.

[0150] In some embodiments, the cytoplasmic tail of the glycoprotein is truncated by 30 amino acids from the C-terminus.

[0151] In some embodiments of any of the above-described recombinant fusion-promoting proteins, the recombinant fusion-promoting protein may also include a cytoplasmic tail from VSV-G or a functional fragment or derivative thereof.

[0152] In some implementations, the cytoplasmic tail of VSV-G contains the sequence CIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 16).

[0153] In some embodiments, the targeting molecule is located at the N-terminus of the glycoprotein or its functional fragments or derivatives.

[0154] In some embodiments, the targeting molecule is attached to the N-terminus of the glycoprotein or its functional fragment or derivative via a linker.

[0155] In some embodiments, the adapter is sensitive to proteolytic cleavage via endogenous or exogenously added proteases.

[0156] In some embodiments, the linker comprises arginine (R) and / or lysine (K) residues.

[0157] In some embodiments, the connector is included in a sequence selected from:

[0158] KRAAASGGS(G4S)2GPK(SEQ ID NO: 174);

[0159] KRAAASGGS(G4S)2(SEQ ID NO:2);

[0160] (EAAAK)3(SEQ ID NO: 3);

[0161] KR(EAAAK)3(SEQ ID NO: 4);

[0162] AAARGSPK(G4S)3(SEQ ID NO:5);

[0163] RAAARGSPK(G4S)3(SEQ ID NO:169);

[0164] AAARGSPK(G4S)3K(SEQ ID NO: 19);

[0165] K(G4S)3(SEQ ID NO: 20);

[0166] KR(G4S)3(SEQ ID NO: 21);

[0167] (G4S)3GPK(SEQ ID NO: 6); and

[0168] AAA(G4S)3K(SEQ ID NO: 7).

[0169] In some embodiments, the adapter is insensitive to proteolytic cleavage by endogenous or exogenously added proteases.

[0170] In some embodiments, the N-terminus of the glycoprotein to which the targeting molecule is attached, or a functional fragment or derivative thereof, does not contain one or more amino acids present at the N-terminus of the mature wild-type fusionin.

[0171] In some embodiments, the target molecule is an antibody or its antigen-binding fragment, affinity compound, darpin, peptide, natural or modified natural receptor ligand, T-cell receptor or its fragment or derivative, or MHC-peptide complex or its fragment or derivative.

[0172] In some embodiments, the antibody or its antigen-binding fragment is a single-chain variable region fragment (scFv), a biantibody, a microantibody, a nanobody, a single-domain antibody (sdAb), or a single-heavy-chain antibody.

[0173] In some implementations, 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.

[0174] In another respect, this paper provides a recombinant polynucleotide encoding the recombinant fusion-promoting protein described herein.

[0175] In some embodiments, the polynucleotide comprises a sequence encoding a signal peptide sequence, wherein such signal sequence is located at the N-terminus of the encoded recombinant fusion protein.

[0176] In some implementations, the polynucleotide is DNA.

[0177] In some implementations, the polynucleotide is RNA.

[0178] In another aspect, this article provides a recombinant polynucleotide, wherein the recombinant polynucleotide is an RNA molecule comprising a nucleotide sequence that serves as a template for a positive transcript encoding the recombinant fusion protein described herein.

[0179] In some embodiments, the positive transcript contains a sequence encoding a signal peptide sequence, wherein such signal sequence is located at the N-terminus of the encoded recombinant fusion protein.

[0180] In some embodiments, the recombinant polynucleotide is an RNA molecule comprising: a nucleotide sequence serving as a template for a positive transcript encoding a vesicular stomatitis virus (VSV) nucleoprotein (N) polypeptide or a functional fragment or derivative thereof; a nucleotide sequence serving as a template for a positive transcript encoding a VSV phosphoprotein (P) polypeptide or a functional fragment or derivative thereof; a nucleotide sequence serving as a template for a positive transcript encoding a VSV matrix (M) polypeptide or a functional fragment or derivative thereof; a nucleotide sequence serving as a template for a positive transcript encoding the fusion-promoting protein described herein; and a nucleotide sequence serving as a template for a positive transcript encoding a VSV large protein (L) polypeptide or a functional fragment or derivative thereof.

[0181] In some embodiments, the VSV M polypeptide is a mutant VSV M polypeptide.

[0182] In some embodiments, the mutant VSV M polypeptide contains a mutation at methionine (M)51.

[0183] In some embodiments, the mutation at methionine (M)51 is a substitution of methionine (M) for arginine (R).

[0184] In some implementations, the polynucleotide is optimized for expression in human cells.

[0185] In another aspect, this document provides a composition comprising the recombinant polynucleotide described herein, as well as a carrier and / or excipient.

[0186] In another aspect, this article provides a host cell containing the recombinant polynucleotides described herein.

[0187] In another aspect, this article provides a recombinant pseudovirus or cell-derived nanovesicle containing the recombinant polynucleotides described herein.

[0188] In another aspect, this article provides a recombinant pseudovirus or cell-derived nanovesicle that contains one or more of the recombinant fusion-promoting proteins described herein.

[0189] In some implementations, the recombinant pseudoviruses or cell-derived nanovesicles described herein may contain two or more different recombinant fusion-promoting proteins described herein.

[0190] In some embodiments, the recombinant fusion-promoting protein forms a chimeric trimer with one or two different fusion-promoting proteins on the surface of the recombinant pseudovirus or cell-derived nanovesicles.

[0191] In some embodiments, the chimeric trimer comprises (i) at least one fusion-promoting protein described herein, and (ii) a fusion-promoting protein comprising a rhabdoviral glycoprotein or a functional fragment or derivative thereof and without a target molecule.

[0192] In some embodiments, the fusion-promoting protein (ii) comprises a fusionin containing the sequence SEQ ID NO: 8 and having amino acid substitutions and / or deletions at one or more positions selected from K47, R354, H8 and Y209.

[0193] In another aspect, this document provides a recombinant pseudovirus or cell-derived nanovesicle comprising a chimeric trimer comprising: (i) one or two monomers of a first fusion-promoting protein, wherein the first fusion-promoting protein comprises a rhabdoviral 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-promoting protein, wherein the second fusion-promoting protein comprises a rhabdoviral glycoprotein or a functional fragment or derivative thereof, but does not contain the targeting molecule. In some embodiments, for example, the targeting molecule may be attached to the N-terminus of the rhabdoviral glycoprotein, its functional fragment, or a derivative thereof.

[0194] In some implementations, in the first fusion-promoting protein, the targeting molecule is linked to the rhabdoviral glycoprotein via a linker.

[0195] In some embodiments, the adapter is insensitive to proteolytic cleavage by endogenous or exogenously added proteases.

[0196] In some embodiments, the adapter is sensitive to proteolytic cleavage via endogenous or exogenously added proteases.

[0197] In some embodiments, the first fusion-promoting protein and / or the second fusion-promoting protein comprises a rhabdoviral glycoprotein containing the sequence SEQ ID NO: 8 and having amino acid substitutions and / or deletions at one or more positions selected from K47, R354, H8 and Y209.

[0198] In some embodiments, the first and / or second fusion-promoting proteins comprise a rhabdoviral glycoprotein, which comprises any of the various rhabdoviral glycoprotein sequences shown herein. In some embodiments, the first and / or second fusion-promoting proteins comprise a rhabdoviral glycoprotein containing one or more amino acid substitutions and / or deletions at positions corresponding to H8, K47, Y209, or R354 in SEQ ID NO: 8. In some embodiments, the first and / or second fusion-promoting proteins comprise a rhabdoviral glycoprotein comprising or consisting of SEQ ID NO: 8, and the one or more amino acid mutations are substitutions at positions K47 and R354. In some embodiments, the one or more amino acid mutations are substitutions at positions K47, R354, and Y209. In some embodiments, the one or more amino acid mutations are substitutions at position H8.

[0199] In some embodiments, the first and / or second fusion-promoting proteins comprise a rhabdoviral glycoprotein that contains 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 and / or second fusion-promoting proteins comprise a rhabdoviral glycoprotein that comprises or is composed of the sequence SEQ ID NO: 8, and the one or more amino acid deletions are deletions at positions H8, K47, Y209, or R354. In some embodiments, the one or more amino acid deletions are deletions at position K47. In some embodiments, the one or more amino acid deletions are deletions at position H8. In some embodiments, the one or more amino acid deletions are deletions at positions H8 and K47.

[0200] In another aspect, this article provides a recombinant pseudovirus or cell-derived nanovesicle containing a glycoprotein (FLAV-G) from Flanders virus or a functional fragment or derivative thereof.

[0201] In some implementations, the FLAV-G contains the sequence SEQ ID NO: 9.

[0202] In some implementations, the FLAV-G consists of the sequence SEQ ID NO: 9.

[0203] In another aspect, this article provides a recombinant pseudovirus or cell-derived nanovesicle containing a glycoprotein (CHPV-G) from Chandipura virus or a functional fragment or derivative thereof.

[0204] In some implementations, the CHPV-G contains the sequence SEQ ID NO: 10.

[0205] In some implementations, the CHPV-G consists of the sequence SEQ ID NO: 10.

[0206] In another aspect, this article provides a recombinant pseudovirus or cell-derived nanovesicle containing a glycoprotein (PERV-G) from the Perinet virus or a functional fragment or derivative thereof.

[0207] In some implementations, the PERV-G contains the sequence SEQ ID NO: 11.

[0208] In some implementations, the PERV-G consists of the sequence SEQ ID NO: 11.

[0209] In another aspect, this article provides a recombinant pseudovirus or cell-derived nanovesicle containing a glycoprotein (PIRYV-G) from Piry virus or a functional fragment or derivative thereof.

[0210] In some implementations, the PIRYV-G contains the sequence SEQ ID NO: 12.

[0211] In some implementations, the PIRYV-G consists of the sequence SEQ ID NO: 12.

[0212] In another aspect, this article provides a recombinant pseudovirus or cell-derived nanovesicle containing a glycoprotein (FUKV-G) from Fukuoka virus or a functional fragment or derivative thereof.

[0213] In some implementations, the FUKV-G contains the sequence SEQ ID NO: 13.

[0214] In some implementations, the FUKV-G consists of the sequence SEQ ID NO: 13.

[0215] In another aspect, this article provides a recombinant pseudovirus or cell-derived nanovesicle containing a glycoprotein (JOIV-G) from Joinjaka virus or a functional fragment or derivative thereof.

[0216] In some implementations, the JOIV-G contains the sequence SEQ ID NO: 14.

[0217] In some implementations, the JOIV-G consists of the sequence SEQ ID NO: 14.

[0218] In another aspect, this article provides a recombinant pseudovirus or cell-derived nanovesicle containing a glycoprotein (KRV-G) from Kumasi virus or a functional fragment or derivative thereof.

[0219] In some implementations, the KRV-G contains the sequence SEQ ID NO: 15.

[0220] In some implementations, the KRV-G consists of the sequence SEQ ID NO: 15.

[0221] In another aspect, this article provides a recombinant pseudovirus or cell-derived nanovesicle containing a glycoprotein (KEUV-G) from Keuraliba virus or a functional fragment or derivative thereof.

[0222] In some implementations, the KEUV-G contains the sequence SEQ ID NO: 17.

[0223] In some implementations, the KEUV-G consists of the sequence SEQ ID NO: 17.

[0224] In some embodiments, the cytoplasmic tail of the glycoprotein has been removed or truncated and optionally replaced by another sequence.

[0225] In some embodiments, the cytoplasmic tail of the glycoprotein is truncated from the C-terminus by up to 40 amino acids.

[0226] In some embodiments, the cytoplasmic tail of the glycoprotein is truncated by 10 to 40 amino acids from the C-terminus.

[0227] In some embodiments, the cytoplasmic tail of the glycoprotein is truncated by 30 amino acids from the C-terminus.

[0228] In some embodiments, the glycoprotein also includes a cytoplasmic tail derived from VSV-G or a functional fragment or derivative thereof.

[0229] In some implementations, the cytoplasmic tail of VSV-G contains the sequence CIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 16).

[0230] In some implementations, the virus is a rhabdovirus.

[0231] In some implementations, the virus is recombinant vesicular stomatitis virus (VSV).

[0232] In some implementations, the virus is a retrovirus.

[0233] In some implementations, the retrovirus is a lentivirus (LV).

[0234] In some implementations, the virus has the ability to replicate.

[0235] In some implementations, the virus is non-replicating.

[0236] In some implementations, the virus also contains molecular cargo.

[0237] 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 one or more components thereof, an antisense oligonucleotide, a ribozyme, or an RNAi molecule.

[0238] 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 one or more components thereof, an antisense oligonucleotide, a ribozyme, or an RNAi molecule.

[0239] In some embodiments, the molecular cargo is a gene-editing ribonucleoprotein complex or one or more components thereof.

[0240] In some embodiments, the molecular cargo is a Cas9 protein complexed with a guide RNA (gRNA) specific to the gene of interest.

[0241] In another aspect, this article provides a composition comprising the recombinant pseudovirus or cell-derived nanovesicles described herein, as well as a carrier and / or excipient.

[0242] In another aspect, this article provides a method for reducing the sensitivity of a subject in need to serum neutralization of recombinant viruses or nanovesicles, comprising administering to the subject the recombinant pseudoviruses or cell-derived nanovesicles or compositions described herein.

[0243] In another aspect, this article provides a method for enhancing resistance to low-density lipoprotein (LDL) and / or very low-density lipoprotein (VLDL)-mediated neutralization in a subject of need, comprising administering to the subject the recombinant pseudovirus or cell-derived nanovesicles described herein or the compositions described herein.

[0244] In another aspect, this article provides a method for treating cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of the recombinant pseudovirus or cell-derived nanovesicles or compositions described herein.

[0245] In some implementations, the method does not include pretreatment with drugs that lower LDL / VLDL.

[0246] In some implementations, the method further includes pretreatment with drugs that lower LDL / VLDL.

[0247] In another aspect, this article provides a method for inducing an immune response in subjects in need, comprising administering to the subject an effective amount of the recombinant pseudovirus or cell-derived nanovesicles or the composition described herein.

[0248] In another aspect, this document provides a method for delivering molecular cargo to cells in the body of a subject in need, comprising administering to the subject an effective amount of the recombinant pseudovirus or cell-derived nanovesicles described herein, or a composition comprising the recombinant pseudovirus or cell-derived nanovesicles and a carrier and / or excipient, wherein the recombinant fusion-promoting protein within the recombinant pseudovirus or cell-derived nanovesicles comprises a targeting molecule that targets the cells.

[0249] In some implementations, the subjects are humans.

[0250] On the other hand, this article provides a method for delivering molecular cargo ex vivo to cells, comprising administering to the cells an effective amount of the recombinant pseudovirus or cell-derived nanovesicles described herein, or a composition comprising the pseudovirus or cell-derived nanovesicles and a carrier and / or excipient, wherein the recombinant fusion-promoting protein within the recombinant pseudovirus or cell-derived nanovesicles comprises a targeting molecule targeting the cells. Brief description of the attached diagram

[0252] Figure 1A-1B This study demonstrates the inhibitory effect of serum on wild-type (WT) vesicular stomatitis virus (VSV). Vero cells were infected with WT (glycoprotein [G]) VSV (VSV-G) encoding a firefly luciferase (Fluc) reporter factor. At different time points during the infection process, escalating concentrations of mixed human serum were added to the cells, including: 1) throughout the entire infection process ( Figure 1A (Left image); 2) Only within the first 4 hours after vaccination, then remove ( Figure 1A (see Chinese image); or 3) only 4 hours after vaccination (see Chinese image); Figure 1A (See right figure). Luciferase activity was measured 16 hours after initial inoculation. In separate experiments, the mixed serum was either left untreated or incubated at 56°C to inactivate complement in the serum. Serum (or culture medium alone) was then added to Vero cells. WT VSV (VSV-GFP), encoding the green fluorescent protein (GFP) reporter factor, was added to the cells. After 16 hours, the culture plates were imaged using an imaging cell analyzer. Figure 1B (Left figure), and quantified the number of GFP-positive cells ( Figure 1B(See right image).

[0253] Figure 2 Human serum inhibits WT VSV in human cell lines. The mixed human serum was combined with VSV-Fluc virus and coated onto human cell lines, such as HT1080 human fibrosarcoma cells. Figure 2 (Left image) Human embryonic kidney (HEK) 293T cells ( Figure 2 (Chinese image) or SKOV3.ip1 human ovarian cancer cells ( Figure 2 (Right figure). The figure shows luciferase activity in three cell lines measured by standard luciferase assay 16 hours after infection.

[0254] Figure 3 This study demonstrates the inhibition of WT VSV in K562 cells by heat-inactivated serum. VSV-GFP was mixed with culture medium, complement-active serum (serum), or heat-inactivated serum (HI serum), and then combined with K562 cells. After 24 hours, the culture plates were imaged using an imaging cell analyzer, and the number of GFP-positive cells in each well was determined.

[0255] Figure 4 This demonstrates the inhibition of WT VSV in human cell lines by heat-inactivated serum. Individual culture media, complement-active human serum (serum), or complement-inactivated human serum (HI serum) were added to human cell lines SKOV3.ip1, HT1080, and K562. VSV-GFP was then added to the cells, and the culture plates were imaged using an imaging cell analyzer. The number of GFP-positive cells in each well was determined 16 hours after infection.

[0256] Figures 5A-5B The results showed that delipoproteinized serum did not have inhibitory activity. Culture media, heat-inactivated serum (serum), human serum albumin (HSA), artificial serum (AF serum), intralipid, or delipoproteinized serum (LD serum) were added to HT1080 or K562 human cell lines. VSV-GFP was then added to the cells. After 16 hours, the culture plates were imaged using an imaging cell analyzer, and the number of GFP-positive cells in each well was determined. Figure 5A Examples of fluorescence microscopy images of HT1080 cells are shown below. Figure 5B As shown.

[0257] Figures 6A-6BThe results showed that low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) inhibited WT VSV, while high-density lipoprotein (HDL) did not. K562 cells were mixed with VSV-GFP and coated into wells under various assay conditions, including individual culture media (M), human serum bank (S), delipoproteinized human serum bank (D), or culture media containing different concentrations of purified human LDL, HDL, or VLDL. After 16 hours ( Figure 6A The image above and Figure 6B (Left image) Cell images were taken using a fluorescence microscope. After 24 hours, the culture plate was imaged using an imaging cell analyzer, and the number of GFP-positive cells in each well was determined. Figure 6A The lower left image and Figure 6B (See right image). Figure 6A The lower right figure further shows the luciferase activity (VSV-Fluc) in Vero cells infected with VSV-Fluc 16 hours prior to the infection under different HDL concentrations.

[0258] Figure 7 The thermostability of LDL and VLDL inhibitory activity was demonstrated. Human LDL ( Figure 7 (left image) or VLDL ( Figure 7 (Right image) Heat-inactivated (HI-LDL or HI-VLDL) or untreated (LDL or VLDL). K562 cells were mixed with VSV-GFP and coated into wells under various test conditions, including culture medium alone (medium) or heat-inactivated (HI) or untreated LDL or VLDL. The culture plates were imaged using an imaging cell analyzer, and the number of GFP-positive cells in each well was determined.

[0259] Figures 8A-8B This explains the mechanism by which serum components activate VSV. Figure 8A This diagram illustrates the inactivation of pathogens (e.g., VSV) by serum (thermally unstable) IgM plus complement. The VSV inactivation mechanism can be attributed to the binding of native IgM to the G protein, which subsequently activates the complement cascade, leading to viral C3b coating, irreversible destruction of viral infectivity, and subsequent cleavage by the membrane attack complex. This viral inactivation mechanism is not instantaneous, but can reduce the infectious titer of VSV formulations or lentiviral vector formulations incorporating the VSV-G protein by up to approximately 10,000-fold within one hour. Figure 8B This diagram illustrates the mechanism by which LDL / VLDL and VSV-G competitively bind to LDLR. The binding site of ApoB-100 to the LDL receptor is the same as the binding site of the VSV-G glycoprotein to the LDL receptor. Lipoproteins containing ApoB-100 include LDL and VLDL, which compete with VSV-G for LDLR binding. Figure 8B ).

[0260] Figures 9A-9B The design for targeting the VSV glycoprotein is described. The VSV-G protein contains a signal peptide (SP) that can be cleaved by proteolytic hydrolysis post-translation. The targeting molecule can be a single-chain variable region fragment (scFv) antibody, such as an antibody 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 attached to the N-terminus of the G protein, for example, with or without a flexible linker. Mutations in the G protein can be incorporated into different amino acid positions, such as amino acid positions 47 and 354 (K47Q / R354Q). Figure 9A The same targeting molecule can be attached to the N-terminus of the alternative rhabdovirus Flanders G (FLAV-G) (with or without a flexible linker). Figure 9B ).

[0261] Figure 10A-10D This demonstrates that VSV-G carrying blinding mutations and anti-HER2 scFv specifically induces fusion of HER2-expressing cell lines. Co-culturing SKOV3.ip1 cells stably expressing the bifidus protein (DSP) DSP-1 or DSP-2 reporter factor... Figure 10A HT1080 cells Figure 10B A549 (human lung adenocarcinoma) cells Figure 10C ) and BHK-21 (young hamster kidney) cells ( Figure 10D The following day, cells were transfected with plasmids expressing GFP, WT VSV-G, or VSV-G with an N-terminal anti-HER2scFv and two mutations (K47Q / R354Q) or three mutations (K47Q / R354Q / E353A) (aimed at eliminating the interaction between the glycoprotein and LDLR). Following transfection, cells were treated at pH 5.0 to mediate fusion, or treated with neutral phosphate-buffered saline (PBS), and then added to fresh medium containing the luciferase substrate EnduRen. Renal luciferase activity was measured 4 hours after substrate addition.

[0262] Figure 11A-11B Infection with blinding and retargeted VSV-G cells was shown to be associated with HER2 receptor levels. HER2 receptor expression in SKOV3.ip1, Vero, and HeLa cells was determined 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 acquired using fluorescence microscopy. Figure 11B ).

[0263] Figure 12 This study demonstrates that soluble αHER2 scFv selectively inhibits the infection and spread of HER2-retargeting LDLR mutant viruses 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 culture media derived from cell cultures transfected with plasmids expressing GFP (mimic-sup) or secreted anti-HER2 or anti-EGFR scFv. Viral infection and spread were monitored by imaging and counting of GFP-positive cells using an imaging cell analyzer.

[0264] Figures 13A-13B This demonstrates that VSV-G can target either the EGF or HER2 receptor in the same cell type. SK-BR-3 cells expressing both EGFR and HER2 were infected with either control VSV-GFP or VSV containing two blinding mutations (K47Q / R354Q) and a targeting molecule for EGFR or HER2 (EGFm123 or scFv for HER2), followed by treatment with culture medium (control) or blocking molecules. Cells were imaged using an imaging cell analyzer 20 hours post-infection. Figure 13A ), and the number of GFP-positive cells was measured ( Figure 13B ).

[0265] Figures 14A-14B The specificity of retargeting VSV-αEGFR-G K47QR354Q-GFP in HT1080-EGFR knockout (KO) cells and HEK-293T-EGFR-KO cells was demonstrated. HT1080 WT or HT1080-EGFR-KO cells were infected with VSV-GFP (control) or VSV-αEGFR-G K47QR354Q-GFP. Figure 14A The GFP image shown in the left image was captured using fluorescence microscopy, and the number of GFP-positive cells was quantified using an imaging cell analyzer. Figure 14A (See right figure). HEK-293T WT or HEK-293T-EGFR-KO cells were infected with VSV-GFP or VSV-αEGFR-G K47QR354Q-GFP. Figure 14B The GFP image shown was taken using an imaging cell analyzer. The numbers on the image represent the number of GFP-positive cells.

[0266] Figures 15A-15BThe specificity of retargeted VSV for K562 cells was demonstrated. K562 parental cells or EGFR / HER2 receptor-expressing cells were infected with retargeted VSV using VSV-GFP or with modified adapter sequences (19 amino acid [aa] adapter: RAAA(G4S)3 (SEQ ID NO: 170); 20 aa adapter: KRAAASGGS(G4S)2GPK (SEQ ID NO: 174)). Figure 15A The GFP image shown was taken using a fluorescence microscope. The number of GFP-positive cells was quantified using an imaging cell analyzer. Figure 15B ).

[0267] Figures 16A-16B The incorporation of the target molecules EGFm123 or hSCF into viral particles was demonstrated. Figure 16A The protein blot of EGFm123 on viral particles is shown. Figure 16B The hSCF image shows a protein blot of viral particles.

[0268] Figures 17A-17B This demonstrates the specificity of retargeting VSV with a smaller targeting molecule. K562 parental, K562-EGFR, or K562-HER2 cells were infected with VSV-GFP (as a control) or the retargeted VSV shown. Figure 17A The GFP image shown was captured using fluorescence microscopy. The number of GFP-positive cells was quantified using an imaging cell analyzer. Figure 17B ).

[0269] Figures 18A-18F This demonstrates VSV-EGFm123 infection in the EGFR KO cell line. (To generate...) Figures 18A-18D The data shown indicate that cells were infected with either VSV-GFP or VSV-EGFm123 virus. Figure 18A As shown, GFP and phase-contrast microscopy images of HeLa WT or HeLa-EGFR-KO cells were obtained. GFP images were captured using an imaging cell analyzer. Figure 18B ), and with HeLa cell group ( Figure 18C ) or HT1080 cell group ( Figure 18D The infection level was quantified by the number of GFP-positive cells. The ability of VSV-EGFm123 to bind to the expected receptor was determined using HeLa, HT1080WT, or EGFR-KO cells. Figure 18E-18F ). 1x10 6 Cells were incubated with VSV-GFP or VSV-EGFm123 virus. After cell fixation, they were stained with PE-conjugated VSV-G antibody. Cell analysis was performed by flow cytometry. Figure 18E-18F ).

