Recombinant rhabdovirus encoding interleukin-12 (il-12)

Abstract

The application relates to a recombinant virus comprising a rhabdovirus genome, e.g., a vesicular stomatitis virus (VSV) genome, and a nucleotide sequence encoding a recombinant protein comprising an interleukin-12 (IL-12) polypeptide, or a functional fragment or derivative thereof. In various aspects, the IL-12 polypeptide, or the functional fragment or derivative thereof, may be membrane bound. In various other aspects, the IL-12 polypeptide, or the functional fragment or derivative thereof, may not be membrane bound, e.g., may be a soluble IL-12 polypeptide, or a functional fragment or derivative thereof. The application further relates to recombinant polynucleotides and recombinant vectors encoding virus polypeptides and the recombinant protein, as well as to host cells and methods for preparing host cells that express the polypeptides. The application further relates to pharmaceutical compositions comprising the recombinant virus, and to methods for treating cancer, e.g., a tumor, using the recombinant virus.

Description

RECOMBINANT RHABDOVIRUS ENCODING INTERLEUKIN- 12 (IL-12)CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 570,881, filed March 28, 2024, and U.S. Provisional Application No. 63 / 660,073, filed June 14, 2024, the disclosure of each of which is incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 27, 2025, is named 250298_00081 l_SL.xml and is 726,236 bytes in size.FIELD

[0003] The application provides compositions and methods related to a recombinant virus comprising a rhabdovirus genome, e.g., a vesicular stomatitis virus (VSV) genome, and a nucleotide sequence encoding a recombinant protein comprising, e.g., an interleukin- 12 (IL-12) polypeptide, or a functional fragment or derivative thereof. In various aspects, the IL- 12 polypeptide, or the functional fragment or derivative thereof, may be membrane bound. In various other aspects, the IL- 12 polypeptide, or the functional fragment or derivative thereof, may not be membrane bound, e.g., the IL-12 polypeptide, or the functional fragment or derivative thereof, may be a soluble IL- 12 polypeptide, or a functional fragment or derivative thereof.BACKGROUND

[0004] Oncolytic virus therapy is an anti-cancer approach that uses viruses to selectively infect and kill cancer cells without harming healthy cells. Vesicular stomatitis virus (VSV), a member of the Rhabdoviridae family, can be a potential oncolytic virus. Oncolytic viruses encoding cytokines have demonstrated strong anti-tumor effects in preclinical models of cancers. Still, systemic accumulation of cytokines after delivery by oncolytic viruses remains potentially lethal to patients. There is a need for approaches to improve properties of cytokines for use in cancer immunotherapy, for example, oncolytic virus therapy.SUMMARY

[0005] Disclosed herein is a recombinant virus comprising a rhabdovirus genome, for example, a vesicular stomatitis virus (VSV) genome, and a recombinant protein comprising, e.g., a nucleotide sequence encoding an IL- 12 polypeptide, or a functional fragment or derivative thereof. In various aspects, the recombinant protein can comprise a transmembrane domain and can, e.g., be membrane bound. In various other aspects, the recombinant protein may not comprise a transmembrane domain and may not be membrane bound (i.e., the recombinant protein may be a soluble and / or secreted protein). Further disclosed are recombinant polynucleotides and recombinant vectors encoding virus polypeptides, as well as host cells that can express the polypeptides and methods for preparing the host cells. Additional disclosure relates to pharmaceutical compositions comprising the recombinant virus, and to methods for treating cancer, e.g., a tumor, and for inducing cancer regression, in a subject in need thereof, using the disclosed recombinant virus.

[0006] In one aspect, provided herein is a recombinant rhabdovirus comprising a nucleotide sequence encoding a recombinant protein, wherein the recombinant protein comprises an IL- 12 polypeptide, or a functional fragment or derivative thereof.

[0007] In some embodiments, the recombinant rhabdovirus comprises a rhabdovirus genome, said rhabdovirus genome comprising a gene encoding a fusogenic polypeptide, or a functional fragment or derivative thereof, and a gene encoding a large protein (L) polypeptide, or a functional fragment or derivative thereof, and the nucleotide sequence encoding the recombinant protein is positioned between the gene encoding the fusogenic polypeptide, or a functional fragment or derivative thereof, and the gene encoding the L polypeptide, or a functional fragment or derivative thereof.

[0008] In some embodiments, the nucleotide sequence encoding the recombinant protein is a template for a positive sense transcript encoding said recombinant protein.

[0009] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises an IL-12 p40 subunit, or a functional fragment or derivative thereof.

[0010] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, further comprises an IL-12 p35 subunit, or a functional fragment or derivative thereof.

[0011] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises an IL-12 p35 subunit, or a functional fragment or derivative thereof.

[0012] In some embodiments, the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence which has at least 80% sequence identity thereto.

[0013] In some embodiments, the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 69.

[0014] In some embodiments, the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 69.

[0015] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence which has at least 80% sequence identity thereto.

[0016] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61.

[0017] In some embodiments, the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 61.

[0018] In some embodiments, the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence which has at least 80% sequence identity thereto.

[0019] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 71.

[0020] In some embodiments, the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 71.

[0021] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence which has at least 80% sequence identity thereto.

[0022] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63.

[0023] In some embodiments, the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 63.

[0024] In some embodiments, the IL-12 p40 subunit, or the functional fragment or derivative thereof, and the IL- 12 p35 subunit, or the functional fragment or derivative thereof, are connected by a linker.

[0025] In some embodiments, the linker is from about 5 amino acids to about 30 amino acids in length.

[0026] In some embodiments, the linker comprises the amino acid sequence (VPGVG) (SEQ ID NO: 17).

[0027] In some embodiments, the linker consists of the amino acid sequence (VPGVG)2 (SEQ ID NO: 17).

[0028] In some embodiments, the recombinant protein further comprises a signal peptide operably linked to the IL- 12 polypeptide.

[0029] In some embodiments, the signal peptide is operably linked to the IL-12 p40 subunit, or the functional fragment or derivative thereof, and / or the IL-12 p35 subunit, or the functional fragment or derivative thereof.

[0030] In some embodiments, the signal peptide is operably linked to the IL-12 p40 subunit, or the functional fragment or derivative thereof, to form an IL- 12 p40 subunit precursor, or a functional fragment or derivative thereof.

[0031] In some embodiments, the signal peptide operably linked to the IL- 12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence that has at least 80% sequence identity thereto.

[0032] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that has at least 80% sequence identity thereto.

[0033] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 27.

[0034] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 27.

[0035] In some embodiments, the IL-12 p40 subunit precursor, or a functional fragment or derivative thereof, comprises one or more amino acid changes at positions E81, F82, K106, or K217 of the amino acid sequence of SEQ ID NO: 27.

[0036] In some embodiments, the one or more amino acid changes comprises one or more alanine (A) substitutions.

[0037] In some embodiments, the signal peptide operably linked to the IL- 12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence that has at least 80% sequence identity thereto.

[0038] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that has at least 80% sequence identity thereto.

[0039] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 12.

[0040] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 12.

[0041] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises one or more amino acid changes at positions E81, F82, KI 06, or K217 of the amino acid sequence of SEQ ID NO: 12.

[0042] In some embodiments, the one or more amino acid changes comprises one or more alanine (A) substitutions.

[0043] In some embodiments, the signal peptide is operably linked to the IL-12 p35 subunit, or the functional fragment or derivative thereof, to form an IL- 12 p35 subunit precursor, or a functional fragment or derivative thereof.

[0044] In some embodiments, the signal peptide operably linked to the IL- 12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that has at least 80% sequence identity thereto.

[0045] In some embodiments, the signal peptide operably linked to the IL- 12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence that has at least 80% sequence identity thereto.

[0046] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence that has at least 80% sequence identity thereto.

[0047] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 159.

[0048] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 159.

[0049] In some embodiments, the IL-12 polypeptide, or a functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 154, or an amino acid sequence that has at least 80% sequence identity thereto.

[0050] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 154.

[0051] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 154.

[0052] In another aspect, provided herein is a recombinant rhabdovirus comprising a nucleotide encoding a recombinant protein comprising an IL- 12 polypeptide, wherein the IL-12 polypeptide comprises an IL-12 p40 subunit, or a functional fragment or derivative thereof, and an IL-12 p35 subunit, or a functional fragment or derivative thereof, wherein the IL-12 p40 subunit, or a functional fragment or derivative thereof, and the IL-12 p35 subunit, or a functional fragment or derivative thereof, are connected by a linker, wherein the recombinant rhabdovirus comprises a rhabdovirus genome, said rhabdovirus genome comprising a gene encoding a fusogenic polypeptide, or a functional fragment or derivative thereof, and a gene encoding a large protein (L) polypeptide, or a functional fragment or derivative thereof, and wherein the nucleotide sequence encoding the recombinant protein is positioned between the gene encoding the fusogenic polypeptide, or a functional fragment or derivative thereof, and the gene encoding the L polypeptide, or a functional fragment or derivative thereof.