[0270] Figures 19A-19B This study demonstrates the resistance of VSV-G-QQ-EGFm123 virus to LDL inhibition. VSV-GFP (VSV-G-wt) or VSV-GFP (EGFR-targeted virus) containing a K47Q / R354Q mutation and G appended to EGFm123 was incubated with separate culture medium, human mixed serum (complement-deficient batch), or a mixture of delipoproteinized human serum. The mixture was then spread onto plated HEK-293T cells. After 24 hours, the culture plates were imaged using an imaging cell analyzer, and the number of GFP-positive cells per well was determined. Figure 19A VSV-GFP (VSV-G-WT) or VSV-GFP (EGFm123-K47Q / R354Q) with a K47Q / R354Q mutation appended to EGFm123 was mixed with K562-EGFR cells and added to wells containing either a single culture medium (control) or progressively increasing concentrations of purified human LDL. After 24 hours, the culture plates were imaged using an imaging cell analyzer, and the number of GFP-positive cells in each well was determined. Figure 19B ).

[0271] Figure 20 This study demonstrates the resistance of VSV-G-QQ-EGFm123 virus to VLDL inhibition. VSV-GFP (WT-G) or VSV-GFP (EGFm123-K47Q / R354Q) with a K47Q / R354Q mutation appended to EGFm123 was mixed with K562-EGFR cells and added to wells containing separate culture medium or a specified concentration of purified human HDL, LDL, or VLDL. The culture plates were imaged using an inverted fluorescence microscope after 24 hours.

[0272] Figure 21 The results showed that binding to WT VSV was reduced by LDL, but binding to EGFR-targeted VSV was not reduced. VSV-GFP (VSV-G-WT) or VSV-GFP with a K47Q / R354Q mutation appended to EGFm123 (VSV-MC11-EGFm123-VSV-G(K47Q / R354Q)-GFP) was mixed with culture medium alone, a mixture of human serum (serum), or LDL and coated onto HT1080 cells. RNA was then extracted from the cells, and the samples were subjected to quantitative reverse transcription polymerase chain reaction (qRT-PCR) using VSV genome-specific primers (IDT). Data represent VSV genome copy number relative to viral copy number from a culture medium-only control.

[0273] Figure 22This study demonstrates the sensitivity of retargeted VSV-G to human serum and LDL. VSV-GFP (VSV-G-WT) or VSV-GFP containing a K47Q / R354Q mutation and attached to human stem cell factor (hSCF) (VSV-GFP(hSCF-G-K47Q / R354Q)) or HER2 scFv (VSV-GFP(G-HER2)) was mixed with various cell lines from the K562 cell group (including K562 parent, K562-HER2, or K562-cKit). The mixture was added to wells containing separate culture media, human mixed serum (serum), human mixed delipoprotein serum (LD serum), LDL-containing culture media (+LDL), or LDL-containing human mixed delipoprotein serum (LD serum +LDL). Fluorescence micrographs were obtained after 24 hours.

[0274] Figure 23 The sensitivity of retargeted VSV-G to human serum and LDL was demonstrated in PC3 cells. VSV-GFP (WT-G) or VSV-GFP carrying G containing the K47Q / R354Q mutation and attached to EGFm123 (G-QQ-EGFm123), hSCF (G-QQ-SCF), or HER2scFv (G-QQ-HER2) was mixed with culture medium alone, fresh (complement-active) human mixed serum (serum), heat-inactivated human mixed serum (HI-serum), or human mixed delipoproteinized serum containing LDL (+LDL). The mixture was applied to PC3 prostate cancer cells. Cells were imaged using an imaging cell analyzer after 24 hours (WT) or 42 hours (retargeted virus).

[0275] Figure 24 The cytoplasmic tail truncation of FLAV-G was shown to enhance its fusion activity. A set of FLAV-G constructs containing anti-EGFRscFv and with progressively shortened cytoplasmic tails were constructed. The fusion activity shown in the figure was determined by DSP intercellular fusion activity assay in SKOV3.ip1 cells.

[0276] Figures 25A-25C This demonstrates improved VSV targeting of the HER2 receptor in PC3 cells. Figure 25A The diagram shows the design of the FLAV-G construct with a 23aa linker and a VSV-G cytoplasmic tail, as well as a construct lacking the VSV-G cytoplasmic tail but with a 19aa linker. Two scFvs targeting HER2 and one scFv targeting EGFR were cloned into the 19aa linker construct. To generate... Figure 25B-25C The data shown evaluated the specificity of two HER2-targeting viruses for a group of PC3 cells, including parental PC3, PC3-EGFR, and PC3-HER2. Figure 25BThe image shows fluorescence microscopy images of infected cells taken using a microscope. The number of infected cells under each condition was determined using an imaging cell analyzer. Figure 25C ).

[0277] Figures 26A-26C FLAV-G is shown to target the insulin-like growth factor 1 (IGF1) receptor. To generate... Figures 26A-26B The data shown indicate that MCF7 breast cancer cells were infected with VSV containing the Flanders viral glycoprotein (FLAV-GΔ30) or VSV without any targeting molecules (non-targeting) or VSV containing the IGF1 receptor (IGF1R) and targeting insulin-like growth factor 1 (IGF1). Infected cells were visualized using fluorescence microscopy (FVM). Figure 26A (see above image) and phase contrast microscopy ( Figure 26A (See image below) was used for imaging. Infected cultures were also imaged using an imaging cell analyzer, and the number of GFP-positive cells was quantified. Figure 26B Flow cytometry was used to determine the expression of the IGF1 receptor in cells labeled with anti-IGF1R antibody or isotype control fluorescently. Figure 26C ).

[0278] Figures 27A-27B The specificity of FLAV-G, which displays modified epidermal growth factor (EGF), was demonstrated. K562 cells stably transduced with the indicated receptor were infected with VSV-GFP (WT) or VSV containing the Flanders viral glycoprotein (FLAV-GΔ30) displaying EGFm123. Infected cells were visualized by fluorescence microscopy (FLV-GΔ30). Figure 27A (see above image) and phase contrast microscopy ( Figure 27A (See image below) Imaging. Infected cultures were also imaged using a cell analyzer, and the number of GFP-positive cells was determined. Figure 27B ).

[0279] Figures 28A-28C The design of VSV-G for retargeting in lentivirus production is shown. Figure 28A A schematic diagram of the generated construct is shown. The VSV-G protein contains a signal peptide (SP) that can be cleaved post-translationally by proteolytic hydrolysis. Exemplary targeting molecules include scFv targeting HER2 or EGFR, natural ligands such as EGFm123 (modified EGF) or hSCF, or nanobodies (e.g., Nb7D12 targeting the EGF receptor), which are attached to the N-terminus of the G protein with or without a flexible linker. A construct without the targeting molecule was generated as a control. Blinding mutations from VSV-G were incorporated at K47 and R354. Figure 28A The figure shows SEQ ID NO: 35, 35, 35 and 236 in the order of appearance. Figure 28BThe illustration shows lentivirus production in HEK-293T cells. Figure 28C The protein blots of the lentiviral particles were displayed.

[0280] Figures 29A-29B This study demonstrates the validation of retargeted lentiviruses in receptor-positive cell lines. Lentiviral viruses, either VSV-G-WT or VSV-G pseudotyped with the K47Q / R354Q (G-QQ) mutation and EGFR scFv, EGFR-E11 scFv, or EGFm123 ligand, were transduced into parental Jurkat or K562 cells or modified forms overexpressing EGFR. Fluorescence microscopy images of Jurkat cells were taken 48 hours post-transfection (hpt). Figure 29A K562 cells were transduced with viral supernatant, and fluorescence microscopy images were taken at 24 hpt. Figure 29B ).

[0281] Figures 30A-30B The study demonstrated that EGFR-targeted lentiviral vectors exhibited low sensitivity to serum and LDL inhibition. K562 parental cells or modified EGFR-overexpressing K562 cells (K562-EGFR) were transduced using a specific volume of GFP-expressing lentiviral vectors containing WT VSV-G (WT-G) or VSV-G with the K47Q / R354Q mutation appended to EGFm123 (EGFm123K47Q / R354Q). Transduction was performed in the presence of separate culture media, mixed human serum (complement deficient), mixed delipoproteinized human serum, or mixed delipoproteinized human serum supplemented with 150 mg / dL LDL. After 40 hours, the results were analyzed by fluorescence microscopy. Figure 30A The cells were imaged using an imaging cell analyzer. The number of GFP-positive cells per well was determined using the cell analyzer software. Figure 30B ).

[0282] Figures 31A-31D This demonstrates that the VSV-G H8Q / K47Q / Y209Q / R354Q mutation enhances the specificity of VSV targeting EGFm123. The construct design including the LDLR blinding mutation in VSV-G is shown below. Figure 31A As shown. For Figures 31B-31C The generated data were used to infect K562 parental cells or K562-EGFR cells with VSV-EGFm123 virus, and GFP images were acquired using fluorescence microscopy. Figure 31A (Left image). Figure 31A The right figure shows the quantification of GFP-positive cells using an imaging cell analyzer. Figure 31DImages of GFP captured using an imaging cell analyzer are shown. These results indicate that combining the Y209Q mutation with VSV-GK47Q / R354Q enhances the specificity of EGFm123-expressing VSV retargeting, while combining the H8Q mutation with VSV-GK47Q / R354Q has little or no effect on enhancing retargeting specificity.

[0283] Figures 32A-32D The study demonstrated that the deletion of the K47 residue in VSV-G eliminated VSV tropism and redirected VSVs to EGF receptor (EGFR)-positive cells. K562 parental cells or K562-EGFR cells were infected with VSV-GFP or G-WT or G-ΔK47 VSVs displaying EGFm123, and fluorescence microscopy images were obtained. Figure 32A (Left image). Figure 32A The right figure shows the quantification of GFP-positive cells using an imaging cell analyzer. Figure 32B An example of a GFP image captured by an imaging cell analyzer is shown. Figure 32C Other constructs containing deletions of H8, K47, Y209, and / or R354 residues designed to weaken the LDLR tropism of VSVs are shown. Figure 32D More instances of constructs containing two-residue deletion mutations are shown.

[0284] Figure 33 The sequence confirmation of the VSV-G-ΔK47 residue in VSV1-409-0 is shown. The viral sequence shows a point mutation at F405I in VSV-G. SEQ ID NOs 213-226 are shown in the figure in order of appearance.

[0285] Figures 34A-34B This demonstrates the use of an alternative rhabdoviral G protein for targeting. Figure 34A Nine glycoproteins selected from nine different rhabdovirus genera are depicted (arrows indicate this). FLAV-G was selected as the lead compound for targeting. A group of K562 cells stably expressing (or not expressing) EGFR or HER2 were infected with VSV containing the GFP reporter gene and scFv targeting EGFR or HER2. Cells were imaged using fluorescence microscopy. Figure 34B ).

[0286] Figure 35 Screening for additional alternative G proteins was described. Twenty additional viral glycoproteins were selected for screening to identify glycoproteins that could retarget to receptors of interest. Descriptions of viral species in the genus *Vesicavipira* of the family Rhabdoviridae are shown in the table. Figure 35(Left figure). The percentage of amino acid sequence identity with vesicular stomatitis Indiana virus (VSIV) (see, for example, SEQ ID NO: 8) containing or not containing a signal peptide sequence was calculated using Clustal Omega. Based on the favorable properties of glycoproteins from Fukuoka virus (Ledantevirus), Flanders (Hapavirus), and bovine transient fever virus (Ephemerovirus), other glycoproteins from Ledantevirus, Hapavirus, and Ephemerovirus genera were selected. Figure 35 (Right image).

[0287] Figure 36 The method of screening for 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 figure). Viral species of the genus *Vesicularvirae* in the family Rhabdoviridae were selected based on a percentage of less than 70% amino acid sequence identity with Indiana vesicular stomatitis virus. Other glycoproteins from the genera *Vesicularvirae*, *Vesicularvirae*, and *Vesicular Fever Virus* were selected based on favorable properties of glycoproteins from Fukuoka virus (*Lydativirae*), Flanders virus (*Hapavivirae*), and bovine transient fever virus (*BFV*) in the first phase of screening. For each glycoprotein, a signal peptide sequence was predicted using SignalP 6.0, and a modified epidermal growth factor (EGFm123) was inserted immediately following the signal peptide sequence. The genes for these modified glycoproteins were synthesized and subcloned into a protein expression vector (pCG). Figure 36 (See right image).

[0288] Figure 37 This demonstrates functional screening for EGFR-targeting alternatives to EGF-displaying rhabdoviral G proteins. A schematic diagram of the DSP cell-cell fusion assay described in this article is shown below. Figure 37 The left figure shows the measurement of the fusion activities of the various glycoproteins described in this article in SKOV3.ip1 cells and EGFR KO SKOV3.ip1 cells. Figure 37 As shown in the middle figure. Figure 37 The right figure shows a bar chart illustrating cell surface expression of EGFm123-G protein as measured by median fluorescence intensity of EGFm123-G protein particles (top) and Western blot analysis of EGFm123 levels in EGFm123-G protein particles (bottom).

[0289] Figure 38This demonstrates the use of targeted G proteins for lentiviral pseudotyping. Lentivirals were generated in HEK 293T cells by expressing a glycoprotein vector (pCG), a packaging plasmid (p8.91), and a GFP-expressing genomic vector (pLV-SFFV-GFP). These lentiviruses were pseudotyped using a set of proteins targeting rhabdoviral glycoproteins (or non-targeted VSV G WT and EGFR-targeted FLAV-GΔ30 as controls). Figure 38 (Left image). The lentiviral supernatant was then added to a monolayer culture of SKOV3.ip1 or SKOV3.ip1 EGFR KO cells. The number of GFP-positive cells was determined using an imaging cell analyzer. Figure 38 (See right image).

[0290] Figure 39 This document describes various features of the bifidus protein (DSP) screening and the lentivirus screening described herein.

[0291] Figure 40 This document describes an exemplary experimental timeline for the VSV-G EGFR scFv transfection screening described in this paper.

[0292] Figure 41 This document presents an example of the EGFR scFv DSP transfection assay screening protocol described in this article.

[0293] Figure 42 A flowchart showing an exemplary analysis of EGFR scFv transfection screening is displayed.

[0294] Figure 43 The EGFR scFv screening in the SKOV3ip.1DSP screening identifies several scFv sequences with improved fusions.

[0295] Figure 44 The results show that αEGFR-VSV-G exhibits a moderate preference for the scFv direction.

[0296] Figure 45 This is a schematic diagram indicating the directional preference for increased function when scFv is located at the N-terminus of the extracellular domain.

[0297] Figure 46 An example of the αEGFR lentivirus production scheme described in this article is shown.

[0298] Figure 47 The description of αEGFR titration via qPCR is shown.

[0299] Figure 48 A flowchart illustrating the exemplary lentivirus screening described in this article is shown.

[0300] Figure 49This study demonstrates that several αEGFR scFv-VSV-G (QQ) lentiviruses exhibit enhanced transduction efficiency in the presence of serum.

[0301] Figure 50 A description of the transduction efficiency of an exemplary αEGFR scFv candidate is shown.

[0302] Figure 51 The comparison of DSP and lentiviral screening for the αEGFR-VSV G construct is shown, including comparison of scFv sequences using log-expectation multiple sequence comparison (MUSCLE), and graphical representation of the alignments in the phylogenetic tree using Jalvuew software and the BLOSUM62 nearest neighbor algorithm.

[0303] Figure 52 VSV construct designs incorporating various adapter sequences are depicted. A retargeted VSV containing a 20aa adapter located between EGFR scFv and VSV-G after proteolytic cleavage is included. Different types of variable-length adapter sequences were cloned between EGFR scFv and VSV-G. EIK, the terminal amino acid sequence of scFv; KFT, the starting amino acid sequence of VSV-G. Adapter sequences are shown in red text. F, flexible adapter; R, rigid adapter; Fm, moderately flexible adapter; F-el, flexible elastin-like adapter; IgG4 h, IgG4 hinge. VSV-G contains blinding K47Q and R354Q mutations against LDLR. SEQ ID NOs: 171-172, 36-39, 3, 173, and 40-41 are disclosed in the figure in order of appearance.

[0304] Figure 53 The rescue of EGFR-targeting VSV with a substitute adapter sequence is shown. The pVSV-MC11-EGFRscFv-VSV-G-GFP plasmid with a substitute adapter sequence was rescued in SKOV3.ip1 cells using a vaccinia virus-based rescue system. Viral supernatant (p0 supernatant) was collected and filtered. The p0 supernatant was then transferred to a monolayer of Vero-EGFR cells, and GFP fluorescence microscopy images shown in the figure were obtained.

[0305] Figures 54A-54C Characterization of EGFR-targeted VSV is shown. Amplification phase, viral titer, and sequencing results are described in... Figure 54A The table shown in the image shows the protein blots of the virus. Figure 54B As shown, the bands indicate intact EGFR scFvs containing G and G cleaved by proteolytic hydrolysis. Figure 54CThis demonstrates the specificity of EGFR targeting VSV. Micrographs show parental K562 or K562-EGFR cells infected with EGFR-targeting VSV and VSV-GFP, along with fluorescence micrographs.

[0306] Figure 55 The 18aaL(F), 15aaL(R), and 16aaL(R) adaptors exhibited proteolytic cleavage. Nine different adaptors ligated to EGFR scFv were selected to identify adaptors that provided specific targeting against EGFR. Figure 55 (Left figure). Adapter sequences were generated in the VSV backbone and then subcloned into the pCG vector backbone for lentiviral production. The construct was transfected into HEK 293T cells, cell lysates were collected, and run on SDS-PAGE gels to analyze protein expression. Figure 55 (See right figure). The figure shows SEQ ID NO: 166-167, 26-30, 168 and 31-32 in the order of appearance.

[0307] Figure 56 The 18aaL(F), 15aaL(R), and 16aaL(R) linkers demonstrate fusion-promoting capabilities. Fusion activity was measured in a mixed population of A549-EGFR-expressing DSP1-7 and DSP8-11 cells containing nine constructs with variable linkers for EGFR scFv. Half of these constructs contained a bifidogenic protein (DSP) reporter gene with splitting GFP, and the other half contained Renilla luciferase.

[0308] Figures 57A-57B The 18aaL(F), 15aaL(R), and 16aaL(R) linkers exhibit proteolytic cleavage in lentivirus production cell lysates and in the lentivirus particles themselves. Figure 57A A schematic diagram of EGFR scFv virus production with a variable linker is depicted. Lentiviral production plasmids were transfected into HEK 293T cells to generate lentivirus. Western blots were performed on cell lysates from cells used for lentivirus production to detect EGFR scFv-linker-VSV-GQQ (full-length) or lysed VSV-GQQ (...). Figure 57B (Left figure). Collect the supernatant containing viral particles and centrifuge using a microcentrifuge. Aspirate the supernatant, lyse the cell pellet, reduce it at 95°C for 5 min, and then run it on an SDS-PAGE gel to detect EGFRscFv-linker-VSV-GQQ (full-length) or lysed VSV-GQQ (…). Figure 57B (middle image). It also includes the titer of each virus as determined by the p24 enzyme-linked immunosorbent assay (ELISA).

[0309] Figures 58A-58B The 18aaL(F), 15aaL(R), and 16aaL(R) linkers exhibiting proteolytic cleavage demonstrate enhanced 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 post-transduction to determine the specificity of each retargeting lentivirus with different linkers. Figure 58A ). Figure 58B A graph showing the number of GFP-positive cells quantified in K562 and K562-EGFR cells transduced with each lentivirus.

[0310] Figure 59 Targeting constructs for assessing virus specificity in the presence or absence of serum are shown. The ligands shown include human EGF, human stem cell factor (hSCF), and scFv targeting EGFR or Her2. The hSCF ligands are displayed on either blinded (VSV-G-QQ)G protein or unblinded (VSV-G-WT)G protein that have a weak interaction with LDLR.

[0311] Figure 60 The diagram depicts the use of inadvertents in the absence (left image) or presence (right image) of 25% heat-inactivated human serum. Figure 59 The images show fluorescence microscopy images of K562 cells (parental or stably expressing HER2, cKit (SCF receptor), or EGFR) infected with recombinant VSV carrying the G protein. 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 the virus displaying the ligand specifically infects only those target cells carrying homologous receptors targeting the ligand it displays. This is evident even for viruses displaying SCF where the G protein is not mutated to eliminate LDLR tropism, indicating that the displayed SCF domain can spatially interfere with G protein-LDLR interactions. The right panel shows that, in the presence of 25% heat-inactivated human serum, entry of the virus carrying the wild-type G protein was blocked in all K562 clones, while entry of the targeting virus via the alternative (i.e., non-LDLR) receptor was not blocked and may even be enhanced in some cases.

[0312] Figure 61 Proof-of-concept data are described that support the retargeting of G pseudotype lentiviral vectors through exhibited domains including EGFm123 and anti-epidermal growth factor receptor (EGFR) scFv(E11).

[0313] Figure 62This indicates 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 an illustrated version of the lentiviral production protocol along with a table showing the amounts of envelope glycoprotein DNA used. The lower right corner contains a Western blot of VSV G (from the cleaved chimera and VSV G-QQ). Rabbit anti-VSV G [8G5F11] antibody was used to detect the protein in the virion pellet.

[0314] Figures 63A-63B The results showed that increasing the ratio of VSV G-QQ to non-lytic EGFR scFv-17aaL(F)-VSV G-QQ improved targeting specificity. Figure 63A The figure above depicts the transduction of K562 and K562-EGFR-expressing cells with lentiviruses in mixed proportions of EGFR scFv-17aaL(F)-VSV G-QQ and VSV G-QQ. Transduction was performed at a multiplicity of infection (MOI) of 5. Bright field (BFR) values ​​for each lentivirus were measured 72 hours post-transduction (hpt). Figure 63A (see image below) and GFP ( Figure 63A The above figure shows the expression for imaging. The amount of DNA corresponding to each scale listed at the top of the figure set is shown in the table below all figures. 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. Reducing the amount of EGFR scFv-17aaL(F)-VSV G-QQ DNA and increasing the amount of VSV G-QQ DNA produces improved targeting specificity. Figure 63B Celigo quantification of the number of GFP-positive cells for each viral mixture ratio is shown.

[0315] Figure 64 This illustrates a potential model for a mixed trimer of non-lytic retargeting VSV G and blinded VSV G. The model on the left depicts the homotrimeric VSV G. Each monomer contains an EGFR scFv retargeting domain, a linker, and blinded VSV G (mutations at K47Q and R354Q - QQ). For initially non-specifically targeted or lytic lentiviruses, mixing non-lytic EGFR scFv retargeting monomers with blinded VSV G-QQ can produce heterotrimers that allow for target specificity.

[0316] Figure 65A-65C This study demonstrated the in vivo targeting of EGFR+ (epidermal growth factor receptor-positive) tumors by VSV, which exhibits EGF (epidermal growth factor), via intravenous (iv) administration in a mouse model of SCID (severe combined immunodeficiency disease). For the study design ( Figure 65AFemale CB17 SCID mice were subcutaneously implanted with mouse myeloma 5TGM1 cells expressing human EGF receptor (hEGFR). When the average tumor volume reached approximately 150 mm², [the cells were implanted]. 3 Mice were randomly divided into groups. They were treated with saline solution and 1x10... 8 TCID 50 VSV-M-RFP-GFP or 1x10 8 TCID 50 Mice were treated intravenously with VSV-M-GqqEGFm123-GFP virus. Adverse clinical symptoms, body weight, and tumor growth in mice were observed until day 40 post-treatment. The 5TGM1-hEGFR+ tumor growth curve is shown below. Figure 65B As shown. Mice treated with VSV-M-RFP-GFP exhibited adverse clinical symptoms, and they were found to be dead or euthanized. On day 21 post-infection, no mice survived. Figure 65B-65C Compared with mice treated with VSV-M-RFP-GFP, VSV-M-GqqEGFm123-GFP virus completely suppressed tumor growth in all six treated mice. No adverse clinical symptoms were observed in the mice. Figure 65B-65C ).

[0317] Figures 66A-66D This study describes the optimization of the cytoplasmic tail of KRV-G. The fusion activity of the KRV-G cytoplasmic tail truncated mutant was also investigated. SKOV3.ip1 cells stably transduced with either the N-terminus or C-terminus half of the bifidus protein GFP-Renilla luciferase reporter were seeded in equal proportions. The next day, plasmids expressing VSV-G WT or EGFm123 modified (at the N-terminus), FLAV-G with 30 amino acids removed from the C-terminus (Δ30), or KRV-G with a truncated cytoplasmic tail (e.g., removal of 10 amino acids (Δ10), 20 amino acids (Δ20), or 30 amino acids (Δ30)) were transfected into the cells. 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 minutes, then the saline was replaced with fresh medium containing EnduRen substrate. The luminescence activity was measured 2 hours later. Figure 66A ) GFP-encoded VSVs with KRV-G or KRV-G with 10 amino acids removed from the C-terminus (Δ10) and the N-terminus modified with EGFm123 were rescued and amplified. (via TCID) 50 The infectious titer of the virus obtained by the assay method ( Figure 66B The specificity of full-length or Δ10KRV-G was determined by monitoring the infection process of recombinant VSV carrying these EGFm123-tagged glycoproteins and green fluorescent protein (GFP). Figures 66C-66DThe recombinant virus is used to infect wild-type (WT) or EGF receptor (EGFR) knockout (KO) versions of SKOV3.ip1 with an MOI of 1. Figure 66C ) or HEK 293T ( Figure 66D The number of infected cells expressing virus-encoded GFP was measured using a Celigo imaging cell analyzer one day post-infection.

[0318] Figures 67A-67B The epidermal growth factor receptor (EGFR)-targeted KRV-G and KEUV-G demonstrated enhanced resistance to serum inhibitory factors and specificity comparable to that of blinding VSV-G. VSVs encoding GFP were generated that possess the WT VSV glycoprotein (VSV-G) or a glycoprotein with one of the following modifications replacing native VSV-G: VSV-G with LDLR receptor blinding glutamine substitution at K47 and R354 (QQ) or K47, Y209, and R354 (QQQ); Kumasi rhabdovirus G (KRV-G); KRV-G (Δ10) with 10 amino acids removed from the C-terminus; Keuraliba virus G (KEUV-G); Perinet virus G (PERV-G); Piry virus G (PIRYV-G); Fukuoka virus G (FUKV-G); or Curionopolis virus G (CURV-G). The virus shown contains a modified epidermal growth factor (EGF) molecule attached to the N-terminus of the encoded glycoprotein. This was achieved by using 1x10... 6 VSV from each infection unit was incubated at 37°C for 1 hour in OptiMEM medium alone, OptiMEM medium containing 50% heat-inactivated human serum, or OptiMEM medium containing 50% complement-active serum. The sensitivity of the modified VSV to complement-active serum was then determined. Standard TCID... 50 In the titration assay, the incubated virus was serially diluted and used to inoculate Vero cells stably expressing human EGFR. The titration plates were scored 3 days post-inoculation. Figure 67A The specificity of VSVs retargeting EGFR using various rhabdoviral glycoproteins was determined by monitoring the infection process of recombinant VSVs encoding the GFP gene over 3 days. HT1080 cells (WT) or HT0180 cells in which the EGFR gene was knocked out (KO) were infected with the VSVs shown at an MOI of 1. The number of infected cells expressing virus-encoded GFP was measured using a Celigo imaging cell analyzer at 1, 2, and 3 days post-infection. Figure 67B ).

[0319] Figures 68A-68BThis demonstrates that the Kumasi rhabdoviral glycoprotein (KRV-G) can retarget EGFR. The infection process of recombinant VSV encoding the GFP gene was monitored over 3 days. Figure 68A The virus shown has its glycoprotein gene replaced by KRV-G in which the C-terminus 10 amino acids are removed (Δ10). At the indicated location, a targeting motif (modified epidermal growth factor (EGF) or an scFv targeting the EGF receptor (EGFR)) is added to the N-terminus of KRV-G. SKOV3.ip1 cells (WT) or SKOV3.ip1 cells in which the EGFR gene is knocked out (KO) are infected with the designated VSV at an MOI of 1, and the number of infected cells expressing virus-encoded GFP is measured using a Celigo imaging cell analyzer at 1, 2, and 3 days post-infection. Figure 68B The virus shown, VSV-G gene, was mutated to include two (QQ) or three (QQQ) mutations to eliminate LDLR binding, or replaced by a glycoprotein from Kumasi rhabdovirus (KRV-G) in which the C-terminal 10 amino acids were removed (Δ10). At the indicated locations, targeting moieties (modified epidermal growth factor (EGF), scFv targeting the EGF receptor (EGFR), or scFv targeting Her2) were added to the N-terminus of KRV-G or VSV-G. SKOV3.ip1 cells (WT) or SKOV3.ip1 cells in which the EGFR gene was knocked out (KO) were infected with the shown KRV or VSV at an MOI of 1, and the number of infected cells expressing virus-encoded GFP was measured using a Celigo imaging cell analyzer at 1, 2, and 3 days post-infection.

[0320] Figure 69 An example of the design of a retargeted mutant VSV-G construct is shown. The VSV-G protein contains a signal peptide (SP) that is cleaved post-translational by proteolytic hydrolysis. The targeting molecule is a natural ligand, such as, but not limited to, EGFm123 (modified EGF), which can attach to the N-terminus of the G protein without a flexible linker. LDLR-binding residues (H8, K47, R354, and Y209) in the G protein are missing in single, double, triple, or quadruple combinations.

[0321] Figure 70 Protein expression of the VSV-G deletion mutant construct was depicted. The plasmid DNA construct was transfected together with the lentiviral transfer and packaging construct to generate lentivirus in HEK-293T cells. For comparison, triple or quadruple substitution mutants combined with G-QQ (K47QR354Q) were also included in this experiment. After collecting the lentiviral supernatant, cell lysates were collected 72 hours post-transfection, and then Western blot analysis was performed using anti-VSV-G and anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibodies.

[0322] Figure 71 This demonstrates the incorporation of the VSV-G deletion mutant into lentiviral particles. Lentiviral supernatant collected 72 hours post-transfection was titrated using a p24 ELISA method. (Total 5 x 10⁻⁶) 5 The physical particles were lysed and loaded into SDS-PAGE, and then subjected to Western blot analysis using anti-VSV-G and anti-p24 antibodies.

[0323] Figures 72A-72D The screening for deletion mutants that eliminate the VSV-G tropism is described. Figures 72A-72B Lentiviral transduction on K562 cell groups is shown. K562 parental cells or K562-EGFR cells were transduced at MOI=20 using a heavily targeted lentivirus displaying EGFm123, as shown. WT-G pseudotyped lentivirus was used as a control. Celigo images were acquired 72 hours post-transduction. Figures 72A-72B ) and Nikon images ( Figure 72C ), and quantify the transduced GFP-positive cells ( Figure 72D ). Invention Details

[0325] Rhabdoviral glycoproteins are the only viral proteins found on the surface of the virion and mediate the binding of viral particles to cellular receptors and subsequent viral entry into cells (infection). The glycoprotein (G) of vesicular stomatitis virus (VSV) mediates infection in multiple cell types across various species by interacting with the relatively ubiquitous low-density lipoprotein receptor (LDLR) and its associated receptor family members (Finkelshtein et al., 2013; Nikolic et al., 2018). VSV-G can be used as a pseudotyped lentivirus for various therapeutic applications, including, for example, chimeric antigen receptor (CAR)-T cells; several VSV-G pseudotyped lentiviruses are currently undergoing clinical trials (Munis et al., 2020).

[0326] CAR-T cells can be used to treat hematologic malignancies. However, the logical challenges associated with in vitro CAR-T cell manufacturing may be a limiting factor for the wider clinical application of this therapy. Currently, CAR-T cells are primarily engineered in vitro using VSV-G pseudotyped lentiviral vectors to deliver CARs to target T cells collected from patients. The modified CAR-T cells are then expanded in vitro and infused back into the patient. In vivo CAR-T therapy involves the direct delivery of CAR-encoding viral vectors to patients to achieve the modification and proliferation of T cells within the patient, effectively overcoming many of the current logical challenges associated with CAR-T therapy. However, in vivo CAR-T therapy requires effective targeting of CAR-encoding viral vectors to T cells while limiting off-target effects. Since most current lentiviral vector therapies for CAR-T cells use VSV-G pseudotyped vectors, solutions for effectively targeting VSV-G are applicable to both oncolytic VSV therapies and CAR-T cells.

[0327] In addition to proper targeting, effective VSV-G-based therapies must also be able to bypass the natural barriers of infection for a sufficient period of time to reach target cells / tissues after delivery into the body. Oncolytic VSVs and VSV-G pseudotyped lentiviral vector therapies can be inactivated by the complement system (DePolo et al., 2000; Mills and Cooper, 1978). Fabrication of VSVs or VSV pseudotyped lentiviral vectors in cells expressing high levels of CD55 significantly enhances the virus / vector's resistance to complement inactivation (Schauber-Plewa, C. et al., 2005; Johnson et al., 2012). However, as described in this disclosure, another natural barrier has been identified in human serum (blood) that blocks the binding of oncolytic VSVs or VSV-G pseudotyped lentiviruses to cells. This barrier is an ApoB-100-containing lipoprotein that outperforms VSV-G in binding to the low-density lipoprotein receptor (LDLR), thereby preventing the efficient delivery of VSV-G-based therapies to cells. Therefore, effective therapy delivery also needs to overcome or circumvent competition with lipoproteins containing ApoB-100.

[0328] The recombinant fusion-promoting protein disclosed herein offers the following advantages: significantly enhanced targeting of cellular receptors highly expressed in various cancers, and also circumvents LDL / VLDL inhibition during therapy delivery. Extensive data on different adaptor combinations highlight the importance of adaptor sequences and lengths in determining the function of retargeted G proteins, and underscore the need for appropriate adaptors to achieve functional G protein-based therapies.

[0329] This disclosure demonstrates retargeting of exemplary VSV-G using LDLR-blinding mutations (e.g., corresponding to the H8, K47, Y209, and / or R354 positions in SEQ ID NO: 8) in combination with scFv antibodies or nanobodies targeting EGFR or HER2, and specific retargeting of VSV-G using modified EGF (EGFm123) or stem cell factor (SCF) without blinding mutations. The distantly related rhabdovirus glycoprotein (FLAV-G) can be retargeted to both EGFR and HER2, with HER2 retargeting requiring optimization of the adapter sequence / length. This disclosure can be extended to cover other glycoproteins from the rhabdovirus family instead of VSV or FLAV-G, such as those detailed below. Receptors other than EGFR and HER2 can also be targeted in a similar manner using, for example, scFv molecules or other ligands. While the data presented herein describe the use of scFv, nanobodies, and natural receptor ligands for targeting G, other targeting molecules, including but not limited to scFv, affibodies, darpins, peptides, nanobodies, and natural or modified natural receptor ligands, can also be used. The data presented herein primarily demonstrate VSV targeting using replicating viruses, but the VSV platform can also be used in situations where a replicating vector is not required, where the retargeted glycoprotein is not encoded in the genome but is provided in trans form. Furthermore, VSV glycoproteins can be used to pseudotype other viruses, including lentiviral vectors for gene therapy applications. In these applications, retargeted rhabdoviral glycoproteins would be highly desirable for tailoring therapies to target cells of interest. Therefore, this disclosure illustrates that strategies for specific retargeting of VSV-G in the context of replicating VSV can be transferred to pseudotyped lentiviral vectors.

[0330] Importantly, the data presented in this paper demonstrate that targeting G proteins not only achieves specific targeting of cells of interest, but also bypasses a previously unrecognized natural barrier to infection: VSV-G competes with LDL / VLDL for LDLR binding. Since LDL / VLDL exhibits a dose-dependent inhibitory response, another approach to counteracting this competition could be to pre-treat patients with drugs that lower LDL / VLDL to limit the amount of these competing molecules in the blood at the time of treatment. However, this alternative requires delaying treatment long enough for blood LDL / VLDL levels to decrease to a level that does not induce inhibition of the delivered therapy.

[0331] Further details of the compositions and methods disclosed herein will be described in the following various exemplary embodiments.

[0332] definition

[0333] 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 pertains.

[0334] Unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” also include plural references. Thus, for example, a reference to “a method” includes one or more methods and / or steps of the type described herein, and / or one or more methods and / or steps that will be apparent to those skilled in the art upon reading this disclosure.

[0335] The terms “about” or “approximately” encompass a range within a statistical sense of a given value. Such a range may be within an order of magnitude, preferably within 50% of a given value or range, more preferably within 20%, even more preferably within 10%, and still more preferably within 5%. The permissible variations covered by the terms “about” or “approximately” depend on the specific system under study and can be readily understood by those skilled in the art.

[0336] The term “antigen” refers to any substance (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, or a combination thereof) that, when introduced into a host, animal, or human with an immune system (either directly or through expression, such as in a DNA vaccine), is recognized by the host’s immune system and is able to elicit or actually elicit an immune response.

[0337] The terms “viral element” and “viral component” are used herein to refer to viral genes (e.g., genes encoding polymerases or structural proteins) or other elements of the viral genome (e.g., packaging signals, regulatory elements, LTRs, ITRs, etc.).

[0338] The term "oncolytic virus" is used herein to refer to a virus capable of infecting and replicating within tumor cells, thereby killing the tumor cells. Oncolytic viruses may possess replication capabilities. As a non-limiting example, oncolytic viruses may include rhabdoviruses, that is, any virus belonging to the Rhabdoviridae family, such as vesicular stomatitis virus (VSV).

[0339] As used herein, the term "vesicular virus" refers to any virus in the genus *VesicularVirula*. Non-limiting examples of vesicular viruses include vesicular stomatitis virus (VSV) (e.g., VSV-New Jersey, VSV-Indiana), Alagoas vesicular virus, Cocal vesicular virus, Jurona vesicular virus, Carajas vesicular virus, Maraba vesicular virus, Piry vesicular virus, Calchaqui vesicular virus, Yug Bogdanovac vesicular virus, Isfahan vesicular virus, Chandipura vesicular virus, Perinct vesicular virus, and Porton-S vesicular virus. Vesicular stomatitis virus (VSV) in the genus *VesicularVirula* is a prototypical rhabdovirus. While VSV is used as an example in this disclosure, it can also be applied to other vesicular viruses and other rhabdoviruses. VSV has two main serotypes: the New Jersey type and the Indiana type, both of which can infect insects and mammals, causing disease in cattle, horses, and pigs. The VSV genome consists of 11-12 kb 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 the large protein) (L). G monomers associate to form trimer spikes anchored to the viral membrane.

[0340] The terms "vector," "expression vector," and "cloning vector" refer to any medium by which a nucleotide sequence encoding, for example, a foreign gene (e.g., an RNA or DNA sequence) can be introduced into a cell (e.g., a host cell) to genetically modify the cell and promote the expression (e.g., transcription and translation) of the introduced nucleotide sequence. Non-limiting examples of vectors include synthetic RNA and DNA molecules, plasmids, viruses, bacteriophages, etc. In some embodiments, the vector may be a viral vector, including but not limited to baculovirus vectors, herpesvirus vectors, lentivirus vectors, retrovirus vectors, vaccinia virus vectors, adeno-associated virus vectors, adenovirus vectors, and alphavirus vectors.

[0341] The term "replicating capability" in this article refers to viruses (including wild-type and recombinant viruses) that are capable of infecting and multiplying within cells.

[0342] The term "pseudotyped" in relation to the viral particles described herein refers to the presence of molecules (e.g., proteins, glycoproteins, etc.) in the lipid envelope or capsid of a viral particle that are mutated and / or heterologous compared to molecules typically found on the surface of the virus from which the viral particle originates, and that may influence, facilitate, guide, redirect, and / or completely alter the tropism of the viral particle compared to the reference wild-type virus from which the viral particle originates. In some embodiments, the viral particle is pseudotyped such that it recognizes, binds to, and / or infects targets (ligands or cells) different from those of the reference wild-type virus from which the viral particle originates. In some embodiments, the viral particle is pseudotyped such that it cannot recognize, bind to, and / or infect targets (ligands or cells) of the reference wild-type virus from which the viral particle originates.

[0343] The term “encoding” can refer to encoding from the (+) or (-) sense strand of a polynucleotide, for example, for expression in viral particles.

[0344] The term "antibody" refers to monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), intracellular antibodies, microantibodies, biantibodies, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against antigen-specific TCRs), as well as epitope-binding fragments of any of the above antibodies. The term "antibody" also refers to covalent biantibodies, 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 available in this disclosure include immunoglobulin molecules and their immunologically active fragments, i.e., molecules containing antigen-binding sites. Immunoglobulin molecules can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass.

[0345] The term “T cell” or “T lymphocyte” is used in its broadest sense herein 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 (TIL; CD8+ T cells), CD4+CD8+ T cells, regulatory T cells (Tregs), and NK-T cells. T cells can include thymocytes, naive T cells, memory T cells, immature T cells, mature T cells, resting T cells, or activated T cells. T cells can also include “γ-δ T cells (γδ T cells),” which refers to a special population of a small subset of T cells with a distinctive TCR on its surface, and unlike most T cells where the TCR is composed of two glycoprotein chains (called α- and β-TCR chains), the TCR in γδ T cells is composed of γ and δ chains.

[0346] 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) chains, β2-microglobulin, MHC class II α chains, MHC class II β chains), individual subunits of such MHC molecule chains (e.g., α1, α2, and / or α3 subunits of MHC class I α chains, α1 and / or α2 subunits of MHC class II α chains, β1 and / or β2 subunits of MHC class II β chains), and their fragments, mutants, and various derivatives (including fusion proteins), wherein such fragments, mutants, and derivatives 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, which can accommodate a peptide 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 openness of the MHC class II peptide-binding groove (where the α1 domain of a class II MHC α peptide binds to the β1 domain of a class II MHC β peptide) allows for a wider range of peptide lengths. MHC class II peptides are typically 13 to 17 amino acids long, but shorter or longer lengths are not uncommon. Therefore, peptides can move within the MHC class II peptide-binding groove, thus altering the decimator located directly within the groove at any given time. This article uses the routine identification of specific MHC variants. For example, HLA-B 17 refers to the human leukocyte antigen from gene locus number 17 in the B gene group (and therefore class I MHC); the gene HLA-DR11 refers to the human leukocyte antigen encoded by a gene from locus number 11 in the DR region (and therefore class II MHC).

[0347] The term "operable link" or similar term refers to a juxtaposition where the described components are positioned to allow them to function in a desired manner. For example, a control sequence "operable link" to a coding sequence is linked in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequence. Sequences of an "operable link" include expression control sequences adjacent to the gene of interest, as well as expression control sequences that act trans- or at a distance to control the gene of interest (or the sequence of interest). The term "expression control sequence" includes polynucleotide sequences that are essential for influencing the expression and processing of the coding sequence linked to them. "Expression control sequences" include: appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences stabilizing cytoplasmic mRNA; sequences enhancing translation efficiency (e.g., Kozak concordant sequences); sequences enhancing peptide stability; and sequences enhancing peptide secretion when needed. The nature of such control sequences varies depending on the host organism. For example, in prokaryotes, such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences, while in eukaryotes, they 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 may also include other components whose presence is advantageous, such as leader sequences and fusion chaperone sequences.

[0348] The term "host cell" refers to any cell containing heterologous nucleic acids. As a non-limiting example, the heterologous nucleic acid can be a vector. The host cell can be, for example but not limited to, a cell from any organism that is used, manipulated, modified, selected, transformed, or grown for the purpose of producing substances by the cell, such as expressing RNA or DNA sequences, genes, proteins, or enzymes by the cell.

[0349] "Individual," "subject," or "animal" refers to humans, veterinary animals (e.g., cats, dogs, cattle, horses, sheep, pigs, etc.), and experimental animal models of diseases (e.g., mice, rats). In a preferred embodiment, the subject is a human.

[0350] The terms “nucleic acid,” “polynucleotide,” and “nucleotide” are used interchangeably herein to refer to polymers of nucleotides of any length, including ribonucleotides (RNA), deoxyribonucleotides (DNA), or their analogues or modified forms. These include single-stranded, double-stranded, 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 derived nucleotide bases. Single-stranded nucleic acids can be sense or antisense strands.

[0351] Nucleic acids are described as having both a "5' end" and a "3' end" because the way mononucleotide reactions produce oligonucleotides is that the 5' phosphate ester of a mononucleotide's pentose ring is linked in one direction to the 3' oxygen atom of its adjacent mononucleotide's pentose ring via a phosphodiester bond. If the 5' phosphate ester of an oligonucleotide is not linked to the 3' oxygen atom of a mononucleotide's pentose ring, the end of that oligonucleotide is called a "5' end." If the 3' oxygen atom of an oligonucleotide is not linked to the 5' phosphate ester of another mononucleotide's pentose ring, the end of that oligonucleotide is called a "3' end." Nucleic acid sequences can also be described as having both a 5' end and a 3' end, even within larger oligonucleotides. In linear or circular DNA molecules, discrete elements are referred to as being "downstream" or "upstream" of 3' elements or 5' elements.

[0352] The term "fragment" when referring to proteins means a protein that is shorter than the full-length protein or has fewer amino acids. A fragment can be, for example, an N-terminal fragment (i.e., a portion of the C-terminus of a protein removed), a C-terminal fragment (i.e., a portion of the N-terminus of a protein removed), or an internal fragment. The term "fragment" when referring to nucleic acids means a nucleic acid that is shorter than the full-length nucleic acid or has fewer nucleotides. A fragment can be, for example, a 5' fragment (i.e., a portion of the 3' end of a nucleic acid removed), a 3' fragment (i.e., a portion of the 5' end of a protein removed), or an internal fragment.

[0353] As used herein, the term "derivative" refers to a nucleic acid, protein, or variant thereof, or an analogue 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 involving nucleic acids include, for example, modifications to the base moiety, sugar moiety, phosphate moiety, phosphate-sugar backbone, or combinations thereof.

[0354] In the context of two polynucleotide or polypeptide sequences, "sequence identity" or "identity" refers to the same residues in two sequences when compared for maximum correspondence within a specified comparison window. When the sequence identity percentage is applied to proteins, dissimilar residue positions are often due to conserved amino acid substitutions, where amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not alter the functional properties of the molecule. When sequences differ in terms of conserved substitutions, the sequence identity percentage can be adjusted upwards to correct for the conservation of the substitution. Sequences that are different due to similar conserved substitutions are thus said to have "sequence similarity" or "identity." Methods for making such adjustments are well known. Typically, this involves counting conserved substitutions as partial mismatches rather than complete mismatches, thereby increasing the sequence identity percentage. Thus, for example, the score for a conserved substitution is between 0 and 1 when the same amino acid scores 1 and a non-conserved substitution scores 0. The score for a conserved substitution is calculated, such as in the PC / GENE program, for example.

[0355] The "Sequence Identity Percentage" is a value determined by comparing two optimally aligned sequences (the maximum number of perfectly matched residues) across a comparison window. The polynucleotide sequence portion within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence used for the optimal alignment of the two sequences (which contains no additions or deletions). This percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears to arrive at the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and then multiplying the result by 100 to obtain the sequence identity percentage. Unless otherwise specified (e.g., the shorter sequence contains linked heterologous sequences), the comparison window is the full length of the shorter of the two sequences being compared.

[0356] The term “treatment” or “curing” for a state, symptom, disease, or condition includes: (1) preventing, delaying, or reducing the incidence and / or likelihood of developing at least one clinical or subclinical symptom of a state, symptom, disease, or condition in a subject who may have or is susceptible to the state, symptom, disease, or condition but has not yet experienced or displayed clinical or subclinical symptoms of the state, symptom, disease, or condition; or (2) suppressing the state, symptom, disease, or condition, i.e., preventing, reducing, or delaying the development of the disease or its recurrence or at least one clinical or subclinical symptom of the disease; or (3) alleviating the state, symptom, disease, or condition, i.e. causing the resolution of the state, symptom, disease, or condition or at least one clinical or subclinical symptom of the state, symptom, disease, or condition. The benefit to the subject receiving treatment is statistically significant, or at least perceptible to the patient or physician.

[0357] When the term "effective" is used in the context of dosage or amount, it refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject in need. It is important to note 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 would be effective when administered individually. The exact dosage required varies from subject to subject, depending on the subject's species, age and general condition, the severity of the condition being treated, the specific one or more medications used, the method of administration, etc.

[0358] The phrase “pharmaceutical acceptable” as used in connection with the compositions described herein means that the molecular entities and other components of such compositions are physiologically tolerable and generally do not produce adverse effects when administered to mammals (e.g., humans). Preferably, the term “pharmaceutical acceptable” means approved by federal or state regulatory agencies or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in mammals, more specifically for use in humans.

[0359] The term "administration" and similar terms refer to and include administering the composition to a subject or system (e.g., to cells, organs, tissues, organisms, or related components or sets of components thereof). Those skilled in the art will understand that the route of administration may vary depending on factors such as the subject or system to which the composition is administered, the nature of the composition, the purpose of administration, etc. For example, in some embodiments, administration to animal subjects (e.g., humans or rodents) may be bronchial administration (including bronchial infusion), oral administration, enteral administration, intradermal administration, intra-arterial administration, intradermal administration, gastric administration, intramedullary administration, intramuscular injection, intranasal administration, intraperitoneal administration, intrathecal administration, intravenous administration, intravenous administration, intraventricular administration, mucosal administration, nasal administration, oral administration, rectal administration, subcutaneous administration, sublingual administration, local administration, tracheal administration (including tracheal infusion), percutaneous administration, vaginal administration, and / or vitreous administration. In some embodiments, administration may involve intermittent dosing. In some embodiments, administration may involve continuous dosing (e.g., infusion) for at least a selected period of time.

[0360] Based on the disclosure herein, conventional molecular biology, microbiology, and recombinant DNA techniques within the scope of this art can be employed. These techniques are 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 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 ToMolecular Cloning (1984); Ausubel, FM et al. (eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994.These techniques include site-directed mutagenesis, as described in Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985), US 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. 24:428-431 (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), US Patent Nos. 5,789,166 and 5,932,419; Hogrefe, Strategies l4.3:74-75 (2001), US 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 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.

[0361] Fusion-promoting proteins

[0362] In one aspect, this disclosure provides a recombinant fusion-promoting protein. Typically, the fusion-promoting protein may comprise a membrane-embedded polypeptide capable of mediating the fusion of two lipid membranes, wherein at least one lipid membrane may be incorporated into the polypeptide. In some embodiments, the fusion-promoting protein described herein may comprise: (i) a rhabdoviral glycoprotein (G) or a functional fragment or derivative thereof, and (ii) a targeting molecule. The targeting molecule may be linked via a linker to the N-terminus of the rhabdoviral glycoprotein or a functional fragment or derivative thereof.

[0363] Rhabdoviruses are members of the Rhabdoviridae family of viruses in the order Mononegavirales, encompassing over 150 viruses from vertebrates, invertebrates, and plants. Examples of rhabdoviruses include rabies virus (RABV) from the genus Lyssavirus, vesicular viruses from the genus Vesiculovirus, viral hemorrhagic septicemia virus (VHSV), and infectious hematopoietic necrosis virus (both from the genus Novirhabdovirus). Members of the genus Lyssavirus can cause fatal meningoencephalitis in humans and animals, while VSV (vesicular virus) can cause symptoms similar to those of foot-and-mouth disease in cattle and occasionally causes limited infections in humans. Dimarhabdoviruses are a supergroup of rhabdoviruses that infect mammals and mosquitoes.