[0053] In some embodiments, the nucleotide sequence encoding the recombinant protein is a template for a positive sense transcript encoding said recombinant protein.

[0054] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence which has at least 80% sequence identity thereto.

[0055] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 69.

[0056] In some embodiments, the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 69.

[0057] In some embodiments, the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence which has at least 80% sequence identity thereto.

[0058] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61.

[0059] In some embodiments, the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 61.

[0060] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence which has at least 80% sequence identity thereto.

[0061] In some embodiments, the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 71.

[0062] In some embodiments, the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 71.

[0063] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence which has at least 80% sequence identity thereto.

[0064] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63.

[0065] In some embodiments, the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 63.

[0066] In some embodiments, the linker is from about 5 amino acids to about 30 amino acids in length.

[0067] In some embodiments, the linker comprises the amino acid sequence (VPGVG)2 (SEQ ID NO: 17).

[0068] In some embodiments, the linker consists of the amino acid sequence (VPGVG) (SEQ ID NO: 17).

[0069] In some embodiments, the recombinant protein further comprises a signal peptide operably linked to the IL- 12 p40 subunit, or the functional fragment or derivative thereof, to form an IL- 12 p40 subunit precursor, or a functional fragment or derivative thereof.

[0070] In some embodiments, the signal peptide operably linked to the IL-12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence that has at least 80% sequence identity thereto.

[0071] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that has at least 80% sequence identity thereto.

[0072] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 27.

[0073] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 27.

[0074] In some embodiments, the IL-12 p40 subunit precursor, or a functional fragment or derivative thereof, comprises one or more amino acid changes at positions E81, F82, K106, or K217 of the amino acid sequence of SEQ ID NO: 27.

[0075] In some embodiments, the one or more amino acid changes comprises one or more alanine (A) substitutions.

[0076] In some embodiments, the signal peptide operably linked to the IL- 12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence that has at least 80% sequence identity thereto.

[0077] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that has at least 80% sequence identity thereto.

[0078] In some embodiments, the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 12.

[0079] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 12.

[0080] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises one or more amino acid changes at positions E81, F82, K106, or K217 of the amino acid sequence of SEQ ID NO: 12.

[0081] In some embodiments, the one or more amino acid changes comprises one or more alanine (A) substitutions.

[0082] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence that has at least 80% sequence identity thereto.

[0083] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 159.

[0084] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 159.

[0085] In some embodiments, the IL- 12 polypeptide, or a functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 154, or an amino acid sequence that has at least 80% sequence identity thereto.

[0086] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 154.

[0087] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 154.

[0088] In some embodiments, the recombinant rhabdovirus is a vesiculovirus.

[0089] In some embodiments, the vesiculovirus is a vesicular stomatitis virus (VSV).

[0090] In some embodiments, the rhabdovirus genome further comprises a gene encoding a VSV nucleoprotein (N) polypeptide, or a functional fragment or derivative thereof.

[0091] In some embodiments, the rhabdovirus genome further comprises a gene encoding a VSV phosphoprotein (P) polypeptide, or a functional fragment or derivative thereof.

[0092] In some embodiments, the rhabdovirus genome further comprises a gene encoding a VSV matrix (M) polypeptide, or a functional fragment or derivative thereof.

[0093] In some embodiments, the L polypeptide is a VSV large protein (L) polypeptide, or a functional fragment or derivative thereof.

[0094] In some embodiments, the rhabdovirus genome comprises a gene encoding a VSV nucleoprotein (N) polypeptide, or a functional fragment or derivative thereof, a gene encoding a VSV phosphoprotein (P) polypeptide, or a functional fragment or derivative thereof, a gene encoding a VSV matrix (M) polypeptide, or a functional fragment or derivative thereof, a gene encoding a fusogenic polypeptide, or a functional fragment or derivative thereof, and a gene encoding a VSV large protein (L) polypeptide, or a functional fragment or derivative thereof.