[0364] Rhabdoviruses are warhead-shaped enveloped viruses with a negative-sense, single-stranded RNA genome of 11-15 kb in length. The rhabdovirus genome can contain up to 10 genes, of which only 5 are common to all members of the family. These 5 common rhabdovirus genes encode nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and viral polymerase (also known as the large protein) (L). The rhabdovirus genome associates with N, L, and P to form a nucleocapsid, which is condensed into a tightly coiled helical structure by the M protein. The condensed nucleocapsid is surrounded by a lipid bilayer containing the viral glycoprotein G, which forms the spikes protruding from the viral surface. Rhabdoviruses enter host cells via endocytosis and subsequently fuse with the cell membrane in the acidic environment of the endosome. Both receptor recognition and membrane fusion are mediated by a single transmembrane viral glycoprotein (G). The fusion between the viral envelope and the endosomal membrane is triggered by low pH-induced (in endosome) G-structure rearrangement, leading to the release of the viral genome and its associated proteins into the cytoplasm of the target cell.

[0365] In some implementations, the rhabdovirus described herein may be a vesicular virus, or a functional fragment or derivative thereof. Examples of vesicular viruses that can be used in this disclosure are listed in Table 1.

[0366] Table 1. Examples of vesicular virus

[0367]

[0368]

[0369] In some embodiments, the bullet-shaped virus disclosed herein may comprise a VSV. In some embodiments, the VSV may be a VSV capable of replication. In some embodiments, the VSV may be a non-replicating VSV.

[0370] "Fusogen" (e.g., a fusion-promoting protein, such as the recombinant fusion-promoting protein described herein) or "fusion-promoting molecule" can refer to any molecule present on the surface of a virus that can trigger membrane fusion. In some embodiments, the fusogen can act on the cell membrane to prevent spontaneous membrane fusion and promote fusion that occurs in a controlled and / or regulated manner. Upon activation, the fusogen can extend a trimer at one end anchored by its transmembrane domain and expose an amphiphilic cyclic or hydrophobic fusion peptide inserted into the target membrane. At this point, the two interacting domains are located in different membranes. The fusion-promoting complex is controlled to refold into a hairpin-like structure, subsequently bringing the fusion peptide and the transmembrane domain to the same end of the molecule, which generates a pull that brings the two membranes together tightly (approximately 1 nanometer). The energy accumulated from this event can drive fusion through the formation of a semi-fusion stalk-like junction, in which only the proximal lobe of the membrane contact fuses, while the inner lobe remains intact. The expansion of the semi-fusion stalk and the subsequent fusion of the distal lobe complete the reaction by opening a fusion pore that allows the contents of the two compartments to mix. Fusion pore expansion is considered the final energy barrier before membrane fusion permanentization. Unbound by theory, there is evidence that viral fusionins mediate fusion via hemifusion. This paper may use a variety of different protein and non-protein fusion-promoting molecules. In some embodiments, the fusion-promoting molecule is a fusion-promoting protein or peptide.

[0371] In some embodiments, the fusionin described herein can drive the fusion of the viral envelope of the virus described herein (e.g., rhabdovirus, such as but not limited to VSV) with the cell membrane of a target cell. In some embodiments, the fusionin can mediate the fusion of the plasma membrane of the target cell and / or one or more cells adjacent to the target cell (i.e., neighboring cells), resulting in the formation of multinucleated cells (i.e., syncytia). In some embodiments, the viruses disclosed herein can trigger cell-cell fusion, for example via fusionin, which can form syncytia. In some embodiments, syncytia formation can occur during viral infection in vitro and / or in vivo. In some embodiments, cell-cell fusion can allow the virus to spread more efficiently to neighboring cells than without such cell-cell fusion. Viral spread to neighboring cells associated with cell-cell fusion may be more efficient due to the virus not being exposed to, for example, neutralizing antibodies and / or other host immune responses and / or molecules. In various embodiments, syncytia formation may allow the virus to evade or partially evade host defense mechanisms, such as, but not limited to, humoral immune responses. In various implementations, the formation of syncytia may allow the virus to evade or partially evade limiting factors that target viral particle assembly and / or release and / or viral entry into target cells.

[0372] In some embodiments, the fusion-promoting molecule is a viral fusion-promoting molecule. Non-limiting examples of viral fusion-promoting molecules include, for example, vesicular virus fusion elements (e.g., vesicular stomatitis virus G glycoprotein), alphavirus fusion elements (e.g., Sindbis virus glycoprotein), orthomyxovirus fusion elements (e.g., influenza HA protein), paramyxovirus fusion elements (e.g., Nipah virus F protein or measles virus F protein), and fusion elements from dengue virus (DV), Lassa virus, tick-borne encephalitis virus, dengue virus, hepatitis B virus, rabies virus, Semliki Forest virus, Ross River virus, Aura virus, Borna virus, Hantaan virus, SARS-CoV virus, and various fragments, mutants, and derivatives thereof.

[0373] In some implementations, the fusion-promoting molecule is heterologous to the virus from which the virus originates. In some implementations, the fusion-promoting molecule is a mutant protein that does not bind to its natural ligand.

[0374] There are two classes of viral fusion-promoting molecules, both of which can be used as targeting molecules. Class I fusion-promoting molecules utilize a helical coil-and-coil structure to trigger membrane fusion, while Class II fusion-promoting molecules utilize 13 barrel-shaped structures to trigger fusion. In some embodiments, Class I fusion-promoting molecules are used. In other embodiments, Class II fusion-promoting molecules are used. In still other embodiments, both Class I and Class II fusion-promoting molecules are used simultaneously.

[0375] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (G) or a functional fragment or derivative thereof. In some embodiments, the fusion-promoting molecule is a vesicular stomatitis virus (VSV) envelope protein. In some embodiments, the recombinant fusion-promoting protein includes the VSV G protein (VSV-G or a fragment, mutant, derivative, or homolog thereof). VSV-G can interact with phospholipid components of the cell membrane to mediate viral entry via membrane fusion.

[0376] In some embodiments, the rhabdovirus G protein described herein may include, but is not limited to, vesicular stomatitis virus glycoprotein (VSV-G), Flanders virus glycoprotein (FLAV-G), Chandipura virus glycoprotein (CHPV-G), Perinet virus glycoprotein (PERV-G), Piry virus glycoprotein (PIRYV-G), Fukuoka virus glycoprotein (FUKV-G), Joinjakaka virus glycoprotein (JOIV-G), Kumasi virus glycoprotein (KRV-G), Isfahan glycoprotein (ISFV-G), Jurona virus glycoprotein (JURV-G), Mediterranean Bat glycoprotein (MBV-G), Malpais Spring glycoprotein (MSPV-G), Radi glycoprotein (RADV-G), Rhinolophus affinis-G, Yug Bugdanavoc glycoprotein (YBV-G), Yinshui Bat glycoprotein (YSBV-G), Keuraliba 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 transient heat glycoprotein (BEFV-G), Curionopolis glycoprotein (CURV-G), Drosophila melanogaster Sigma virus glycoprotein (DMelSV-G), Niakha glycoprotein (NIAV-G), Puerto almandras glycoprotein (PTAMV-G), or Tupaia rhabdovirus (TUPTV-G), or functional fragments or derivatives thereof. Non-limiting examples of the amino acid sequences of rhabdovirus G described herein are shown in SEQ ID NO: 8-15, 17, 60-108, 157.

[0377] In some embodiments, the fusion-promoting protein may comprise the targeting molecule described herein. In some embodiments, the targeting molecule is attached to the N-terminus of the rhabdoviral glycoprotein. In some embodiments, the targeting molecule attached to the N-terminus can be attached to the rhabdoviral glycoprotein via a linker described herein.

[0378] In some embodiments, the N-terminus of the rhabdovirus G or a functional fragment or derivative thereof to which the targeting molecule can be attached (e.g., 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 the targeting molecule can be attached is mature rhabdovirus G that does not contain, for example, a rhabdovirus glycoprotein signal sequence. In some embodiments, the rhabdovirus glycoprotein signal sequence may be necessary to ensure that rhabdovirus G containing the signal sequence (e.g., neonatal rhabdovirus G) can enter the endoplasmic reticulum (ER). In some embodiments, the rhabdovirus glycoprotein signal sequence is cleaved from neonatal rhabdovirus G. Non-limiting examples of the amino acid sequence of the rhabdovirus G signal sequence are shown in SEQ ID NO: 109-137. In some embodiments, rhabdovirus G contains a signal sequence. Non-limiting examples of the amino acid sequence of the rhabdovirus glycoprotein containing the signal sequence are shown in SEQ ID NO: 80-108.

[0379] In some embodiments, the N-terminus of the rhabdoviral glycoprotein or a functional fragment or derivative thereof to which the targeting molecule can be attached (e.g., via a linker) does not contain one or more amino acids present at the N-terminus of the mature wild-type (WT) rhabdoviral glycoprotein described herein. For example, in some embodiments, the N-terminus of the mature WT rhabdoviral glycoprotein or a functional fragment or derivative thereof to which the targeting molecule can be attached does not contain 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 the mature WT rhabdoviral glycoprotein described herein. In some embodiments, the N-terminus of the mature WT rhabdoviral glycoprotein or its functional fragment or derivative to which the targeting molecule can be attached does not contain 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 rhabdoviral glycoprotein described herein. In some embodiments, the mature WT rhabdoviral glycoprotein or its functional fragment or derivative may be the mature WT vesicular stomatitis virus glycoprotein (VSV-G) or its functional fragment or derivative described herein.

[0380] In some embodiments, the N-terminus of a rhabdoviral glycoprotein or a functional fragment or derivative thereof to which the targeting molecule may be attached (e.g., via a linker) does not contain one or more amino acids encoded by the 5' end of a wild-type (WT) glycoprotein gene, such as the signal sequence described herein (e.g., the rhabdoviral glycoprotein signal sequence) or a fragment or derivative thereof encoded by the 5' end of a wild-type (WT) glycoprotein gene. In some embodiments, when the N-terminus of a rhabdoviral glycoprotein or a functional fragment or derivative thereof does not contain one or more amino acids (e.g., the signal sequence) encoded by the 5' end of a WT glycoprotein gene, the rhabdoviral glycoprotein may comprise a mature rhabdoviral glycoprotein, i.e., a rhabdoviral glycoprotein that does not contain the signal peptide described herein. In some embodiments, the N-terminus of the mature WT rhabdoviral glycoprotein to which the targeting molecule can be attached does not contain 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 the mature WT rhabdoviral glycoprotein to which the targeting molecule can be attached does not contain approximately 10-32 amino acids encoded by the 5' end of the WT glycoprotein gene. In some embodiments, the N-terminus of the mature WT rhabdoviral glycoprotein or its functional fragment or derivative to which the targeting molecule can be attached does not contain 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 a wild-type glycoprotein gene, the rhabdovirus glycoprotein may comprise a mature rhabdovirus glycoprotein that does not contain the signal peptide sequence described herein (e.g., the signal sequence shown in SEQ ID NO: 109-137). Non-limiting examples of the amino acid sequence of a mature rhabdovirus glycoprotein that does not contain the signal sequence are shown in SEQ ID NO: 8-15, 17, 60-79. In some embodiments, the mature wild-type rhabdovirus glycoprotein or a functional fragment or derivative thereof may be the mature wild-type vesicular stomatitis virus glycoprotein (VSV-G) or a functional fragment or derivative thereof described herein.

[0381] In some embodiments, the fusion-promoting protein 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 with SEQ ID NO: 8 or 80. In some embodiments, the nucleotide sequence encoding VSV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 8 or 80. In some embodiments, VSV-G comprises the amino acid sequence of SEQ ID NO: 8 or 80.

[0382] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from VSV-G. In some embodiments, the VSV-G comprises the sequence SEQ ID NO: 8. In some embodiments, the VSV-G consists of the sequence SEQ ID NO: 8.

[0383] In some embodiments, the VSV-G described herein may be derived from, for example, the VSV-G described in U.S. Patent Publication No. 2020 / 0216502, the entire contents of which are incorporated herein by reference for all purposes. As an example (but not limited to), VSV-G or a functional fragment or derivative thereof may comprise 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 with SEQ ID NO: 157.

[0384] In some embodiments, the fusion-promoting protein or its functional fragment or derivative described herein may comprise a rhabdoviral glycoprotein (G) containing one or more mutations compared to the corresponding WT sequence of the rhabdoviral glycoprotein.

[0385] In some embodiments, the rhabdoviral glycoprotein described herein, or a functional fragment or derivative thereof, may contain amino acid mutations at one or more sites. Non-limiting examples of amino acid mutations include amino acid substitutions, insertions, and / or deletions. An amino acid substitution may refer to the replacement of an amino acid residue at the same site with another amino acid residue. An inserted amino acid residue may be inserted at any site and may be inserted such that some or all of the inserted amino acid residues are adjacent to each other, or may be inserted such that none of the inserted amino acid residues are adjacent to another inserted amino acid residue.

[0386] In some embodiments, the rhabdoviral glycoprotein or its functional fragment or derivative may contain amino acid mutations at one or more positions. As a non-limiting example, the rhabdoviral glycoprotein or its functional fragment or derivative may contain 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, 4 2, 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, 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, 3 Amino acid mutations at positions of 90, 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.

[0387] In some embodiments, compared to the amino acid sequence of wild-type rhabdovirus protein, the rhabdovirus protein or a fragment or derivative thereof may contain one or more mutations at one or more positions in its amino acid sequence, namely amino acid substitutions, insertions, or deletions, or combinations thereof. For example, the VSV-G protein described herein may include one or more amino acids in the amino acid sequence of the parental VSV-G protein substituted with similar or homologous or dissimilar amino acids.

[0388] In some embodiments, the rhabdoviral G protein described herein may include a protein having a percentage of at least about 50% or higher, about 60% or higher, about 70% or higher, 71% or higher, 72% or higher, 73% or higher, 74% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, ... Any amino acid sequence with 9% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, 99.9% or higher, or 100% identity, and having the activity of the WT sequence.

[0389] In some embodiments, the VSV-G polypeptide or its functional fragment or derivative described herein may contain one or more mutations compared to its corresponding wild-type VSV-G sequence (see, for example, SEQ ID NO: 8). In some embodiments, one or more mutations in VSV-G may, for example, reduce or eliminate the binding of the VSV-G protein or its functional fragment or derivative to the low-density lipoprotein receptor (LDLR).

[0390] In some embodiments, one or more mutations in the VSV-G peptide or its functional fragments or derivatives include one or more amino acid substitutions and / or deletions at positions H8, K47, Y209, and / or R354 in SEQ ID NO: 8. In some embodiments, one or more mutations in the VSV-G peptide or its functional fragments or derivatives may include, for example, one or more amino acid substitutions and / or deletions at positions corresponding to the following positions in SEQ ID NO: 8: position 8, or 47, or 209, or 354; or both positions 8 and 47; or both positions 8 and 209; or both positions 8 and 354; or both positions 47 and 209; or both positions 47 and 354; or both positions 209 and 354; or a combination of positions 8 and 47 and 209; a combination of positions 8, 47, and 354; or a combination of positions 8 and 209 and 354; or a combination of positions 47 and 209 and 354; or a combination of positions 8 and 47 and 209 and 354.

[0391] In various embodiments, the mutant VSV-G polypeptide may contain, for example, the mutation in SEQ ID NO: 8, wherein the amino acid at position 8 may be substituted with any amino acid other than H and preferably other than Y; the amino acid at position 47 may be substituted with any amino acid other than K; the amino acid at position 209 may be substituted with any amino acid other than Y and preferably other than H; and / or the amino acid at position 354 may be substituted with any amino acid other than R.

[0392] In some embodiments, the mutant VSV-G polypeptide described herein may comprise a fusionin comprising the sequence SEQ ID NO: 8, wherein an amino acid substitution is contained at (i) K47, (ii) R354 and (iii) H8 or Y209.

[0393] In some embodiments, the mutant VSV-G polypeptide described herein may consist of the sequence SEQ ID NO: 8, wherein an amino acid substitution is contained at positions (i) K47, (ii) R354 and (iii) H8 or Y209.

[0394] In some embodiments, the mutant VSV-G polypeptide described herein may comprise a fusionin comprising the sequence SEQ ID NO: 8, wherein amino acid substitutions are present at positions K47, R354, H8, and Y209.

[0395] In some embodiments, the mutant VSV-G polypeptide described herein may consist of the sequence SEQ ID NO: 8, wherein amino acid substitutions are present at positions K47, R354, H8, and Y209.

[0396] In some embodiments, one or more mutations in the VSV-G peptide or its functional fragments or derivatives include the deletion of amino acids at one or more positions corresponding to H8, K47, Y209 and / or R354 in SEQ ID NO: 8.

[0397] In some embodiments, the mutant VSV-G polypeptide described herein may include SEQ ID NO: 8 or a functional fragment or derivative thereof, wherein an amino acid is deleted at (i) K47, (ii) R354 and (iii) H8 or Y209.

[0398] In some embodiments, the mutant VSV-G polypeptide described herein may consist of the sequence SEQ ID NO: 8, wherein there are amino acid deletions at (i) K47, (ii) PR54 and (iii) H8 or Y209.

[0399] In some embodiments, the mutant VSV-G peptide described herein may comprise the sequence SEQ ID NO: 8, wherein an amino acid is deleted at position K47.

[0400] In some embodiments, the mutant VSV-G polypeptide described herein may consist of the sequence SEQ ID NO: 8, wherein an amino acid is deleted at position K47.

[0401] Other exemplary mutations in VSV-G that could reduce or eliminate the binding of the VSV-G protein or its functional fragments or derivatives to the low-density lipoprotein receptor (LDLR) are described in US2020 / 0216502, which is incorporated herein by reference in its entirety.

[0402] In some embodiments, VSV-G or a functional fragment or derivative thereof may contain one or more mutations that increase viral titer. Non-limiting examples of mutations that increase viral titer are M184T and F250L, specified relative to their positions in SEQ ID NO: 8. In some embodiments, VSV-G or a functional fragment or derivative thereof may contain one or more mutations in VSV-G that increase viral titer, such as, but not limited to, M184T and / or F250L, specified relative to their positions in SEQ ID NO: 8.

[0403] Other examples of mutations that increase viral titer include those described in US2022 / 0162266, which is incorporated herein by reference in its entirety, such as H22N and S422I (or similar substitutions, such as S422F, S422M, S422L or S422V, or equivalent substitutions at equivalent positions in the G extracellular domain of other VSV strains) in the extracellular domain of VSVIndiana glycoprotein G.

[0404] In various embodiments, the recombinant fusion-promoting protein of this disclosure comprises a fusion element having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% amino acid sequence identity with the vesicular stomatitis virus glycoprotein (VSV-G) comprising the sequence SEQ ID NO: 8. In various embodiments, the recombinant fusion-promoting protein of this disclosure comprises a fusion element containing the vesicular stomatitis virus glycoprotein (VSV-G) comprising the sequence SEQ ID NO: 8. In various embodiments, the recombinant fusion-promoting protein of this disclosure comprises a fusion element having at least 60% amino acid sequence identity with the vesicular stomatitis virus glycoprotein (VSV-G) comprising the sequence SEQ ID NO: 8. In such embodiments, the fusion element or its functional fragment or derivative may contain 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 fusion element comprises SEQ ID NO: 8 or a functional fragment or derivative thereof, wherein one or more amino acid deletions are included at one or more positions selected from H8, K47, Y209, and / or R354. In some embodiments, the fusion element or a functional fragment or derivative thereof may also comprise one or more viral titer-enhancing mutations, such as, but not limited to, viral titer-enhancing mutations at one or more positions corresponding to positions M184 and F250 in SEQ ID NO: 8. In some embodiments, one or more viral titer-enhancing mutations in VSV-G or a functional fragment or derivative thereof may be, for example, M184T and / or F250L, specified relative to positions in SEQ ID NO: 8.

[0405] In some embodiments, the recombinant fusion-promoting protein described herein comprises a fusionin having an N-terminus that can be linked, for example, to a target molecule via a linker. In some embodiments, the recombinant fusion-promoting protein further comprises a target molecule located at the N-terminus of the fusionin. In some embodiments, the target molecule is linked via a linker to the N-terminus of the fusionin or a functional or derivative thereof.

[0406] In some embodiments, the N-terminus of the fusionin does not contain the signal sequence described herein and / or does not contain one or more amino acids present on the N-terminus of the mature WT fusionin. As a non-limiting example, the N-terminus of the mature WT fusionin or its functional fragment or derivative to which the targeting molecule may be attached does not contain 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 on the N-terminus of the mature WT fusionin described herein. In some embodiments, the N-terminus of the mature WT fusion protein to which the targeting molecule can be attached does not contain 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 or more amino acids present at the N-terminus of the mature WT fusion protein described herein.

[0407] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (FLAV-G) derived from Flanders virus. In some embodiments, FLAV-G comprises the amino acid sequence of SEQ ID NO: 9 or 81, 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: 9 or 81. In some embodiments, the nucleotide sequence encoding FLAV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 9 or 81. In some embodiments, FLAV-G comprises the amino acid sequence of SEQ ID NO: 9 or 81.

[0408] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from FLAV-G. In some embodiments, FLAV-G comprises the sequence SEQ ID NO: 9. In some embodiments, FLAV-G consists of the sequence SEQ ID NO: 9.

[0409] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral 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 a 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: 10 or 82. In some embodiments, the nucleotide sequence encoding CHPV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 10 or 82. In some embodiments, CHPV-G comprises the amino acid sequence of SEQ ID NO: 10 or 82.

[0410] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from CHPV-G. In some embodiments, CHPV-G comprises the sequence SEQ ID NO: 10. In some embodiments, CHPV-G consists of the sequence SEQ ID NO: 10.

[0411] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (PERV-G) derived from Perinet virus. In some embodiments, PERV-G comprises the amino acid sequence of SEQ ID NO: 11 or 87, or a 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 some embodiments, the nucleotide sequence encoding PERV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 11 or 87. In some embodiments, PERV-G comprises the amino acid sequence of SEQ ID NO: 11 or 87.

[0412] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from PERV-G. In some embodiments, PERV-G comprises the sequence SEQ ID NO: 11. In some embodiments, PERV-G consists of the sequence SEQ ID NO: 11.

[0413] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (PIRYV-G) derived from Piry virus. In some embodiments, PIRYV-G comprises the amino acid sequence of SEQ ID NO: 12 or 88, or a 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 some embodiments, the nucleotide sequence encoding PIRYV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 12 or 88. In some embodiments, PIRYV-G comprises the amino acid sequence of SEQ ID NO: 12 or 88.

[0414] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from PIRYV-G. In some embodiments, PIRYV-G comprises the sequence SEQ ID NO: 12. In some embodiments, PIRYV-G consists of the sequence SEQ ID NO: 12.

[0415] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral 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 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 some embodiments, the nucleotide sequence encoding FUKV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 13 or 93. In some embodiments, FUKV-G comprises the amino acid sequence of SEQ ID NO: 13 or 93.

[0416] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from FUKV-G. In some embodiments, FUKV-G comprises the sequence SEQ ID NO: 13. In some embodiments, FUKV-G consists of the sequence SEQ ID NO: 13.

[0417] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (JOIV-G) derived from Joinjakaka virus. In some embodiments, JOIV-G comprises the amino acid sequence of SEQ ID NO: 14 or 94, or a 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 some embodiments, the nucleotide sequence encoding JOIV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 14 or 94. In some embodiments, JOIV-G comprises the amino acid sequence of SEQ ID NO: 14 or 94.

[0418] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from JOIV-G. In some embodiments, JOIV-G comprises the sequence SEQ ID NO: 14. In some embodiments, JOIV-G consists of the sequence SEQ ID NO: 14.

[0419] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral 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 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: 15 or 97. In some embodiments, the nucleotide sequence encoding KRV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 15 or 97. In some embodiments, KRV-G comprises the amino acid sequence of SEQ ID NO: 15 or 97.

[0420] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from KRV-G. In some embodiments, KRV-G comprises the sequence SEQ ID NO: 15. In some embodiments, KRV-G consists of the sequence SEQ ID NO: 15.

[0421] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (KEUV-G) derived from Keuraliba virus. In some embodiments, KEUV-G comprises the amino acid sequence of SEQ ID NO: 17 or 95, or a 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 some embodiments, the nucleotide sequence encoding KEUV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 17 or 95. In some embodiments, KEUV-G comprises the amino acid sequence of SEQ ID NO: 17 or 95.

[0422] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein from KEUV-G. In some embodiments, KEUV-G comprises the sequence SEQ ID NO: 17. In some embodiments, KEUV-G consists of the sequence SEQ ID NO: 17.

[0423] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral 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 a 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: 60 or 83. In some embodiments, the nucleotide sequence encoding ISFV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 60 or 83. In some embodiments, ISFV-G comprises the amino acid sequence of SEQ ID NO: 60 or 83.

[0424] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from ISFV-G. In some embodiments, ISFV-G comprises the sequence SEQ ID NO: 60. In some embodiments, ISFV-G consists of the sequence SEQ ID NO: 60.

[0425] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (JURV-G) derived from Jurona virus. In some embodiments, JURV-G comprises the amino acid sequence of SEQ ID NO: 61 or 84, or a 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 some embodiments, the nucleotide sequence encoding JURV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 61 or 84. In some embodiments, JURV-G comprises the amino acid sequence of SEQ ID NO: 61 or 84.

[0426] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from JURV-G. In some embodiments, JURV-G comprises the sequence SEQ ID NO: 61. In some embodiments, JURV-G consists of the sequence SEQ ID NO: 61.

[0427] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral 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 some embodiments, the nucleotide sequence encoding MBV-G comprises the amino acid sequence encoding SEQ ID NO: 62 or 85, 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 with SEQ ID NO: 62 or 85. In some embodiments, MBV-G comprises the amino acid sequence of SEQ ID NO: 62 or 85.

[0428] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from MBV-G. In some embodiments, MBV-G comprises the sequence SEQ ID NO: 62. In some embodiments, MBV-G consists of the sequence SEQ ID NO: 62.

[0429] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (MSPV-G) derived from Malpais Spring virus. In some embodiments, MSPV-G comprises the amino acid sequence of SEQ ID NO: 63 or 86, or a 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 some embodiments, the nucleotide sequence encoding MSPV-G comprises the amino acid sequence encoding SEQ ID NO: 63 or 86, 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 with SEQ ID NO: 63 or 86. In some embodiments, MSPV-G comprises the amino acid sequence of SEQ ID NO: 63 or 86.

[0430] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from MSPV-G. In some embodiments, MSPV-G comprises the sequence SEQ ID NO: 63. In some embodiments, MSPV-G consists of the sequence SEQ ID NO: 63.

[0431] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (RADV-G) derived from Radivirus. In some embodiments, RADV-G comprises the amino acid sequence of SEQ ID NO: 64 or 89, or a 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: 64 or 89. In some embodiments, the nucleotide sequence encoding RADV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 64 or 89. In some embodiments, RADV-G comprises the amino acid sequence of SEQ ID NO: 64 or 89.

[0432] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from RADV-G. In some embodiments, RADV-G comprises the sequence SEQ ID NO: 64. In some embodiments, RADV-G consists of the sequence SEQ ID NO: 64.

[0433] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (Rhinolophus G) derived from Rhinolophus virus. In some embodiments, Rhinolophus G comprises the amino acid sequence of SEQ ID NO: 65 or 90, or a 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: 65 or 90. In some embodiments, the nucleotide sequence encoding *Rhinolophus griseus* G comprises the amino acid sequence encoding 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 with SEQ ID NO: 65 or 90. In some embodiments, *Rhinolophus griseus* G comprises the amino acid sequence of SEQ ID NO: 65 or 90.

[0434] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from *Rhinolophus grusonii* G. In some embodiments, *Rhinolophus grusonii* G comprises the sequence SEQ ID NO: 65. In some embodiments, *Rhinolophus grusonii* G consists of the sequence SEQ ID NO: 65.

[0435] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (YBV-G) derived from Yug Bugdanavoc virus. In some embodiments, YBV-G comprises the amino acid sequence of SEQ ID NO: 66 or 91, or a 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 some embodiments, the nucleotide sequence encoding YBV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 66 or 91. In some embodiments, YBV-G comprises the amino acid sequence of SEQ ID NO: 66 or 91.

[0436] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from YBV-G. In some embodiments, YBV-G comprises the sequence SEQ ID NO: 66. In some embodiments, YBV-G consists of the sequence SEQ ID NO: 66.

[0437] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (YSBV-G) derived from Yinshui Bat virus. In some embodiments, YSBV-G comprises the amino acid sequence of SEQ ID NO: 67 or 92, or a 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: 67 or 92. In some embodiments, the nucleotide sequence encoding YSBV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 67 or 92. In some embodiments, YSBV-G comprises the amino acid sequence of SEQ ID NO: 67 or 92.

[0438] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from YSBV-G. In some embodiments, YSBV-G comprises the sequence SEQ ID NO: 67. In some embodiments, YSBV-G consists of the sequence SEQ ID NO: 67.

[0439] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (KIMV-G) derived from Kimberley virus. In some embodiments, KIMV-G comprises the amino acid sequence of SEQ ID NO: 68 or 96, or a 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: 68 or 96. In some embodiments, the nucleotide sequence encoding KIMV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 68 or 96. In some embodiments, KIMV-G comprises the amino acid sequence of SEQ ID NO: 68 or 96.

[0440] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from KIMV-G. In some embodiments, KIMV-G comprises the sequence SEQ ID NO: 68. In some embodiments, KIMV-G consists of the sequence SEQ ID NO: 68.

[0441] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (KYAV-G) derived from Kanyawara virus. In some embodiments, KYAV-G comprises the amino acid sequence of SEQ ID NO: 69 or 98, or a 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 some embodiments, the nucleotide sequence encoding KYAV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 69 or 98. In some embodiments, KYAV-G comprises the amino acid sequence of SEQ ID NO: 69 or 98.

[0442] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from KYAV-G. In some embodiments, KYAV-G comprises the sequence SEQ ID NO: 69. In some embodiments, KYAV-G consists of the sequence SEQ ID NO: 69.

[0443] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (LJV-G) derived from La Joya 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 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: 70 or 99. In some embodiments, the nucleotide sequence encoding LJV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 70 or 99. In some embodiments, LJV-G comprises the amino acid sequence of SEQ ID NO: 70 or 99.

[0444] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from LJV-G. In some embodiments, LJV-G comprises the sequence SEQ ID NO: 70. In some embodiments, LJV-G consists of the sequence SEQ ID NO: 70.

[0445] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (MQOV-G) derived from Mosquiero virus. In some embodiments, MQOV-G comprises the amino acid sequence of SEQ ID NO: 71 or 100, or a 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: 71 or 100. In some embodiments, the nucleotide sequence encoding MQOV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 71 or 100. In some embodiments, MQOV-G comprises the amino acid sequence of SEQ ID NO: 71 or 100.

[0446] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from MQOV-G. In some embodiments, MQOV-G comprises the sequence SEQ ID NO: 71. In some embodiments, MQOV-G consists of the sequence SEQ ID NO: 71.

[0447] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral 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 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: 72 or 101. In some embodiments, the nucleotide sequence encoding PCV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 72 or 101. In some embodiments, PCV-G comprises the amino acid sequence of SEQ ID NO: 72 or 101.

[0448] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from PCV-G. In some embodiments, PCV-G comprises the sequence SEQ ID NO: 72. In some embodiments, PCV-G consists of the sequence SEQ ID NO: 72.

[0449] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (BASV-G) derived from Bas Congo virus. In some embodiments, BASV-G comprises the amino acid sequence of SEQ ID NO: 73 or 102, or a 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: 73 or 102. In some embodiments, the nucleotide sequence encoding BASV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 73 or 102. In some embodiments, BASV-G comprises the amino acid sequence of SEQ ID NO: 73 or 102.

[0450] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from BASV-G. In some embodiments, BASV-G comprises the sequence SEQ ID NO: 73. In some embodiments, BASV-G consists of the sequence SEQ ID NO: 73.

[0451] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (BEFV-G) derived from bovine short-term fever virus. In some embodiments, BEFV-G comprises the amino acid sequence of SEQ ID NO: 74 or 103, or a 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 some embodiments, the nucleotide sequence encoding BEFV-G comprises the amino acid sequence encoding SEQ ID NO: 74 or 103, 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 with SEQ ID NO: 74 or 103. In some embodiments, BEFV-G comprises the amino acid sequence of SEQ ID NO: 74 or 103.

[0452] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from BEFV-G. In some embodiments, BEFV-G comprises the sequence SEQ ID NO: 74. In some embodiments, BEFV-G consists of the sequence SEQ ID NO: 74.

[0453] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (CURV-G) derived from Curionopolis virus. In some embodiments, CURV-G comprises the amino acid sequence of SEQ ID NO: 75 or 104, or a 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: 75 or 104. In some embodiments, the nucleotide sequence encoding CURV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 75 or 104. In some embodiments, CURV-G comprises the amino acid sequence of SEQ ID NO: 75 or 104.

[0454] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from CURV-G. In some embodiments, CURV-G comprises the sequence SEQ ID NO: 75. In some embodiments, CURV-G consists of the sequence SEQ ID NO: 75.

[0455] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral 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 some embodiments, the nucleotide sequence encoding DMelSV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 76 or 105. In some embodiments, DMelSV-G comprises the amino acid sequence of SEQ ID NO: 76 or 105.

[0456] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from DMelSV-G. In some embodiments, DMelSV-G comprises the sequence SEQ ID NO: 76. In some embodiments, DMelSV-G consists of the sequence SEQ ID NO: 76.

[0457] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (NIAV-G) derived from Niakha virus. In some embodiments, NIAV-G comprises the amino acid sequence of SEQ ID NO: 77 or 106, or a 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: 77 or 106. In some embodiments, the nucleotide sequence encoding NIAV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 77 or 106. In some embodiments, NIAV-G comprises the amino acid sequence of SEQ ID NO: 77 or 106.

[0458] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from NIAV-G. In some embodiments, NIAV-G comprises the sequence SEQ ID NO: 77. In some embodiments, NIAV-G consists of the sequence SEQ ID NO: 77.

[0459] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (PTAMV-G) derived from Puerto almandras virus. In some embodiments, PTAMV-G comprises the amino acid sequence of SEQ ID NO: 78 or 107, or a 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: 78 or 107. In some embodiments, the nucleotide sequence encoding PTAMV-G comprises the amino acid sequence encoding SEQ ID NO: 78 or 107, 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 with SEQ ID NO: 78 or 107. In some embodiments, PTAMV-G comprises the amino acid sequence of SEQ ID NO: 78 or 107.

[0460] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from PTAMV-G. In some embodiments, PTAMV-G comprises the sequence SEQ ID NO: 78. In some embodiments, PTAMV-G consists of the sequence SEQ ID NO: 78.

[0461] In some embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (TUPTV-G) derived from Tupaia virus. In some embodiments, TUPTV-G comprises the amino acid sequence of SEQ ID NO: 79 or 108, or a 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 some embodiments, the nucleotide sequence encoding TUPTV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 79 or 108. In some embodiments, TUPTV-G comprises the amino acid sequence of SEQ ID NO: 79 or 108.

[0462] In some embodiments, the recombinant fusion-promoting protein described herein comprises a rhabdoviral glycoprotein derived from TUPTV-G. In some embodiments, TUPTV-G comprises the sequence SEQ ID NO: 79. In some embodiments, TUPTV-G consists of the sequence SEQ ID NO: 79.

[0463] In some embodiments, the recombinant fusion-promoting protein described herein may comprise the rhabdoviral glycoprotein described herein, and the rhabdoviral glycoprotein may be truncated, for example at the N-terminus and / or C-terminus, thereby resulting in a truncated rhabdoviral glycoprotein, such as a truncated functional fragment, which may retain at least the ability to confer activity upon the rhabdoviral glycoprotein.

[0464] In some embodiments, the recombinant fusion-promoting protein described herein may be a fragment of the rhabdoviral glycoprotein described herein, and the cytoplasmic tail of the rhabdoviral glycoprotein has been removed or truncated, and / or optionally replaced by another sequence.

[0465] In some embodiments, the recombinant fusion-promoting protein described herein may be a fragment of the rhabdoviral glycoprotein described herein, and the rhabdoviral glycoprotein fragment may include, but is not limited to, a truncated cytoplasmic tail. As a non-limiting example, the cytoplasmic tail of the rhabdoviral glycoprotein may be truncated from its C-terminus by 2 to 80 amino acids, 3 to 75 amino acids, 4 to 70 amino acids, 5 to 65 amino acids, 6 to 60 amino acids, 7 to 55 amino acids, 8 to 50 amino acids, 9 to 45 amino acids, 10 to 40 amino acids, 11 to 35 amino acids, 12 to 30 amino acids, 13 to 25 amino acids, 14 to 20 amino acids, or 15 to 35 amino acids. In some embodiments, the truncated cytoplasmic tail of the rhabdoviral glycoprotein may be truncated from its C-terminus by 10 to 40 amino acids. In some embodiments, the cytoplasmic tail of the rhabdoviral glycoprotein can be truncated. 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, or 80 or more amino acids. In some embodiments, the cytoplasmic tail of the rhabdoviral glycoprotein can be truncated by 30 amino acids from the C-terminus.

[0466] In some embodiments, the cytoplasmic tail of the rhabdoviral glycoprotein described herein may be truncated by up to 40 amino acids from its C-terminus. In some embodiments, the cytoplasmic tail of the rhabdoviral glycoprotein may be truncated by 10 to 40 amino acids from its C-terminus. In some embodiments, the cytoplasmic tail of the rhabdoviral glycoprotein may be truncated by 30 amino acids from its C-terminus.

[0467] In some embodiments, the recombinant fusion-promoting protein described herein may also 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 CIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 16). In some embodiments, the cytoplasmic tail of the rhabdoviral glycoprotein described herein comprises the amino acid sequence of SEQ ID NO: 16, 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: 16. In some embodiments, the nucleotide sequence encoding the cytoplasmic tail of the rhabdoviral glycoprotein comprises the amino acid sequence encoding SEQ ID NO: 16 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 with SEQ ID NO: 16. In some embodiments, the cytoplasmic tail of the rhabdoviral glycoprotein comprises the amino acid sequence of SEQ ID NO: 16.

[0468] In some embodiments, the fusion-promoting proteins contained in the recombinant virus of this disclosure may also include other fusion proteins. For example, a form of hemagglutinin (HA) from influenza A / fowl cholera virus / Rostock / 34 (FPV), which is a class I fusion protein, may be used. In some embodiments, a form of FPV HA may be used. HA-mediated fusion is generally independent of receptor binding. Another example is the use of Sindbis viral glycoprotein (a class II fusion protein) from the alphavirus family.

[0469] In some implementations, the fusion-promoting molecule is the Sindbis viral envelope protein (SIN). SINdbis virus transfers its RNA into the cell via low-pH-mediated membrane fusion. SIN comprises five structural proteins: E1, E2, E3, 6K, and the capsid. E2 contains a receptor-binding sequence that allows wild-type SIN to bind, while E1 is known to possess properties essential for membrane fusion. E1, E2, and E3 are encoded by polyproteins, whose amino acid sequences are provided, for example, by accession numbers VHWVB, VHWVB2, and P03316; their nucleic acid sequences are provided, for example, by accession numbers SVU90536 and V01403.

[0470] In some embodiments, the Sindbis viral envelope protein is mutated (SINmu). In some embodiments, this mutation reduces the natural affinity of the Sindbis virus. In some embodiments, SINmu comprises SIN proteins E1, E2, and E3, wherein 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 may be mutated at one or more amino acid positions. Furthermore, the fusion proteins described herein encompass combinations of E1, E2, and E3 mutations, such as E1 and E2 mutations, or E2 and E3 mutations, or E3 and E1 mutations, or E1, E2, and E3 mutations. In some embodiments, at least E2 is mutated.

[0471] In some embodiments, SINmu contains the following envelope protein mutations compared to the wild-type Sindbis virus envelope protein, such as the deletion of E3 amino acids 61-64; (ii) E2 KE159-160AA; and (iii) E2 SLKQ68-71AAAA. In some embodiments, SINmu contains the envelope protein mutation E1 AK226-227SG.

[0472] Other enveloped viruses of the Variviridae family (e.g., from the genus Alphavirus), such as Semliki Forest virus, Ross River virus, and equine encephalitis virus, can also be used for pseudotyping of the vectors described herein. The envelope protein sequences of such Alphaviruses are known in the art.

[0473] connector

[0474] In some aspects, this disclosure provides a recombinant fusion-promoting protein comprising a rhabdoviral glycoprotein (G) or a functional fragment or derivative thereof, and a targeting molecule that can be linked to the N-terminus of the rhabdoviral glycoprotein or its functional fragment or derivative via a linker. In some embodiments, the linker may be sensitive to proteolytic cleavage, for example, by naturally occurring cell-associated proteases. In some embodiments, the linker may be sensitive to proteolytic cleavage, for example, by endogenous proteases. In some embodiments, the linker may be sensitive to proteolytic cleavage, for example, by exogenously added proteases. In some embodiments, the linker comprises, for example, but not limited to, arginine (R) and / or lysine (K) residues.

[0475] In some embodiments, the adapters of this disclosure may be insensitive to proteolytic cleavage by endogenous proteases. In some embodiments, the adapters of this disclosure may be insensitive to proteolytic cleavage by exogenously added proteases.

[0476] In some embodiments, the length of the linker can be 1-50 amino acids. As a non-limiting example, the length of 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 some embodiments, the length of the linker can be 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-35, 1-40, 1-50 or more amino acids.

[0477] In some implementations, the connector can be optimized so that it does not impose any restrictions on the conformation and / or interaction of the connected partner.

[0478] In some embodiments, the connector may be a flexible connector. Suitable connectors can be readily selected and can have any suitable length, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. An exemplary flexible connector includes a glycine polymer (G). n Glycine-serine polymer (GS) n(where n is an integer of at least 1, such as 1-20), glycine-alanine polymers, alanine-serine polymers, and other flexible joints known in the art.

[0479] In some implementations, the connector may include a 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.

[0480] In some implementations, the connector may include a 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.

[0481] In some implementations, the joint may be a rigid joint.

[0482] In some embodiments, the connectors of this disclosure may include any connector listed in Table 2, or variations 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 with the connector sequences listed in Table 2. Connector sequences are shown in underlined text.

[0483] Table 2. Examples of connectors

[0484]

[0485]

[0486]

[0487] Further non-limiting examples of usable connectors include any of the various connector sequences listed in Table 5, or variations 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 with the connector sequences listed in Table 5.

[0488] In some embodiments, the adapter sequence described herein may comprise the adapter sequence shown in the amino acid sequence of SEQ ID NO: 1-7, 18-57 or 164-174, 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 with the adapter sequence shown in the amino acid sequence of SEQ ID NO: 1-7, 18-57 or 164-174.

[0489] In some embodiments, the adapter sequence described herein may be contained within an amino acid sequence comprising one or more amino acids of the antibody sequence or its antigen-binding fragment 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, the single-chain variable region fragment (scFv) described herein.

[0490] In some embodiments, the adapter sequence and / or the amino acid sequence containing the adapter sequence described herein may comprise one or more amino acids comprising the amino acid sequence EIKR (SEQ ID NO: 58). In some embodiments, the adapter sequence and / or the amino acid sequence containing the adapter sequence described herein may comprise the amino acid sequence EIK of the antibody or its antigen-binding fragment described herein. In some embodiments, the adapter sequence and / or the amino acid sequence containing the adapter sequence described herein may comprise the amino acid sequence EI of the antibody or its antigen-binding fragment described herein. In some embodiments, the adapter sequence and / or the amino acid sequence containing the adapter sequence described herein may comprise any one or any combination of amino acids E, I, K, and R of the antibody or its antigen-binding fragment described herein.

[0491] In some embodiments, the adapter sequence and / or the amino acid sequence containing the adapter sequence described herein may comprise one or more amino acids comprising the amino acid sequence LGAK (SEQ ID NO: 59). In some embodiments, the adapter sequence and / or the amino acid sequence containing the adapter sequence described herein may comprise the sequence LGAK (SEQ ID NO: 59) of the antibody or its antigen-binding fragment described herein. In some embodiments, the adapter sequence and / or the amino acid sequence containing the adapter sequence described herein may comprise the amino acid sequence LG of the antibody or its antigen-binding fragment described herein. In some embodiments, the adapter sequence and / or the amino acid sequence containing the adapter sequence described herein may comprise amino acids L or G of the antibody or its antigen-binding fragment described herein, or any combination thereof.

[0492] In some embodiments, the adapter sequence described herein may be contained within an amino acid sequence comprising one or more amino acids of the starting amino acid sequence of the VSV-G protein or a fragment or derivative thereof described herein. In some embodiments, the adapter sequence described herein and / or the amino acid sequence containing the adapter sequence may comprise one or more amino acids of the amino acid sequence KFT of the VSV-G protein. In some embodiments, the adapter sequence described herein and / or the amino acid sequence containing the adapter sequence may comprise one or more amino acids of the amino acid sequence FT of the VSV-G protein. In some embodiments, the adapter sequence described herein and / or the amino acid sequence containing the adapter sequence may comprise any one amino acid K, F, or T of the VSV-G protein, or any combination thereof.

[0493] In some embodiments, the adapters described herein may be cleavable or non-cleavable adapters. In some embodiments, the adapter is a cleavable adapter. In other embodiments, the adapter is a non-cleavable adapter. Cleavable adapters may be protease-sensitive, pH-sensitive, or glutathione-sensitive adapters. These adapters are typically cleavable only within cells and are preferably stable in the extracellular environment.

[0494] Protease-sensitive linkers can be cleaved by the enzymatic activity of proteases. These linkers typically comprise peptide sequences of lengths ranging from 2 to 10 amino acids, about 2 to 5 amino acids, about 5 to 10 amino acids, about 5 amino acids, about 3 amino acids, or about 2 amino acids. In some embodiments, the peptide sequence may comprise naturally occurring amino acids, such as cysteine ​​or alanine, or non-natural or modified amino acids. Non-natural amino acids include 3-amino acids, homoamino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids 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 (e.g., cathepsin B) and / or endosomal proteases.

[0495] pH-sensitive linkers are covalently bonded and readily degrade in high or low pH environments. In some embodiments, pH-sensitive linkers can be cleaved in a pH range of 4 to 6. In some embodiments, pH-sensitive linkers comprise hydrazones or cyclic acetals. In some embodiments, pH-sensitive linkers are cleaved in endosomes or lysosomes.

[0496] In some embodiments, the glutathione-sensitive linker includes a disulfide bond moiety. In some embodiments, the glutathione-sensitive linker is cleaved by a disulfide bond exchange reaction with intracellular glutathione. In some embodiments, the disulfide bond moiety also includes at least one amino acid, such as a cysteine ​​residue.

[0497] Non-limiting examples of cleavable adapters are shown in the amino acid sequences of SEQ ID NO: 1-7, 18-26, 30, 169, 174. In some embodiments, the cleavable adapter may comprise SEQ ID NO: 1-7, 18-26, 30 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 with SEQ ID NO: 1-7, 18-26, 30, 169, 174. In some embodiments, the cleavable adapter may be sensitive to cleavage via proteolytic cleavage. In some embodiments, proteolytic cleavage may be carried out by naturally occurring cell-associated proteases (e.g., endogenous proteases) and / or by exogenously added proteases. In some embodiments, proteolytic cleavage may be carried out by endogenous proteases. In some embodiments, proteolytic cleavage may be carried out by exogenous proteases.

[0498] In some embodiments, the protease capable of proteolytic cleavage (e.g., the proteolytic cleavage of the linker described herein) may belong to a class of proteases, such as, but not limited to, serine proteases, cystine proteases, or metalloproteinases. In some embodiments, endogenous proteases may be present in, for example (but not limited to), the endoplasmic reticulum, the Golgi apparatus, the cell surface from which viruses can be released, supernatants (or body fluids), the cell surface from which viruses can be targeted, or the endocytic compartment of the target cell. Protease cleavage signals for various proteases have been extensively studied, for example using high-throughput proteomics methods known in the art. Those skilled in the art will understand that for a given protease, there are multiple cleavage signal options, which depend heavily on the specific circumstances. In some embodiments, protease cleavage signal pairings may have different cleavage kinetics, and many membrane proteins may only partially detach from the cell surface, for example, due to the slower kinetics and / or regulated activity of their cleaving proteases (e.g., exfoliases). In some embodiments, the protease described herein may include any one or a variant of a variety of proteases, for example, as described in Encyclopedia of Cell Biology. 2016:650-60; Int. J. Mol. Sci. 2020, 21(18), 6805 and / or Molecular Neurobiology Vol. 56, pp. 3090-3112, 2019, both of which are incorporated herein by reference in their entirety for all purposes.

[0499] In some embodiments, the linker may be a non-cleavable linker (also known as a non-cleavable linker). Typically, non-cleavable linkers are not easily degraded in cellular or physiological environments. Non-limiting embodiments of non-cleavable linkers include the amino acid sequences shown in SEQ ID NO: 27-29, 31, 32, 36, 37. In some embodiments, a cleavable linker may comprise SEQ ID NO: 27-29, 31, 32, 36, 37 or variations 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 with SEQ ID NO: 27-29, 31, 32.

[0500] In various embodiments, the connector described herein may be included within a sequence comprising, for example but not limited to, the following sequences: 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:174); KRAAASGGS(G4S)2 ... NO:6); or AAA(G4S)3K(SEQ ID NO:7).

[0501] In some embodiments, the connector described herein may be contained in the sequence shown in SEQ ID NO: 1-7, 18-57 or 164-174 or a variant thereof, or in 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 with the sequence shown in SEQ ID NO: 1-7, 18-57 or 164-174.

[0502] In some embodiments, the connector described herein may be contained in the sequences listed in Table 5 or variations thereof, or in sequences 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 with the sequences listed in Table 5.

[0503] In some embodiments, the connector described herein may be contained in the sequences listed in Table 2 or variations thereof, or in sequences 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 with the sequences listed in Table 2.

[0504] In some embodiments, the linker sequence described herein and / or the amino acid sequence containing the linker sequence may contain the amino acid sequence alanine-alanine-alanine (AAA), which may be replaced by the amino acid sequence glycine-glycine-glycine (GGG) (i.e., the linker and / or the amino acid sequence containing the linker may contain a mutation from AAA to GGG), which may, for example, allow the display domain (e.g., the target molecule described herein) to move more freely on the linker described herein.

[0505] While both alanine (A) and glycine (G) are the simplest nonpolar neutral amino acids, glycine (G) is hydrophilic, while alanine (A) is hydrophobic. In some embodiments, the linker described herein may be located between the N-terminus of a VSV-G (e.g., blinded VSV-G) and a display domain (e.g., the target molecule described herein), which may bind to a receptor on a target cell, for example. Therefore, since the linker is exposed to the external environment, placing a hydrophilic amino acid (e.g., glycine (G)) in the linker sequence can improve the flexibility and mobility of the display domain, thereby enhancing its targeting. Hydrophobic amino acids (e.g., alanine (A)) can affect the tertiary conformation of the protein in an external hydrophilic environment and drive the linker (in addition to the display domain) to move inward. This may reduce the ability of the display domain to move toward the target receptor. In some implementations, the presence of hydrophilic amino acids (glycine (G)) can make the engineering of the viral particles described herein easier, for example, if the folding is impaired by the presence of hydrophobic amino acids (e.g., alanine (A)).

[0506] As a non-limiting example, the adapter sequence described herein may comprise the amino acid sequence RAAASGGS(G4S)2 (SEQ ID NO: 172), and when mutated to include the AAA to GGG mutations described herein, may comprise the amino acid sequence RGGGSGGS(G4S)2 (SEQ ID NO: 176). As another non-limiting example, the amino acid sequence comprising the adapter sequence may comprise the amino acid sequence KRAAASGGS(G4S)2 (SEQ ID NO: 2), and when mutated to include the AAA to GGG mutations described herein, may comprise the amino acid sequence KRGGGSGGS(G4S)2 (SEQ ID NO: 175). In some embodiments, the adapter sequence described herein may comprise the amino acid sequence RGGGSGGS(G4S)2 (SEQ ID NO: 176). In some embodiments, the amino acid sequence comprising the adapter sequence described herein may comprise the amino acid sequence KRGGGSGGS(G4S)2 (SEQ ID NO: 175).

[0507] As another non-limiting example, the adapter sequence described herein may comprise the amino acid sequence RAAASGGS(G4S)2GP (SEQ ID NO: 171), and when mutated to include the AAA to GGG mutations described herein, may comprise the amino acid sequence RGGGSGGS(G4S)2GP (SEQ ID NO: 178). As another non-limiting example, the amino acid sequence comprising the adapter sequence may comprise the amino acid sequence KRAAASGGS(G4S)2GPK (SEQ ID NO: 174), and when mutated to include the AAA to GGG mutations described herein, may comprise the amino acid sequence KRGGGSGGS(G4S)2GPK (SEQ ID NO: 177). In some embodiments, the adapter sequence described herein may comprise the amino acid sequence RGGGSGGS(G4S)2GP (SEQ ID NO: 178). In some embodiments, the amino acid sequence comprising the adapter sequence described herein may comprise the amino acid sequence KRGGGSGGS(G4S)2GPK (SEQ ID NO: 177).

[0508] In some embodiments, the adapter sequence described herein may comprise the amino acid sequence (EAAAK)3 (SEQ ID NO: 3), and 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, the adapter sequence described herein may comprise the amino acid sequence (EGGGK)3 (SEQ ID NO: 205).

[0509] In some embodiments, the adapter sequence described herein may comprise the amino acid sequence R(EAAAK)3 (SEQ ID NO: 173), and when mutated to include the AAA to GGG mutations described herein, may comprise the amino acid sequence R(EGGGK)3 (SEQ ID NO: 207). In some embodiments, the amino acid sequence comprising the adapter sequence may comprise the amino acid sequence KR(EAAAK)3 (SEQ ID NO: 4), and when mutated to include the AAA to GGG mutations described herein, may comprise the amino acid sequence KR(EGGGK)3 (SEQ ID NO: 206). In some embodiments, the adapter sequence described herein may comprise the amino acid sequence R(EGGGK)3 (SEQ ID NO: 207). In some embodiments, the amino acid sequence comprising the adapter sequence described herein may comprise the amino acid sequence KR(EGGGK)3 (SEQ ID NO: 206).

[0510] In some embodiments, the adapter sequence described herein may comprise the amino acid sequence AAARGSPK(G4S)3 (SEQ ID NO: 5), and 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, the adapter sequence described herein may comprise the amino acid sequence GGGRGSPK(G4S)3 (SEQ ID NO: 208).

[0511] In some embodiments, the adapter sequence described herein may comprise the amino acid sequence RAAARGSPK(G4S)3 (SEQ ID NO: 169), and when mutated to include the AAA to GGG mutations described herein, may comprise the amino acid sequence RGGGRGSPK(G4S)3 (SEQ ID NO: 209). In some embodiments, the adapter sequence described herein may comprise the amino acid sequence RGGGRGSPK(G4S)3 (SEQ ID NO: 209).

[0512] In some embodiments, the amino acid sequence containing the adapter sequence may comprise the amino acid sequence AAARGSPK(G4S)3K (SEQ ID NO: 19), and when mutated to include the AAA to GGG mutation described herein, may comprise the amino acid sequence GGGRGSPK(G4S)3K (SEQ ID NO: 210). In some embodiments, the amino acid sequence containing the adapter sequence described herein may comprise the amino acid sequence GGGRGSPK(G4S)3K (SEQ ID NO: 210).

[0513] In some embodiments, the adapter sequence described herein may comprise the amino acid sequence AAA(G4S)3 (SEQ ID NO: 34), and when mutated to include the AAA to GGG mutations described herein, may comprise the amino acid sequence GGG(G4S)3 (SEQ ID NO: 212). In some embodiments, the amino acid sequence comprising the adapter sequence may comprise the amino acid sequence AAA(G4S)3K (SEQ ID NO: 7), and when mutated to include the AAA to GGG mutations described herein, may comprise the amino acid sequence GGG(G4S)3K (SEQ ID NO: 211). In some embodiments, the adapter sequence described herein may comprise the amino acid sequence GGG(G4S)3 (SEQ ID NO: 212). In some embodiments, the amino acid sequence comprising the adapter sequence described herein may comprise the amino acid sequence GGG(G4S)3K (SEQ ID NO: 211).

[0514] Targeted molecules

[0515] In various embodiments, the recombinant fusion-promoting protein described herein may comprise a rhabdoviral glycoprotein (G) or a functional fragment or derivative thereof, and a targeting molecule. The targeting molecule may be located at the N-terminus of the rhabdoviral glycoprotein. In some aspects, the targeting molecule may be linked to the N-terminus of the rhabdoviral glycoprotein or a functional fragment or derivative thereof, for example, through any or a combination of the various linkers described herein. In some embodiments, the N-terminus of the rhabdoviral glycoprotein or a functional fragment or derivative thereof to which the targeting molecule is linked via a linker does not contain one or more amino acids present at the N-terminus of the mature wild-type rhabdoviral glycoprotein. In some embodiments, the rhabdoviral glycoprotein (G) linked to the targeting molecule may comprise any of the various rhabdoviral glycoproteins described herein, or a functional fragment or derivative thereof. For example, as described in this article, rhabdoviral glycoproteins can be derived from vesicular stomatitis virus glycoprotein (VSV-G), Flanders virus glycoprotein (FLAV-G), Chandipura virus glycoprotein (CHPV-G), Perinet virus glycoprotein (PERV-G), Piry virus glycoprotein (PIRYV-G), Fukuoka virus glycoprotein (FUKV-G), Joinjakaka 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 fasciatus-G, YugBugdanavoc glycoprotein (YBV-G), and Yinshui Bat glycoprotein (YSBV-G), Keuraliba 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 Transient Fever glycoprotein (BEFV-G), Curionopolis glycoprotein (CURV-G), Drosophila melanogaster Sigma virus glycoprotein (DMelSV-G), Niakha glycoprotein (NIAV-G), Puerto almandras glycoprotein (PTAMV-G), or Tupaia rhabdovirus (TUPTV-G), or functional fragments or derivatives thereof.

[0516] In some embodiments, when the rhabdoviral G protein contains VSV-G or a functional fragment or derivative thereof, the targeting molecule is capable of interfering with the ability of VSV-G or a functional fragment or derivative thereof to interact with the low-density lipoprotein receptor (LDLR). In some embodiments, the targeting molecule can interfere with the ability of VSV-G or a functional fragment or derivative thereof to interact with the LDLR, for example, through steric hindrance.

[0517] Non-limiting examples of targeting molecules include antibodies or antigen-binding fragments thereof, affinity molecules, 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.

[0518] In some implementations, the targeting molecules target, for example, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), mucin 16 (MUC16), cKit, α-vβ-3 (αVβ3) integrin, insulin-like growth factor 1 receptor (IGF1R), B cell maturation antigen (BCMA), Nectin-4, mitogen-activated protein kinase kinase (MEK), differentiation cluster 44 (CD44), CD3, CD4, CD28, stem cell factor (SCF), thrombopoietin, c-Met, CXCR4, IL2R, or interleukin-3 (IL-3).

[0519] In some implementations, the targeting molecules described herein target cells, such as cancer cells, including but not limited to tumor cells. Examples of cancer cells include those originating from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testes, tongue, or uterus. In addition, cancer can specifically be, but is not limited to, the following histological types: growths, malignant; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilonidal stromal 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 in adenomatous polyps; adenocarcinoma, familial adenomatous polyposis; solid carcinoma; carcinoid tumor, malignant; bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; oxyadenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular adenocarcinoma. Cellular carcinoma; Follicular adenocarcinoma; Papillary and follicular adenocarcinoma; Non-capsulated sclerosing carcinoma; Adrenocortical carcinoma; Endometrioid carcinoma; Skin appendage carcinoma; Apocrine gland adenocarcinoma; Sebaceous gland carcinoma; Penile gland adenocarcinoma; Mucoepidermoid carcinoma; Cystic adenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous gland carcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease, breast; Acinar cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Thymoma, malignant; Ovarian stromal tumor, malignant; Theca cell tumor, malignant; Granular cell tumor, malignant; Male cell tumor, malignant; Sertoli cell carcinoma; Leydig cell carcinoma Cellular tumor, malignant; Lipiocytoma, malignant; Paraganglioma, malignant; Extramammary paraganglioma, malignant; Pheochromocytoma; Balloon sarcoma; Malignant melanoma; Amelanoma; Superficial diffuse melanoma; Malignant melanoma in giant nevus; Epithelioid cell melanoma; Blue nevus, malignant; Sarcoma; Fibrosarcoma; Fibrohistocytoma, malignant; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor, malignant; Müllerian mixed tumor; Nephroblastoma; Hepatoblastoma carcinoma; Carcinosarcoma; Stromal tumor, malignant; Brenner's tumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; ovarian goiter, malignant; choriocarcinoma; mesonephric tumor, malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic odontosarcoma; pineal tumor, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma;Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal; Cerebellar sarcoma; Ganglioblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granulosarcoma, malignant; Malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; Paragranuloma; Malignant lymphoma, small lymphocyte; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular lymphoma; mycosis fungoides; other designated non-Hodgkin lymphoma; malignant histiocytic proliferative disorders; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel 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.

[0520] In some implementations, the targeting molecule is selected from humanized antibodies or their antigen-binding fragments, human antibodies or their antigen-binding fragments, mouse antibodies or their antigen-binding fragments, chimeric antibodies or their antigen-binding fragments, monovalent Fab', bivalent Fab2, F(ab)'3 fragments, single-chain variable region fragments (scFv), biscFv, (scFv)2, biantibodies, microantibodies, nanobodies, triantibodies, tetraantibodies, disulfide-stabilized Fv proteins (dsFv), single-domain antibodies (sdAb), IgNAR, single heavy chain antibodies, bispecific antibodies or their binding fragments, bispecific T-cell adaptors (BiTE), trispecific antibodies or their chemically modified derivatives.

[0521] The antibodies described herein may include immunoglobulin molecules comprising four polypeptide chains, namely two heavy chains (HC) and two light chains (LC), interconnected by disulfide bonds (e.g., IgG). In various embodiments, each antibody heavy chain (HC) contains a heavy chain variable region (“HCVR” or “V”). H Each antibody light chain (LC) contains a light chain variable region (“LCVR” or “V”) and a heavy chain constant region; each antibody light chain (LC) contains a light chain variable region (“LCVR” or “V”). L ") and the light chain constant region (CL). V H and V L The region can be further subdivided into high-variability regions, called complementary determinant regions (CDRs), interspersed with more conservative regions, called frame regions (FRs).

[0522] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant domain, such as a heavy chain constant domain of the type 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. In some embodiments, the antibody or its antigen-binding fragment may comprise a light chain constant domain, such as a light chain constant domain of the type κ or λ. In one embodiment of this disclosure, V H It can be linked to the human heavy chain constant domain (e.g., IgG), V L It can be connected to the constant structural domain of human light chains (e.g., κ).

[0523] In some implementations, the amino acid assignment of each framework domain or CDR domain conforms to the definition in Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publication 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. Therefore, this disclosure includes antibody and antigen-binding fragments comprising a CDR of VH and a CDR of VL, wherein VH and VL comprise the amino acid sequences (or variants thereof) shown herein, and wherein the CDR is defined, for example, according to Kabat and / or Chothia.

[0524] In some embodiments, the antibody or antigen-binding fragment may include a heavy chain constant domain, such as a heavy chain constant domain of the type 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. In one embodiment of this disclosure, the antigen-binding protein, such as an antibody or antigen-binding fragment, includes a light chain constant domain, such as a light chain constant domain of the type κ or λ.

[0525] As used herein, the term "human antibody" or antigen-binding fragment includes antibodies and fragments having human amino acid sequences; for example, variable and constant regions derived from human germline immunoglobulin sequences, whether in human cells or transplanted into non-human cells (e.g., mouse cells). In one embodiment of this disclosure, the human antibody and antigen-binding fragment of this disclosure may contain amino acid residues encoded by non-human germline immunoglobulin sequences (e.g., with mutations introduced by in vitro random or site-directed mutagenesis or in vivo somatic mutations), for example in CDRs, particularly CDR3. However, the term "human antibody" as used herein is not intended to include mAbs in which a CDR sequence derived from another mammalian species (e.g., mouse) has been transplanted onto a human FR sequence. The term includes antibodies recombinantly generated in non-human mammals or non-human mammalian cells. The term is not intended to include antibodies isolated or generated from human subjects.

[0526] This disclosure includes chimeric antibodies and their antigen-binding fragments, and methods of use thereof. As used herein, "chimeric antibody" refers to an antibody having a variable domain derived from a first antibody and a constant domain derived from a second antibody, wherein the first and second antibodies are from different species. (See, for example, US4816567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855). This disclosure includes chimeric antibodies comprising a variable domain and a non-human constant domain.

[0527] In various embodiments, the antigen-binding fragment of the antibody will contain a portion less than that of the complete antibody but still specifically binding to the antigen, for example, containing at least one variable domain. The variable domain can be of any size or amino acid composition and typically contains at least one (e.g., three) CDRs, which are adjacent to or within one or more frame sequences. L V of domain association H In the antigen-binding fragment of the domain, V H and V L Domains can be positioned relative to each other in any suitable arrangement. For example, a variable region can be a dimer and contain V. H -V H V H -V L or V L -V L Dimer. Alternatively, the antigen-binding fragment of the antibody may contain a non-covalently bound monomer V. H and / or V L Structural domain.

[0528] In some embodiments, the antigen-binding fragment of the antibody may include at least one variable domain covalently linked to at least one constant domain. Non-limiting examples of configurations of variable and constant domains that can be found in the antigen-binding fragment of the antibody of this disclosure include: (i) 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 configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly connected to each other or connected via complete or partial hinge or linker regions. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in flexible or semi-flexible connections between adjacent variable and / or constant regions in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of this disclosure may comprise connections between and / or with one or more monomers V. H or V L Homodimers or heterodimers (or other polymers) of any of the above variable and constant region configurations, which are non-covalently linked by structural domains (e.g., via disulfide bonds).

[0529] Antibodies and their antigen-binding fragments can be monospecific or multispecific (e.g., bispecific).

[0530] The antibody and antigen-binding fragments described herein may be fused with other polypeptide molecules, such as, but not limited to, epitopes (e.g., FLAG) or tag sequences (e.g., His tag sequences, etc.), to allow detection and / or separation of antibody or antigen-binding fragments; ligands or portions thereof that bind to transmembrane receptor proteins; enzymes or portions thereof that have catalytic activity; polypeptides or peptides that promote oligomerization, such as leucine zipper domains; polypeptides or peptides that increase stability, such as immunoglobulin constant regions (e.g., Fc domains); polypeptides that extend half-life (e.g., albumin or albumin-binding peptide / protein); functional or non-functional antibodies, or their heavy or light chains; and / or polypeptides that have activities different from those of the antibody or antigen-binding fragments disclosed herein.

[0531] In some embodiments, the antibodies or antigen-binding fragments described herein may be post-translational modified, including, for example: N-terminal Glu or Gln cyclization; loss of N-terminal positive charge; C-terminal Lys variants; deamidation (Asn to Asp); isomerization (Asp to isoAsp); deamidation (Gln to Glu); oxidation (Cys, His, Met, Tyr, Trp); and / or disulfide heterogeneity (reorganization, thioether and trisulfide bond formation).

[0532] 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 variable region fragment (scFv). In some embodiments, the scFv comprises domains arranged from the N-terminus to the C-terminus in the following orientation: HCVR-LCVR. In some embodiments, the scFv comprises domains arranged from the N-terminus to the C-terminus in the following orientation: LCVR-HCVR. The scFv variable regions are connected by adapters, such as, but not limited to, any of the various adapters described herein.

[0533] The term "specific binding" refers to a target molecule (e.g., an antibody or its antigen-binding fragment) that has binding affinity for an antigen. This disclosure includes target molecules that specifically bind to, 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 target molecules disclosed herein may comprise scFvs that target the tumor antigen HER2 (also known 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 from the N-terminus to the C-terminus in the following orientation: HCVR-LCVR. In some embodiments, the anti-HER2 scFv comprises domains arranged from the C-terminus to the N-terminus in the following orientation: LCVR-HCVR. In some embodiments, the variable region of the anti-HER2 scFv is connected via a adapter, such as, but not limited to, the adapter described herein. In some embodiments, the anti-HER2 scFv specifically binds to human HER2. For example, an anti-HER2 scFv that can be used to implement the present disclosure may comprise any anti-HER2 scFv, such as those described by Shier et al. (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 entirety). In some embodiments, the anti-HER2 scFv may comprise, for example, the clone C6-B1D2 described by Shier et al. (1996) and may bind to human HER2, and in some cases, K d Approximately, for example, 0.15 x 10 -10 M.

[0534] In some embodiments, the anti-HER2 scFv described herein is derived from antibody C6B1D2. In some embodiments, the anti-HER2 scFv derived from antibody C6B1D2 comprises the amino acid sequence of SEQ ID NO: 158, or an amino acid sequence having at least 80% sequence identity with it. In some embodiments, the nucleotide sequence encoding the anti-HER2 scFv derived from 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% sequence identity with it.

[0535] In some embodiments, the anti-HER2 scFv derived from antibody C6B1D2 includes a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence having at least 80% sequence identity with it. In some embodiments, the nucleotide sequence encoding the HCVR of the anti-HER2 scFv derived from antibody C6B1D2 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence having at least 80% sequence identity with it. In some embodiments, the anti-HER2 scFv derived from antibody C6B1D2 includes a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 160, or an amino acid sequence having at least 80% sequence identity with it. In some embodiments, the nucleotide sequence encoding the LCVR of the anti-HER2 scFv derived from antibody C6B1D2 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 160, or an amino acid sequence having at least 80% sequence identity with it. In some embodiments, the anti-HER2 scFv derived from antibody C6B1D2 includes a linker sequence between HCVR and LCVR, the linker sequence comprising the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence having at least 80% identity with it. In some embodiments, the nucleotide sequence encoding the linker between HCVR and LCVR in the anti-HER2 scFv derived from antibody C6B1D2 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence having at least 80% identity with it. In some embodiments, the anti-HER2 scFv derived from antibody C6B1D2 includes a domain arranged from the N-terminus to the C-terminus in the direction of HCVR-LCVR. In some embodiments, the anti-HER2 scFv derived from antibody C6B1D2 includes a domain arranged from the N-terminus to the C-terminus in the direction of LCVR-HCVR, and the variable region of the anti-HER2 scFv is connected by a linker, such as, but not limited to, any of the various linkers described herein.

[0536] In some embodiments, the anti-HER2 scFv described herein is derived from antibody C6.5. In some embodiments, the anti-HER2 scFv derived from antibody C6.5 comprises the amino acid sequence of SEQ ID NO: 192, or an amino acid sequence having at least 80% sequence identity with it. In some embodiments, the nucleotide sequence encoding the anti-HER2 scFv derived from 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 with it.

[0537] In some embodiments, the anti-HER2 scFv derived from antibody C6.5 includes a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 193, or an amino acid sequence having at least 80% sequence identity with it. In some embodiments, the nucleotide sequence encoding the HCVR of the anti-HER2 scFv derived from antibody C6.5 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 193, or an amino acid sequence having at least 80% sequence identity with it. In some embodiments, the anti-HER2 scFv derived from antibody C6.5 includes a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 194, or an amino acid sequence having at least 80% sequence identity with it. In some embodiments, the nucleotide sequence encoding the LCVR of the anti-HER2 scFv derived from antibody C6.5 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 194, or an amino acid sequence having at least 80% sequence identity with it. In some embodiments, the anti-HER2 scFv encoding antibody C6.5 includes a linker sequence between HCVR and LCVR, the linker sequence comprising the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence having at least 80% identity with it. In some embodiments, the nucleotide sequence encoding the linker between HCVR and LCVR in the anti-HER2 scFv derived from antibody C6.5 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence having at least 80% identity with it. In some embodiments, the anti-HER2 scFv derived from antibody C6.5 includes a domain arranged from the N-terminus to the C-terminus in the direction of HCVR-LCVR. In some embodiments, the anti-HER2 scFv derived from antibody C6.5 includes a domain arranged from the N-terminus to the C-terminus in the direction of LCVR-HCVR, and the variable region of the anti-HER2 scFv is connected by a linker, such as, but not limited to, any of the various linkers described herein.

[0538] In some embodiments, the targeting molecules disclosed herein may comprise scFvs targeting the tumor antigen EGFR (also known as epidermal growth factor receptor, ERBB1, receptor tyrosine protein kinase ErbB-1, etc.). In some embodiments, the anti-EGFR scFv comprises domains arranged from the N-terminus to the C-terminus in the following orientation: HCVR-LCVR. In some embodiments, the anti-EGFR scFv comprises domains arranged from the C-terminus to the N-terminus in the following orientation: LCVR-HCVR. In some embodiments, the scFv variable region of the anti-EGFR scFv is connected by a linker, such as, but not limited to, the linker described herein. In some embodiments, the anti-EGFR scFv specifically binds to human EGFR. For example, an anti-EGFR scFv that can be used to implement the present disclosure may comprise any anti-EGFR scFv, such as those described by Nakamura et al. (Nakamura et al., Antibody-targetedcell fusion, Nature Biotechnology volume 22, pages 331-336 (2004), the contents of which are incorporated herein by reference in their entirety).

[0539] 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 with it. 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% sequence identity with it.

[0540] In some embodiments, the anti-EGFR scFv described herein comprises a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 162, or an amino acid sequence having at least 80% sequence identity with it. 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 with it. In some embodiments, the anti-EGFR scFv comprises a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 163, or an amino acid sequence having at least 80% sequence identity with it. 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 with it. In some embodiments, the anti-EGFR scFv comprises a linker sequence between the HCVR and the LCVR, the linker sequence containing the amino acid sequence shown in SEQ ID NO: 37, or an amino acid sequence having at least 80% sequence identity with it. 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 shown in SEQ ID NO: 37 or an amino acid sequence having at least 80% sequence identity with it. In some embodiments, the anti-EGFR scFv comprises a domain arranged from the N-terminus to the C-terminus in the direction of HCVR-LCVR. In some embodiments, the anti-EGFR scFv comprises a domain arranged from the N-terminus to the C-terminus in the direction of LCVR-HCVR, and the variable region of the anti-EGFR scFv is connected by a linker, such as, but not limited to, any of the various linkers described herein.

[0541] In some embodiments, the targeted molecules disclosed herein may comprise scFvs 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 virus oncogene homolog, mast / stem cell growth factor receptor Kit, CD117, PBT, tyrosine protein kinase Kit, mottled trait protein, proto-oncogene C-Kit, P145C-Kit, C-Kit, V-Kit Hardy-Zuckerman 4 feline sarcoma virus oncogene-like protein, proto-oncogene tyrosine protein kinase Kit, C-Kit proto-oncogene, mottled trait, CD117 antigen, MASTC, C-KIT, etc.). In some embodiments, the anti-cKit scFv comprises domains arranged from the N-terminus to the C-terminus in the following orientation: HCVR-LCVR. In some embodiments, the anti-cKit scFv comprises domains arranged from the C-terminus to the N-terminus in the following orientation: LCVR-HCVR. In some implementations, the scFv variable region of the anti-cKit scFv is connected via a connector, such as, but not limited to, the connector described herein. In some implementations, the anti-cKit scFv specifically binds to human cKit.

[0542] 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 with it. 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 with it.

[0543] In some embodiments, the anti-cKit scFv described herein comprises a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 196, or an amino acid sequence having at least 80% sequence identity with it. 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 with it. In some embodiments, the anti-cKit scFv comprises a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 197, or an amino acid sequence having at least 80% sequence identity with it. 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 with it. In some embodiments, the anti-cKit scFv comprises a linker sequence between the HCVR and the LCVR, the linker sequence containing the amino acid sequence shown in SEQ ID NO: 55, or an amino acid sequence having at least 80% sequence identity with it. 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 shown in SEQ ID NO: 55 or an amino acid sequence having at least 80% sequence identity with it. In some embodiments, the anti-cKit scFv comprises a domain arranged from the N-terminus to the C-terminus in the direction of HCVR-LCVR. In some embodiments, the anti-cKit scFv comprises a domain arranged from the N-terminus to the C-terminus in the direction of LCVR-HCVR, and the variable region of the anti-cKit scFv is connected by a linker, such as, but not limited to, any of the various linkers described herein.

[0544] In some embodiments, the targeting molecules disclosed herein may include nanobodies targeting the tumor antigen EGFR. A non-limiting example of an EGFR-targeting nanobody is the nanobody Nb 7D12 (anti-EGFR) (also known as EGFRNb). In some embodiments, the 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 with it. Another non-limiting example of an EGFR-targeting nanobody is the nanobody Nb 9G8 (anti-EGFR). In some embodiments, the nanobody Nb 9G8 (anti-EGFR) may comprise the amino acid sequence EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSALE (SEQ ID NO: 188), or an amino acid sequence having at least 80% identity with it.

[0545] In some embodiments, the targeting molecule may 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, the EGFm123 of this disclosure may comprise the amino acid sequence NSYSECPPSYDGYCLHDGVCRYIEALDSYACNCVVGYAGERCQYRDLRWWGRR (SEQ ID NO: 186), or an amino acid sequence having at least 80% identity with it. In some embodiments, the human stem cell factor (hSCF) of this disclosure may comprise the amino acid sequence EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAA (SEQ ID NO: 189), or an amino acid sequence having at least 80% identity with it. In some embodiments, the human insulin-like growth factor 1 (hIGF1) of this disclosure may comprise the amino acid sequence GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA (SEQ ID NO: 190), or an amino acid sequence having at least 80% identity with it.

[0546] In some embodiments, the targeting molecules disclosed herein may comprise a T-cell receptor (TCR) or a fragment or derivative thereof. The TCR can recognize peptides presented in the context of a major histocompatibility complex (MHC) molecule. Peptide-MHC (pMHC) complexes can be recognized by the TCR, where the peptide (antigenic determinant) and the TCR idiotype provide specificity for the interaction. Therefore, the antigens described herein can encompass peptides presented in the context of an MHC molecule. Peptides that can be displayed on an MHC molecule may also comprise the epitopes described herein. In some embodiments, the epitopes may not only encompass epitopes naturally presented by antigen-presenting cells (APCs), but may also be any desired peptide, as long as it can be recognized by immune cells, for example, when appropriately presented to cells of the immune system. For example, peptides having artificially prepared amino acid sequences can also be used as epitopes. In some embodiments, in addition to fusionins, such as rhabdoviral glycoprotein (G), such as, but not limited to, VSV-G or a functional fragment or derivative thereof described herein, the viral particles described herein may also display TCR-binding molecules. In some embodiments, the TCR-binding molecules and fusionins may be included in the recombinant fusion proteins described herein. In some implementations, the TCR-binding molecule may contain a TCR-specific antibody or a portion thereof.

[0547] In various embodiments, the TCR-binding molecules described herein may comprise peptides presented in the MHC molecular environment, such as antigenic determinants associated with the peptide-binding groove of the MHC. In some embodiments, the viral particles described herein, such as viral particles comprising the recombinant fusion-promoting protein described herein, are capable of binding to antigen-specific T-cell receptors (TCRs). These recombinant viral particles comprise a lipid envelope containing (i) a peptide (p) presented in the major histocompatibility complex (MHC) molecule, i.e., the pMHC complex, and (ii) a fusionin. In some embodiments, the antigen-specific TCR specifically binds to the pMHC complex.

[0548] MHC molecules are generally classified into two classes: class I and class II. Class I MHC molecules are integrated membrane proteins containing a single glycoprotein heavy chain, also referred to herein as the α chain. This heavy chain has three extracellular domains (α1, α2, and α3) and two intracellular domains (a transmembrane domain (TM) and a cytoplasmic domain (CYT)). The heavy chain is non-covalently bound to a soluble subunit called β2-microglobulin (β2m or B2M). Class II MHC proteins are heterodimeric integrated membrane proteins composed of a non-covalently bound α chain and a β chain. The α chain has two extracellular domains (α1 and α2) and two intracellular domains (one TM domain and one CYT domain). The β chain contains two extracellular domains (β1 and β2) and two intracellular domains (one TM domain and one CYT domain).

[0549] Class I and Class II MHC domains organize to form antigenic determinant binding sites, or peptide-binding grooves. A peptide-binding groove is a cavity-like portion of an MHC protein into which peptides (such as antigenic determinants) can bind. The conformation of the peptide-binding groove can change after peptide binding, thereby correctly aligning the amino acid residues essential for the binding of the TCR to the peptide-MHC (pMHC) complex.

[0550] The MHCs described herein include MHC chain fragments sufficient to form peptide-binding grooves. For example, the peptide-binding groove of a class I protein may contain portions of the heavy chain α1 and α2 domains, which are capable of forming two β-sheets and two α-helices. The addition of a portion of the β2 microglobulin chain stabilizes the complex. While for most class II MHC molecules, the interaction between the α and β chains can occur without a peptide, the double-stranded complex of class I MHC is unstable until the binding groove is filled with a peptide. The peptide-binding groove of a class II protein may contain portions of the α1 and β1 domains, which are capable of forming two β-sheets and two α-helices. The first portion of the α1 domain forms the first β-sheet, and the second portion forms the first α-helix. The first portion of the β1 domain forms the second β-sheet, and the second portion forms the second α-helix. X-ray crystal structures of class II proteins with peptides bound to protein-binding grooves show that one or both ends of the binding peptide may protrude beyond the MHC protein. Therefore, the ends of the α1 and β1α helices of class II proteins form an open cavity, preventing the peptide ends that bind to the binding groove from being buried within the cavity. Furthermore, X-ray crystallography of class II proteins shows that the N-terminus of the MHC β chain appears to protrude from the side of the MHC protein in a non-structural manner, as the first four amino acid residues of the β chain cannot be localized by X-ray crystallography.

[0551] Many human and other mammalian MHC molecules are well known in the field, and any MHC class I or II molecule can be part of the TCR-binding molecules described herein.

[0552] The MHC molecules available in viral particles described herein include naturally occurring full-length MHC molecules and single chains of MHC molecules (e.g., MHC class I α (heavy) chains, β2-microglobulin, MHC class II α chains, and MHC class II β chains), individual subunits of such MHC chains (e.g., α1, α2, and / or α3 subunits of MHC class I α chains, α1 and / or α2 subunits of MHC class II α chains, and β1 and / or β2 subunits of MHC class II β chains), as well as fragments, mutants, and various derivatives thereof, wherein such fragments, mutants, and derivatives retain the ability to display antigenic determinants for recognition by antigen-specific TCRs. In one specific embodiment, the MHC comprises transmembrane domains embedded in the lipid envelope of the viral particle.

[0553] Naturally occurring MHC molecules are encoded by a set of genes on human chromosome 6 or mouse chromosome 17. These MHC molecules (known as H-2 in mice and HLA (human leukocyte antigen) in humans) are classified into 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 molecules include, but are not limited to, HLA-specific molecules 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, HLA specificity includes 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.

[0554] The MHC described in this article can come from any mammal or bird, such as primates (e.g., humans), rodents, rabbits, horses, cattle, dogs, cats, pigs, etc.

[0555] Naturally occurring MHC class I molecules bind to peptides derived from proteins (especially endogenously synthesized proteins) resulting from cellular proteolytic degradation. The resulting short peptides are transported to the endoplasmic reticulum, where they bind to nascent MHC class I molecules, then cross the Golgi apparatus and are displayed on the cell surface for recognition by cytotoxic T lymphocytes.

[0556] Naturally occurring MHCI molecules consist of an α (heavy) chain bound to β2-microglobulin. The heavy chain comprises α1-α3 subunits. β2-microglobulin binds to the α3 subunit of the heavy chain. In some embodiments, β2-microglobulin is covalently bound to the α3 subunit. In other embodiments, β2-microglobulin is non-covalently bound to the α3 subunit. The α1 and α2 subunits of the heavy chain fold to form a groove for the display of peptides (e.g., antigenic determinants) and recognition by TCRs.

[0557] Class I molecules bind to peptides that are approximately 8-9 amino acids long. All humans have 3 to 6 different Class I molecules, each of which can bind to many different types of peptides.

[0558] In some embodiments, MHC comprises (i) a class I MHC polypeptide or a fragment, mutant, or derivative thereof, and optionally (ii) a β2-microglobulin polypeptide or a fragment, mutant, or derivative thereof. In one specific embodiment, the class I MHC polypeptide is linked to the β2-microglobulin polypeptide via a peptide linker.

[0559] In some embodiments, the class I MHC peptide is a human class I MHC peptide selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In another specific embodiment, the class I MHC peptide is a mouse class I MHC peptide selected from H-2K, H-2D, H-2L, H2-IA, H2-IB, H2-IJ, H2-IE, and H2-IC.

[0560] 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 chain is fully human. In some embodiments, the MHC class I α heavy chain is humanized. Humanized MHC class I α heavy chains are described, for example, in U.S. Patent Publications 2013 / 0111617, 2013 / 0185819, and 2014 / 0245467, all of which are incorporated herein by reference in their entirety. In some embodiments, the MHC class I α heavy chain comprises human extracellular domains (human α1, α2, and / or α3 domains) and cytoplasmic domains from 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 may be the HLA-A*0201 sequence. In various respects, the peptide-MHC may comprise all domains of the MHC class I heavy chain.

[0561] In some embodiments, the viral particles contain 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 Publications 2013 / 0111617 and 2013 / 0185819, both of which are incorporated herein by reference in their entirety.

[0562] In some implementations, MHCI-class molecules contain mutations in the β2-microglobulin (β2m or B2M) polypeptide and heavy chain sequences, thereby affecting the disulfide bond between B2M and the heavy chain. In some cases, the heavy chain is HLA, and the disulfide bond connects 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; β2M residue Trp60, HLA residue Gln96; β2M residue Trp60, HLA residue Aspl22; β2M residue Tyr63, HLA residue Tyr27; β2M residue Lys6, HLA residue Glu232; β2M residue Gln8, HLA residue Arg234; β2M residue Tyrl0, HLA residue Pro235; β2M residues Ser1, HLA residue Gln242; β2M residue Asn24, HLA residue Ala236; β2M residue Ser28, HLA residue Glu232; β2M residue Asp98, HLA residue His192; and β2M residue Met99, HLA residue Arg234, first linker position Gly2, heavy chain (HLA) position Tyr84; light chain (β2M) position Arg12, HLA Ala236; and / or β2M residue Argl2, HLA residue Gly237. See, for example, International Patent Application Publication WO2015 / 195531.

[0563] In some embodiments, the antigenic determinant amino acid sequence may be a sequence of a peptide that can be presented by MHC class I molecules. In some embodiments, the sequence may comprise about 8 to about 15 consecutive amino acids. In some embodiments, the peptide sequence may be a sequence of a protein fragment derived from, for example, an infectious agent or a cellular protein (e.g., a protein expressed by cancer cells), and wherein the peptide may bind to an MHC class I heavy chain.

[0564] In some embodiments, at least one chain of the MHC and the peptide is included in the fusion protein described herein. In one specific embodiment, the MHC and the peptide are separated by a linker sequence. For example, the single-chain molecule may include an antigenic determinant, a β2-microglobulin sequence, and a class I α (heavy) chain sequence from the N-terminus to the C-terminus. Alternatively, the single-chain molecule may include an antigenic determinant, a class I α (heavy) chain sequence, and a β2-microglobulin sequence from the N-terminus to the C-terminus. The single-chain molecule may also include a signal peptide sequence at the N-terminus. In some embodiments, a linker sequence may be present between the peptide sequence and the β2-microglobulin sequence. In some embodiments, a linker sequence may be present between the β2-microglobulin sequence and the class I α (heavy) chain sequence. The single-chain molecule may also include a signal peptide sequence at the N-terminus, as well as 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 some embodiments, the β2-microglobulin and class I α (heavy) chain sequences may be human, mouse, or porcine.

[0565] In some embodiments, the single-chain molecule may include a first flexible linker located between the peptide ligand fragment and the β2-microglobulin fragment. For example, the linker may extend from the carboxyl terminus of the peptide ligand fragment and connect to the amino terminus of the β2-microglobulin fragment. Preferably, the structure of the linker allows the linked peptide ligand to fold into the binding groove, thereby generating a functional MHC antigenic peptide. In some embodiments, the linker may contain at least about 10 amino acids and at most about 15 amino acids. In some embodiments, the single-chain molecule may include a second flexible linker inserted between the β2-microglobulin fragment and the heavy chain fragment. For example, the linker may extend from the carboxyl terminus of the β2-microglobulin fragment and connect to the amino terminus of the heavy chain fragment. In some embodiments, the β2-microglobulin and the heavy chain may fold into the binding groove, thereby generating a molecule capable of promoting T cell proliferation.

[0566] Suitable linkers used in MHC can be of any of a variety of suitable lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Non-limiting examples of linkers include, for example, glycine polymers (G). n Glycine-serine polymers (including, for example, (GS)) n (GSGGS (SEQ ID NO: 44)) and (GGGS (SEQ ID NO: 45)), where n is an integer of at least 1, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Glycine and glycine-serine polymers can be used; both Gly and Serine are relatively unstructured and can therefore act as neutral linkers between components. Glycine polymers can be used; glycine can access more phi-psi spaces than alanine and is much less restricted than residues with longer side chains. Exemplary linkers may comprise amino acid sequences, including but not limited to GGSG (SEQ ID NO:46), GGSGG (SEQ ID NO:47), GGSG (SEQ ID NO:48), GGSGG (SEQ ID NO:49), GGGSG (SEQ ID NO:50), GSSSG (SEQ ID NO:51), GCGASGGGGSGGGGS (SEQ ID NO:52), GGGGSGGGGS (SEQ ID NO:53), GGGAASGGGGSGGGGS (SEQ ID NO:54), GGGGSGGGGSGGGGS (SEQ ID NO:55), GGGAASGGGGS (SEQ ID NO:56), or GGGGSGGGGSGGGGSGGGS (SEQ ID NO:57). In some embodiments, the linker peptide comprises a cysteine ​​residue capable of forming a disulfide bond with a cysteine ​​residue present in the second peptide.

[0567] In some embodiments, the single-chain molecule may comprise a peptide covalently linked to an MHC class I α (heavy) chain via a disulfide bond (i.e., a disulfide bond between two cysteine ​​residues). In some embodiments, the disulfide bond comprises a first cysteine ​​residue contained by a linker extending from the carboxyl terminus of the antigenic peptide, and a second cysteine ​​residue contained by an MHC class I heavy chain (e.g., an MHC class I α (heavy) chain having a non-covalent binding site for the antigenic peptide). In some embodiments, the second cysteine ​​residue may be a mutation (addition or substitution) in the MHC class I α (heavy) chain. In some embodiments, the single-chain molecule may comprise a continuous polypeptide chain and a disulfide bond. In some embodiments, the single-chain molecule may comprise two continuous polypeptide chains linked by a disulfide bond as the sole covalent bond. In some embodiments, the linking sequence may comprise at least one amino acid in addition to cysteine ​​residues, including one or more glycines, one or more alanines, and / or one or more serines.

[0568] In some embodiments, if the pMHC complex contains a first cysteine ​​residue in the Gly-Ser linker extending between the C-terminus of the peptide and the β2 microglobulin, and a second cysteine ​​residue in the proximal heavy chain position, then a disulfide bond can link the antigenic peptide in the class I groove of the pMHC complex.

[0569] In some embodiments, the β2-microglobulin sequence may comprise the full-length β2-microglobulin sequence. In some embodiments, the β2-microglobulin sequence lacks a leader peptide sequence. Therefore, in some configurations, the β2-microglobulin sequence may comprise approximately 99 amino acids and may be a mouse β2-microglobulin sequence (e.g., Genebank X 01838). In some other configurations, the β2-microglobulin sequence may comprise approximately 99 amino acids and may be a human β2-microglobulin sequence (e.g., Genebank AF072097.1).

[0570] In some embodiments, the pMHC complex sequence may be a sequence disclosed in the following: U.S. Patent Nos. 4,478,82; 6,011,146; 8,518,697; 8,895,020; 8,992,937; WO 96 / 04314; Mottez et al., J. Exp. Med. 181:493-502, 1995; Madden et al., Cell 70:1035-1048, 1992; Matsumura et al., Science 257:927-934, 1992; Mage et al., Proc. Natl. Acad. Sci. USA 89:10658-10662, 1992; Toshitani et al., Proc. Natl. Acad. Sci. 93:236-240, 1996; Chung et al., J. Immunol. 163:3699-3708, 1999; Uger and Barber, J. Immunol. 160:1598-1605, 1998; Uger et al., J. Immunol. 162, pp.6024-6028, 1999; White et al., J. Immunol. 162:2671-2676, 1999; Yu et al., J. Immunol. 168:3145-3149, 2002; Truscott et al., J. Immunol. 178:6280-6289, 2007. All of the above references are incorporated herein by reference in their entirety for all purposes.

[0571] In some embodiments, the MHC comprises a class II MHC polypeptide or a fragment, mutant, or derivative thereof. In one specific embodiment, the MHC comprises α and β polypeptides of a class II MHC complex or a fragment, mutant, or derivative thereof. In one specific embodiment, the α and β polypeptides are linked by a peptide linker. In one specific embodiment, the MHC comprises α and β polypeptides of a human class II MHC complex selected from HLADP, HLA-DR, HLA-DQ, HLA-DM, and HLA-DO. In another specific embodiment, the MHC comprises α and β polypeptides of a mouse H-2A or H-2E class II MHC complex.

[0572] Naturally occurring MHC class II molecules consist of two polypeptide chains, α and β. These chains may originate from the DP, DQ, or DR genome. Approximately 40 different human MHC class II molecules are known. They all share the same basic structure but differ slightly in their molecular structure. MHC class II molecules bind peptides of 13–18 amino acids in length.

[0573] In some embodiments, the viral particle comprises one or more MHC class II α chains. In some embodiments, the MHC class II α chain is fully human. In some embodiments, the MHC class II α chain is humanized. Humanized MHC class II α chains are described, for example, in U.S. Patent Nos. 8,847,005 and 9,043,996 and U.S. Patent Publication No. 2014 / 0245467, the full text of which is incorporated herein by reference for all purposes. In some embodiments, the humanized MHC class II α chain polypeptide comprises human extracellular domains and cytoplasmic domains of other species. In some embodiments, the class II α chain is HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, or HLA-DRA. In some embodiments, the class II α chain polypeptide is humanized HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, and / or HLA-DRA.

[0574] In some embodiments, the viral particle comprises one or more MHC class II β chains. In some embodiments, the MHC class II β chain is fully human. In some embodiments, the MHC class II β chain polypeptide is humanized. Humanized MHC class II β chain polypeptides are described, for example, in U.S. Patent Nos. 8,847,005 and 9,043,996 and U.S. Patent Publication No. 2014 / 0245467, the full text of which is incorporated herein by reference for all purposes. In some embodiments, the humanized MHC class II β chain comprises human extracellular domains and cytoplasmic domains of other species. In some embodiments, the class II β chain is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, or HLA-DRB. In some embodiments, the class II β chain is humanized HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, and / or HLA-DRB.

[0575] In some embodiments, the peptides described herein that can be used to target rhabdoviral glycoprotein (G) or functional fragments or derivatives thereof can be generated synthetically or by hydrolysis. Synthetically generated peptides may include randomly generated peptides, specially designed peptides, and peptides in which at least some amino acid positions are conserved in a plurality of peptides while the remaining positions are random. Alternatively, the peptides of this disclosure can be generated by expression in a heterologous host cell.

[0576] In some embodiments, the peptides of this disclosure may have about 5 to about 40 amino acid residues, more preferably about 6 to about 30 amino acid residues, even more preferably about 8 to about 20 amino acid residues, and even more preferably about 9 to 11 amino acid residues. In some embodiments, the peptides may include peptides of any size, for example, with a length between 5 and 200 amino acids in integer increments (i.e., 5, 6, 7, 8, 9…200).

[0577] The peptides disclosed herein may contain one or more reverse peptide bonds, one or more non-peptide bonds, one or more chemical modifications, one or more D-isomers of amino acids, or any combination thereof.

[0578] The peptides described herein may contain one or more (e.g., 1, 2, 3, or 4) amino acid substitutions and / or insertions and / or deletions. An amino acid substitution refers to the replacement of one amino acid residue with another amino acid residue at the same position. Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are adjacent to each other, or may be inserted such that none of the inserted amino acid residues are adjacent to any other inserted amino acid residue.

[0579] The inserted and substituted amino acids can be naturally occurring or non-natural, for example, they may contain non-natural side chains and / or be linked together by non-natural peptide bonds. If multiple amino acid residues are substituted and / or inserted, the substituted / inserted amino acid residues may be the same as or different from each other. Each substituted amino acid may have a different side chain than the amino acid it was substituted.

[0580] It is possible to develop and test a variety of combinations of substitutions / additions / deletions at multiple positions to determine whether the combination will produce an additive or synergistic effect on the peptide.

[0581] The multiple peptides described herein can be operatively linked together. For example, such multi-epitope peptides or polypeptides may comprise 2 to 37, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 single-epitope peptides. These single-epitope peptides can be linked by the connectors described herein or other such connectors known to those skilled in the art to which this disclosure pertains.

[0582] A non-limiting example of a peptide that can be used to practice the present disclosure is peptide 27-24M, which targets the tumor antigen HER2. In some embodiments, peptide 27-24M comprises the amino acid sequence NKFNKGMRGYWGALGGGNGKRGIMGYD (SEQ ID NO: 191), or an amino acid sequence having at least 80% identity with it.

[0583] Recombinant polynucleotides

[0584] In one aspect, this disclosure provides recombinant polynucleotide molecules encoding one or more of the aforementioned polypeptides. In some embodiments, the recombinant polynucleotide molecule may encode the fusion-promoting protein described herein. In some embodiments, the polynucleotide may comprise a sequence encoding a signal peptide sequence, and the signal peptide sequence may be located at the N-terminus of the encoded recombinant fusion-promoting protein. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA.

[0585] In one embodiment, this document provides a recombinant polynucleotide that is an RNA molecule comprising a nucleotide sequence that serves as a template for a positive transcript encoding the recombinant fusion-promoting protein described herein. In some embodiments, the positive transcript may comprise a sequence encoding a signal peptide sequence, and the signal peptide sequence may be located at the N-terminus of the encoded recombinant fusion-promoting protein.

[0586] In one embodiment, this document provides a recombinant polynucleotide, which is an RNA molecule comprising: a nucleotide sequence serving as a template for a positive transcript encoding a vesicular stomatitis virus (VSV) nucleoprotein (N) polypeptide or a functional fragment or derivative thereof; a nucleotide sequence serving as a template for a positive transcript encoding a VSV phosphoprotein (P) polypeptide or a functional fragment or derivative thereof; a nucleotide sequence serving as a template for a positive transcript encoding a VSV matrix (M) polypeptide or a functional fragment or derivative thereof; a nucleotide sequence serving as a template for a positive transcript encoding the fusionin described herein; and a nucleotide sequence serving as a template for a positive transcript encoding a VSV large protein (L) polypeptide or a functional fragment or derivative thereof.

[0587] In some embodiments, the VSV nucleoprotein (N) polypeptide comprises the amino acid sequence of SEQ ID NO: 180, 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 with SEQ ID NO: 180. In some embodiments, the nucleotide sequence encoding the VSV nucleoprotein (N) polypeptide comprises the amino acid sequence encoding SEQ ID NO: 180 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 with SEQ ID NO: 180. In some embodiments, the nucleotide sequence encoding the VSV nucleoprotein (N) polypeptide comprises the nucleotide sequence of SEQ ID NO: 181, or a nucleotide sequence 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: 181. In some embodiments, the VSV nucleoprotein (N) polypeptide comprises the amino acid sequence of SEQ ID NO: 180. In some embodiments, the nucleotide sequence encoding the VSV nucleoprotein (N) polypeptide comprises the nucleotide sequence of SEQ ID NO: 181.

[0588] In some embodiments, the VSV nucleoprotein (N) polypeptide comprises the amino acid sequence of SEQ ID NO: 180 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 180.

[0589] In some embodiments, the VSV phosphoprotein (P) polypeptide comprises the amino acid sequence of SEQ ID NO: 182, 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 with SEQ ID NO: 182. In some embodiments, the nucleotide sequence encoding the VSV phosphoprotein (P) polypeptide comprises the amino acid sequence encoding SEQ ID NO: 182 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 with SEQ ID NO: 182. In some embodiments, the nucleotide sequence encoding the VSV phosphoprotein (P) polypeptide comprises the nucleotide sequence of SEQ ID NO: 183, or a nucleotide sequence 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: 183. In some embodiments, the VSV phosphoprotein (P) polypeptide comprises the amino acid sequence of SEQ ID NO: 182. In some embodiments, the nucleotide sequence encoding the VSV phosphoprotein (P) polypeptide comprises the nucleotide sequence of SEQ ID NO: 183.

[0590] In some embodiments, the VSV phosphoprotein (P) polypeptide comprises the amino acid sequence of SEQ ID NO: 182 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 182.

[0591] In some embodiments, the VSV M peptide is a mutant VSV M peptide. In some embodiments, the mutant VSV M peptide contains a mutation at methionine (M)51. In some embodiments, the mutation at methionine (M)51 is a substitution of methionine (M) with arginine (R). In some embodiments, the mutant VSV M peptide may contain a deletion at methionine (M)51 (ΔM51).

[0592] In some embodiments, the wild-type VSV matrix (M) polypeptide comprises the amino acid sequence of SEQ ID NO: 138, 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 with SEQ ID NO: 138. In some embodiments, the nucleotide sequence encoding the wild-type VSV matrix (M) polypeptide comprises the amino acid sequence encoding SEQ ID NO: 138 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 with SEQ ID NO: 138. In some embodiments, the nucleotide sequence encoding the wild-type VSV matrix (M) polypeptide comprises the nucleotide sequence of SEQ ID NO: 139, or a nucleotide sequence 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: 139. In some embodiments, the wild-type VSV matrix (M) polypeptide comprises the amino acid sequence of SEQ ID NO: 138. In some embodiments, the nucleotide sequence encoding the wild-type VSV matrix (M) polypeptide comprises the nucleotide sequence of SEQ ID NO: 139.

[0593] In some embodiments, the VSV M polypeptide comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 138.

[0594] In some embodiments, the mutant VSV matrix (M) polypeptide M51R comprises the amino acid sequence of SEQ ID NO: 140, 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 with SEQ ID NO: 140. In some embodiments, the nucleotide sequence encoding the mutant VSV matrix (M) polypeptide M51R comprises the amino acid sequence encoding SEQ ID NO: 140 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 with SEQ ID NO: 140. In some embodiments, the nucleotide sequence encoding the mutant VSV matrix (M) polypeptide M51R comprises the nucleotide sequence of SEQ ID NO: 141, or a nucleotide sequence 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: 141. In some embodiments, the mutant VSV matrix (M) polypeptide M51R comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, the nucleotide sequence encoding the mutant VSV matrix (M) polypeptide M51R comprises the nucleotide sequence of SEQ ID NO: 141.

[0595] In some embodiments, the VSV M polypeptide comprises the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 104.

[0596] In some embodiments, the mutant VSV matrix (M) polypeptide ΔM51 comprises the amino acid sequence of SEQ ID NO: 142, 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 with SEQ ID NO: 142. In some embodiments, the nucleotide sequence encoding the mutant VSV matrix (M) polypeptide ΔM51 comprises the amino acid sequence encoding SEQ ID NO: 142 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 with SEQ ID NO: 142. In some embodiments, the nucleotide sequence encoding the mutant VSV matrix (M) polypeptide ΔM51 comprises the nucleotide sequence of SEQ ID NO: 143, or a nucleotide sequence 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: 143. In some embodiments, the mutant VSV matrix (M) polypeptide ΔM51 comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the nucleotide sequence encoding the mutant VSV matrix (M) polypeptide ΔM51 comprises the nucleotide sequence of SEQ ID NO: 143.

[0597] In some embodiments, the VSV M polypeptide comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 142.

[0598] In some embodiments, the VSV large protein (L) polypeptide comprises the amino acid sequence of SEQ ID NO: 184, 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 with SEQ ID NO: 184. In some embodiments, the nucleotide sequence encoding the VSV large protein (L) polypeptide comprises an amino acid sequence encoding SEQ ID NO: 184 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 with SEQ ID NO: 184. In some embodiments, the nucleotide sequence encoding the VSV large protein (L) polypeptide comprises the nucleotide sequence of SEQ ID NO: 185, or a nucleotide sequence 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: 185. In some embodiments, the VSV large protein (L) polypeptide comprises the amino acid sequence of SEQ ID NO: 184. In some embodiments, the nucleotide sequence encoding the VSV large protein (L) polypeptide comprises the nucleotide sequence of SEQ ID NO: 185.

[0599] In some embodiments, the VSV large protein (L) polypeptide comprises the amino acid sequence of SEQ ID NO: 184 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 184.

[0600] In some embodiments, the polynucleotide encoding the polypeptide disclosed herein may include one or more regulatory elements. These regulatory elements may be capable of regulating polypeptide expression. Non-limiting examples of regulatory elements include promoters, initiation sites, polyadenylation (polyA) tails, IRES elements, enhancers, responsive elements, and termination signals. In some embodiments, the promoter is an inducible promoter.

[0601] The flanking regions of the nucleic acid inserted into the VSV genome can be intragenic regions of the virus, which contain the gene transcription start and stop codons required for the viral polymerase to transcribe the inserted nucleic acid sequence.

[0602] In some implementations, the polynucleotides described herein are optimized for expression in human cells.

[0603] Recombinant pseudotyped viruses and cell-derived nanovesicles

[0604] In some respects, this disclosure provides a recombinant pseudovirus or cell-derived nanovesicle comprising one or more of the recombinant fusion-promoting proteins or recombinant polynucleotides described herein.

[0605] In some embodiments, the recombinant fusion-promoting protein forms a chimeric trimer with one or two different fusion-promoting proteins on the surface of a recombinant pseudovirus or cell-derived nanovesicle. In some embodiments, the chimeric trimer comprises (i) at least one fusion-promoting protein described herein, and (ii) a wild-type rhabdovirus glycoprotein and / or a recombinant fusion-promoting protein comprising a rhabdovirus glycoprotein or a functional fragment or derivative thereof, and a targeting molecule. These chimeric trimers may be distributed on the surface of the recombinant pseudovirus or cell-derived nanovesicle such that all trimers contain at least one targeting molecule, or not all trimers contain at least one targeting molecule. In the latter case, some trimers may exhibit one targeting molecule, others may exhibit two targeting molecules, and still others may not. In some embodiments, the chimeric trimer may comprise (i) at least one fusion-promoting protein described herein, and (ii) a fusion-promoting protein comprising a rhabdovirus glycoprotein or a functional fragment or derivative thereof, but without a targeting molecule. In some embodiments, the chimeric trimer described herein may comprise two or more different recombinant fusion-promoting proteins described herein.

[0606] In some embodiments, the fusion-promoting protein described herein may comprise a fusion-promoting protein containing the sequence SEQ ID NO: 8, wherein there are amino acid substitutions and / or deletions at one or more positions selected from K47, R354, H8 and Y209.

[0607] In some embodiments, the recombinant pseudovirus or cell-derived nanovesicles described herein may comprise a chimeric trimer comprising (i) one or two monomers of a first fusion-promoting protein, wherein the first fusion-promoting protein comprises a rhabdoviral glycoprotein or a functional fragment or derivative thereof; and a target molecule or a functional fragment or derivative thereof; and (ii) one or two monomers of a second fusion-promoting protein, wherein the second fusion-promoting protein comprises a rhabdoviral glycoprotein or a functional fragment or derivative thereof, but does not comprise a target molecule.

[0608] In some embodiments, the targeting molecule can be linked to a rhabdoviral glycoprotein via a linker. In some embodiments, the linker is insensitive to proteolytic cleavage via endogenous or exogenously added proteases. In some embodiments, the linker is sensitive to proteolytic cleavage via endogenous or exogenously added proteases. In some embodiments, the linker can be any of the various linkers described herein.

[0609] In some embodiments, the first fusion-promoting protein and / or the second fusion-promoting protein may comprise a rhabdoviral glycoprotein comprising the sequence SEQ ID NO: 8 and having amino acid substitutions and / or deletions at one or more positions selected from K47, R354, H8 and Y209.

[0610] Cell-derived nanovesicles can be natural or engineered nanoscale vesicles that can carry a variety of biomaterials, such as proteins, polynucleotides, or lipids. One example of a cell-derived nanovesicle is the gesicle. Gesicles are cell-derived nanovesicles released from the cytoplasmic membrane and are typically generated through overexpression of the vesicular stomatitis virus G (VSV-G) glycoprotein (Campbell, LA et al., Mol. Ther., 2019, 27, pp. 151-163; Mangeot, PE et al., Mol. Ther., 2011, 19, pp. 1656-1666, both incorporated herein by reference in their entirety). With an average diameter of 100 nanometers, gesicles are heterogeneous in size, shape, and payload, and can be used for the rapid and direct transfer of membrane proteins, cytoplasmic proteins, and nuclear proteins to recipient cells. Gesicles could be a viable method for delivering genome editing agents, such as CRISPR / Cas9 ribonucleoproteins (RNPs), to target cells. Unlike gene delivery via viral vectors, protein delivery via gesicles allows for rapid intracellular transfer of function without involving transcriptional mechanisms or any viral integration processes that might limit viral transduction in specific cell types.

[0611] Another example of cell-derived nanovesicles is the Nanoblade. Nanoblades are engineered particles loaded with genome editing agents (such as Cas9-gRNA ribonucleoprotein (RNP)) (Mangeot, PE et al., Nat. Commun., 2019, 45, pp. 1-15; Gutierrez-Guerrero, A. et al., Front. Genome Ed., 2021, 3, pp. 1-21, both articles are incorporated herein by reference in their entirety). Nanoblades are formed when viral structural proteins (such as the MLV protein Gag) polymerize and spontaneously assemble into particles on the cell membrane. These particles can incorporate one or more guide RNAs by associating with Cas9 and act as delivery agents into target cells. To alter the cytotropism of Nanoblades, different viral envelope proteins can be expressed to form pseudo-Nanoblades. For example, Nanoblade pseudotyped with a mixture of VSV-G and baboon endogenous retrovirus Rless glycoprotein (BaEVRless) has been shown to have a high transduction rate in recipient cells (Mangeot, PE et al., Nat. Commun., 2019, 45, p. 1-15).

[0612] In one aspect, this disclosure also provides a recombinant pseudovirus or cell-derived nanovesicle comprising glycoproteins or functional fragments or derivatives thereof from various viruses. In some embodiments, the recombinant pseudovirus is a rhabdovirus, such as vesicular stomatitis virus (VSV). In some embodiments, the rhabdovirus may be replicative. In some embodiments, the rhabdovirus may be replication-deficient. In some embodiments, the rhabdovirus may be non-replicating.

[0613] In some embodiments, the recombinant pseudovirus is a retrovirus, such as a lentivirus (LV). In some embodiments, the LV may be replicative. In some embodiments, the LV may be replication-deficient. In some embodiments, the LV may be non-replicative. In some embodiments, the LV described herein may not contain the gp120 surface envelope protein and / or the gp41 transmembrane envelope protein. In some embodiments, the LV described herein may contain a mutant gp120 surface envelope protein and / or a mutant gp41 transmembrane envelope protein. In some embodiments, the LV described herein may not bind to cells, for example, in the absence of the TCR described herein.

[0614] In some embodiments, the recombinant pseudoviruses described herein may include, for example, but not limited to, components from viruses selected from: human immunodeficiency virus (e.g., HIV-1 or HIV-2), bovine immunodeficiency virus (BIV), feline immunodeficiency virus (FIV), simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV), mouse stem cell virus (MSCV), murine leukosis virus (MLV), avian leukosis virus (ALV), feline leukosis virus (FLV), bovine leukosis virus (BLV), human T-lymphocyte virus (HTLV), feline sarcoma virus, avian reticuloendotheliosis virus, caprine arthritis encephalitis virus (CAEV), and Visna-Maedi virus (VMV).

[0615] In some implementations, the recombinant pseudoviruses or cell-derived nanovesicles of this disclosure may be derived from viruses of the Retroviridae family.

[0616] In some embodiments, the recombinant pseudoviruses or cell-derived nanovesicles of this disclosure may comprise glycoproteins derived from rhabdoviruses, such as, but not limited to, vesicular stomatitis virus glycoprotein (VSV-G), Flanders virus glycoprotein (FLAV-G), Chandipura virus glycoprotein (CHPV-G), Perinet virus glycoprotein (PERV-G), Piry virus glycoprotein (PIRYV-G), Fukuoka virus glycoprotein (FUKV-G), Joinjakaka virus glycoprotein (JOIV-G), Kumasi virus glycoprotein (KRV-G), Isfahan glycoprotein (ISFV-G), Jurona virus glycoprotein (JURV-G), Mediterranean Bat glycoprotein (MBV-G), Malpais Spring glycoprotein (MSPV-G), Radi glycoprotein (RADV-G), Rhinolophus affinis-G, Yug Bugdanavoc glycoprotein (YBV-G), Yinshui Bat glycoprotein (YSBV-G), Keuraliba glycoprotein (KEUV-G), Kimberley glycoprotein (KIMV-G), Kanyawara glycoprotein (KYAV-G), LaJoya glycoprotein (LJV-G), Mosquiero glycoprotein (MQOV-G), Parry Creek glycoprotein (PCV-G), Bas Congo glycoprotein (BASV-G), bovine transient heat glycoprotein (BEFV-G), Curionopolis glycoprotein (CURV-G), Drosophila melanogaster Sigma virus glycoprotein (DMelSV-G), Niakha glycoprotein (NIAV-G), Puerto almandras glycoprotein (PTAMV-G), or Tupaia rhabdovirus (TUPTV-G), or functional fragments or derivatives thereof. Non-limiting examples of the amino acid sequences of rhabdovirus G described herein are shown in SEQ ID NO: 8-15, 17, 60-108, 157.

[0617] In one embodiment, the recombinant pseudovirus or cell-derived nanovesicles of this disclosure comprise a glycoprotein (FLAV-G) from Flanders virus, or a functional fragment or derivative thereof. In some embodiments, FLAV-G comprises the amino acid sequence of SEQ ID NO: 9 or 81, or a functional fragment or derivative thereof having sequence identity with SEQ ID NO: 9 or 81 of 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, the nucleotide sequence encoding FLAV-G comprises the amino acid sequence encoding 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 with SEQ ID NO: 9 or 81. In some embodiments, FLAV-G comprises the amino acid sequence of SEQ ID NO: 9 or 81.

[0618] In some embodiments, the recombinant pseudovirus or cell-derived nanovesicles described herein comprise a rhabdoviral glycoprotein from FLAV-G. In some embodiments, FLAV-G comprises the sequence SEQ ID NO: 9. In some embodiments, FLAV-G consists of the sequence SEQ ID NO: 9.

[0619] In one embodiment, the recombinant pseudovirus or cell-derived nanovesicles of this disclosure comprise a glycoprotein (CHPV-G) from Chandipura virus, or a functional fragment or derivative thereof. In some embodiments, CHPV-G comprises the amino acid sequence of SEQ ID NO: 10 or 82, or a 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: 10 or 82. In some embodiments, the nucleotide sequence encoding CHPV-G comprises the amino acid sequence encoding 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...

Claims

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

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

3. The recombinant fusion-promoting protein of claim 1, wherein the linker comprises a sequence selected from: 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).

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

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

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

8.

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

8.

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

9. The recombinant fusion-promoting protein of any one of claims 5-8, wherein the VSV-G or its functional fragment or derivative comprises one or more mutations, wherein the one or more mutations reduce or eliminate the binding of the VSV-G polypeptide or its functional fragment or derivative to LDLR.

10. The recombinant fusion-promoting protein of claim 9, wherein the one or more mutations in the VSV-G or its functional fragments or derivatives 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. The recombinant fusion-promoting 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. The recombinant fusion-promoting 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. The recombinant fusion-promoting 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 the H8 position.

14. The recombinant fusion-promoting protein of claim 10, wherein the one or more mutations in the VSV-G or its functional fragments or derivatives comprise the deletion of one or more amino acids at positions corresponding to H8, K47, Y209 or R354 in SEQ ID NO:

8.

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

8.

16. The recombinant fusion-promoting protein of claim 15, wherein the one or more deletions are deletions at the K47 position.

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

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

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

20. The recombinant fusion-promoting protein of claim 19, wherein the one or more viral titer-enhancing mutations in said VSV-G or its functional fragments or derivatives are M184T and / or F250L as specified at the position in SEQ ID NO:

8.

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

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

9.

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

9.

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

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

10.

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

10.

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

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

11.

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

11.

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

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

12.

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

12.

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

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

13.

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

13.

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

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

14.

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

14.

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

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

15.

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

15.

42. The recombinant fusion-promoting protein of any one of claims 1-4, wherein the rhabdoviral glycoprotein is a glycoprotein (KEUV-G) derived from Keuraliba virus.

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

17.

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

17.

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

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

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

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

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

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

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

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

54. The recombinant fusion-promoting protein of claim 52, wherein the fusionin comprises or consists of the sequence SEQ ID NO: 8, and contains an amino acid deletion at positions H8 and K47.

55. The recombinant fusion-promoting protein of claim 52, wherein the fusionin comprises the sequence SEQ ID NO: 8 and contains an amino acid deletion at (i) K47, (ii) R354 and (iii) H8 or Y209.

56. The recombinant fusion-promoting protein of claim 55, wherein the fusionin consists of the sequence SEQ ID NO: 8 and contains an amino acid deletion at (i) K47, (ii) R354 and (iii) H8 or Y209.

57. The recombinant fusion-promoting protein of claim 52, wherein the fusionin comprises the sequence SEQ ID NO: 8 and contains an amino acid deletion at position K47.

58. The recombinant fusion-promoting protein of claim 57, wherein the fusionin consists of the sequence SEQ ID NO: 8 and contains an amino acid deletion at position K47.

59. A recombinant fusion-promoting protein comprising a fusionin comprising the sequence SEQ ID NO: 8, and comprising an amino acid substitution at (i) K47, (ii) R354 and (iii) H8 or Y209.

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

61. A recombinant fusion-promoting protein comprising a fusionin, the fusionin comprising the sequence SEQ ID NO: 8, and comprising amino acid substitutions at positions K47, R354, H8 and Y209.

62. The recombinant fusion-promoting protein of claim 61, wherein the fusionin comprises the sequence SEQ ID NO: 8 and contains amino acid substitutions at positions K47, R354, H8 and Y209.

63. The recombinant fusion-promoting protein of any one of claims 51-62, wherein the fusionin or its functional fragment or derivative further comprises one or more mutations that increase viral titer.

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

8.

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

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

67. The recombinant fusion-promoting protein of claim 66, wherein the linker is sensitive to proteolytic cleavage via endogenous or exogenously added proteases.

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

69. The recombinant fusion-promoting protein of claim 67, wherein the linker comprises a sequence selected from: 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).

70. The recombinant fusion-promoting protein of claim 66, wherein the linker is insensitive to proteolytic cleavage via endogenous or exogenously added proteases.

71. The recombinant fusion-promoting protein of any one of claims 65-70, wherein the N-terminus of the fusionin to which the targeting molecule is attached, or a functional fragment or derivative thereof, does not contain one or more amino acids present at the N-terminus of the mature wild-type fusionin.

72. A recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises: (i) glycoprotein (FLAV-G) from Flanders virus or its functional fragments or derivatives, and (ii) Targeting molecules.

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

9.

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

9.

75. A recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises: (i) glycoprotein (CHPV-G) from Chandipura virus or its functional fragments or derivatives, and (ii) Targeting molecules.

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

10.

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

10.

78. A recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises: (i) glycoprotein (PERV-G) from the Perinet virus or its functional fragments or derivatives, and (ii) Targeting molecules.

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

11.

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

11.

81. A recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises: (i) glycoprotein (PIRYV-G) from Piry virus or its functional fragments or derivatives, and (ii) Targeting molecules.

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

12.

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

12.

84. A recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises: (i) A glycoprotein (FUKV-G) or a functional fragment or derivative thereof derived from Fukuoka virus, and (ii) Targeting molecules.

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

13.

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

13.

87. A recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises: (i) glycoprotein (JOIV-G) or its functional fragments or derivatives derived from Joinjakaka virus, and (ii) Targeting molecules.

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

14.

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

14.

90. A recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises: (i) Glycoprotein (KRV-G) from Kumasi virus or its functional fragments or derivatives, and (ii) Targeting molecules.

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

15.

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

15.

93. A recombinant fusion-promoting protein, wherein the fusion-promoting protein comprises: (i) A glycoprotein (KEUV-G) or a functional fragment or derivative thereof derived from the Keuraliba virus, and (ii) Targeting molecules.

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

17.

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

17.

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

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

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

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

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

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

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

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

104. The recombinant fusion-promoting protein of claim 103, wherein the linker is sensitive to proteolytic cleavage via endogenous or exogenously added proteases.

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

106. The recombinant fusion-promoting protein of claim 104, wherein the linker comprises a sequence selected from: 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).

107. The recombinant fusion-promoting protein of claim 103, wherein the linker is insensitive to proteolytic cleavage via endogenous or exogenously added proteases.

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

109. The recombinant fusion protein of any one of claims 1-50 and 65-108, wherein the targeting molecule is an antibody or an antigen-binding fragment thereof, an affinity compound, 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. The recombinant fusion protein of claim 109, wherein the antibody or its antigen-binding fragment is a single-chain variable region fragment (scFv), a biantibody, a microantibody, a nanobody, a single-domain antibody (sdAb), or a single-heavy-chain antibody.

111. The recombinant fusion-promoting 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 a recombinant fusion-promoting protein of any one of claims 1-111.

113. The recombinant polynucleotide of claim 112, wherein the polynucleotide comprises a sequence encoding a signal peptide sequence, wherein such signal sequence is located at the N-terminus of the encoded recombinant fusion protein.

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

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

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

117. The recombinant polynucleotide of claim 116, wherein the positive transcript comprises a sequence encoding a signal peptide sequence, wherein such signal sequence is located at the N-terminus of the encoded recombinant fusion protein.

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

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

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

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

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

123. A composition comprising the recombinant polynucleotide of any one of claims 112-122, and a carrier and / or excipient.

124. A host cell comprising the recombinant polynucleotide of any one of claims 112-122.

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

126. A recombinant pseudovirus or cell-derived nanovesicle comprising one or more recombinant fusion-promoting proteins according to any one of claims 1-111.

127. The recombinant pseudovirus or cell-derived nanovesicle of claim 126, comprising two or more different recombinant fusion-promoting proteins of any one of claims 1-111.

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

129. The recombinant pseudovirus or cell-derived nanovesicle of claim 128, wherein the chimeric trimer comprises (i) at least one fusion-promoting protein of any one of claims 1-111, and (ii) a fusion-promoting protein comprising a rhabdoviral glycoprotein or a functional fragment or derivative thereof and free of a targeting molecule.

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

131. A recombinant pseudovirus or cell-derived nanovesicle comprising a chimeric trimer comprising: (i) one or two monomers of a first fusion-promoting protein, wherein the first fusion-promoting protein comprises a rhabdoviral 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-promoting protein, wherein the second fusion-promoting protein comprises a rhabdoviral glycoprotein or a functional fragment or derivative thereof, but does not contain a targeting molecule.

132. The recombinant pseudovirus or cell-derived nanovesicle of claim 131, wherein in the first fusion-promoting protein, the targeting molecule is linked to the rhabdovirus glycoprotein via a linker.

133. The recombinant pseudovirus or cell-derived nanovesicle of claim 132, wherein the adapter is insensitive to proteolytic cleavage via endogenous or exogenously added proteases.

134. The recombinant pseudovirus or cell-derived nanovesicle of claim 132, wherein the adapter is sensitive to proteolytic cleavage via endogenous or exogenously added proteases.

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

136. The recombinant pseudovirus or cell-derived nanovesicle of claim 135, wherein the first fusion-promoting protein and / or the second fusion-promoting protein comprises a rhabdoviral glycoprotein, the rhabdoviral glycoprotein comprising the sequence shown in any one of claims 10-18 or 52-54.

137. A recombinant pseudovirus or cell-derived nanovesicle comprising a glycoprotein (FLAV-G) from Flanders virus or a functional fragment or derivative thereof.

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

9.

139. The recombinant pseudovirus or cell-derived nanovesicle of claim 138, wherein the FLAV-G comprises the sequence SEQ ID NO:

9.

140. A recombinant pseudovirus or cell-derived nanovesicle comprising a glycoprotein (CHPV-G) from Chandipura virus or a functional fragment or derivative thereof.

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

10.

142. The recombinant pseudovirus or cell-derived nanovesicle of claim 141, wherein the CHPV-G comprises the sequence SEQ ID NO:

10.

143. A recombinant pseudovirus or cell-derived nanovesicle comprising a glycoprotein (PERV-G) from Perinet virus or a functional fragment or derivative thereof.

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

11.

145. The recombinant pseudovirus or cell-derived nanovesicle of claim 144, wherein the PERV-G comprises the sequence SEQ ID NO:

11.

146. A recombinant pseudovirus or cell-derived nanovesicle comprising a glycoprotein (PIRYV-G) from Piry virus or a functional fragment or derivative thereof.

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

12.

148. The recombinant pseudovirus or cell-derived nanovesicle of claim 147, wherein the PIRYV-G comprises the sequence SEQ ID NO:

12.

149. A recombinant pseudovirus or cell-derived nanovesicle comprising a glycoprotein (FUKV-G) from Fukuoka virus or a functional fragment or derivative thereof.

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

13.

151. The recombinant pseudovirus or cell-derived nanovesicle of claim 150, wherein the FUKV-G comprises the sequence SEQ ID NO:

13.

152. A recombinant pseudovirus or cell-derived nanovesicle comprising a glycoprotein (JOIV-G) from Joinjaka virus or a functional fragment or derivative thereof.

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

14.

154. The recombinant pseudovirus or cell-derived nanovesicle of claim 153, wherein the JOIV-G comprises the sequence SEQ ID NO:

14.

155. A recombinant pseudovirus or cell-derived nanovesicle comprising a glycoprotein (KRV-G) from Kumasi virus or a functional fragment or derivative thereof.

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

15.

157. The recombinant pseudovirus or cell-derived nanovesicle of claim 156, wherein the KRV-G comprises the sequence SEQ ID NO:

15.

158. A recombinant pseudovirus or cell-derived nanovesicle comprising a glycoprotein (KEUV-G) from Keuraliba virus or a functional fragment or derivative thereof.

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

17.

160. The recombinant pseudovirus or cell-derived nanovesicle of claim 159, wherein the KEUV-G comprises the sequence SEQ ID NO:

17.

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

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

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

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

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

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

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

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

169. The recombinant pseudovirus of any one of claims 125-166, wherein the virus is a retrovirus.

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

171. A recombinant pseudovirus of any one of claims 125-170, wherein the virus has the ability to replicate.

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

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

174. The recombinant pseudovirus 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 one or more components thereof, an antisense oligonucleotide, a ribozyme, or an RNAi molecule.

175. The recombinant pseudovirus 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 one or more components thereof, an antisense oligonucleotide, a ribozyme, or an RNAi molecule.

176. The recombinant pseudovirus or cell-derived nanovesicle of claim 173, wherein the molecular cargo is a gene-edited ribonucleoprotein complex or a component thereof.

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

178. A composition comprising a recombinant pseudovirus or cell-derived nanovesicle of any one of claims 125-177, and a carrier and / or excipient.

179. A method for reducing the sensitivity of a subject in need to serum neutralization of a recombinant virus or nanovesicles, comprising administering to the subject the recombinant pseudovirus or cell-derived nanovesicles of any one of claims 125-177 or the composition of claim 178.

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

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

182. The method of claim 181, wherein the method does not include pretreatment with a drug that lowers LDL / VLDL.

183. The method of claim 181, wherein the method further comprises pretreatment with a drug that lowers LDL / VLDL.

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

185. A method of delivering molecular cargo to cells in a subject in need, comprising administering to the subject an effective amount of a recombinant pseudovirus or cell-derived nanovesicle of any one of claims 125-177, or a composition comprising the pseudovirus or cell-derived nanovesicle and a carrier and / or excipient, wherein the recombinant fusion-promoting protein within the recombinant pseudovirus or cell-derived nanovesicle comprises a targeting molecule targeting the cells.

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

187. A method of delivering molecular cargo ex vivo to cells, comprising administering to the cells an effective amount of a recombinant pseudovirus or cell-derived nanovesicle of any one of claims 125 to 177, or a composition comprising the pseudovirus or cell-derived nanovesicle and a carrier and / or excipient, wherein the recombinant fusion-promoting protein within the recombinant pseudovirus or cell-derived nanovesicle comprises a targeting molecule targeting the cells.

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