[0095] In some embodiments, the recombinant rhabdovirus comprises an RNA molecule, wherein the RNA molecule comprises a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, or a functional fragment or derivative thereof, and a nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein.

[0096] In some embodiments, the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein is positioned between the nucleotide sequence that is a template for a positive sense transcript encoding the fusogenic polypeptide, or the functional fragment or derivative thereof, and the nucleotide sequence that is a template for a positive sense transcript encoding the VSV L polypeptide, or the functional fragment or derivative thereof.

[0097] In some embodiments, the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein comprises, from the 3’ end to the 5’ end of said nucleotide sequence, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein, and a nucleotide sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, or a functional fragment or derivative thereof.

[0098] In some embodiments, the VSV M polypeptide is a wild-type VSV M polypeptide, or a functional fragment or derivative thereof.

[0099] In some embodiments, the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that has at least 80% sequence identity thereto.

[0100] In some embodiments, the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 3.

[0101] In some embodiments, the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 3.

[0102] In some embodiments, the VSV M polypeptide is a mutant VSV M polypeptide, or a functional fragment or derivative thereof.

[0103] In some embodiments, the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises an amino acid change at position M51 of the amino acid sequence of SEQ ID NO: 3.

[0104] In some embodiments, the amino acid change comprises a methionine (M) substitution.

[0105] In some embodiments, the methionine (M) substitution comprises a methionine (M) to an arginine (R) substitution (M51R).

[0106] In some embodiments, the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that has at least 80% sequence identity thereto.

[0107] In some embodiments, the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 5.

[0108] In some embodiments, the mutant VSV M polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 5.

[0109] In some embodiments, the fusogenic polypeptide is a VSV glycoprotein (G) polypeptide, or a functional fragment or derivative thereof.

[0110] In some embodiments, the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence that has at least 80% sequence identity thereto.

[0111] In some embodiments, the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 49.

[0112] In some embodiments, the recombinant rhabdovirus consists of the amino acid sequence of SEQ ID NO: 49.

[0113] In some embodiments, the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 113, or an amino acid sequence that has at least 80% sequence identity thereto.

[0114] In some embodiments, the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 113.

[0115] In some embodiments, the recombinant rhabdovirus consists of the amino acid sequence of SEQ ID NO: 113.

[0116] In some embodiments, upon infection of a cell with the recombinant rhabdovirus, the cell expresses the recombinant protein comprising the IL-12 polypeptide, or the functional fragment or derivative thereof.

[0117] In some embodiments, the cell is a human cell.

[0118] In some embodiments, the cell is a cancer cell.

[0119] In some embodiments, the recombinant rhabdovirus is a replication-competent virus.

[0120] In some embodiments, the recombinant rhabdovirus is a non-replicative.

[0121] In some embodiments, the recombinant rhabdovirus is an oncolytic virus.

[0122] In a further aspect, provided herein is a recombinant polynucleotide comprising: i. a nucleotide sequence encoding a vesicular stomatitis virus (VSV) nucleoprotein (N) polypeptide, or a functional fragment or derivative thereof; ii. a nucleotide sequence encoding a VSV phosphoprotein (P) polypeptide, or a functional fragment or derivative thereof; iii. a nucleotide sequence encoding a VSV matrix (M) polypeptide, or a functional fragment or derivative thereof; iv. a nucleotide sequence encoding a fusogenic polypeptide, or a functional fragment or derivative thereof; v. a nucleotide sequence encoding a VSV large protein (L) polypeptide, or a functional fragment or derivative thereof; and vi. a nucleotide sequence encoding a recombinant protein comprising an IL- 12 polypeptide, or a functional fragment or derivative thereof.

[0123] In some embodiments, the nucleotide sequence encoding the recombinant protein is positioned between the nucleotide sequence encoding the fusogenic polypeptide, or a functionalfragment or derivative thereof, and the nucleotide sequence encoding the L polypeptide, or a functional fragment or derivative thereof.

[0124] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises an IL- 12 p40 subunit, or a functional fragment or derivative thereof.

[0125] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, further comprises an IL-12 p35 subunit, or a functional fragment or derivative thereof.

[0126] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises an IL-12 p35 subunit, or a functional fragment or derivative thereof.

[0127] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence which has at least 80% sequence identity thereto.

[0128] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 69.

[0129] In some embodiments, the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 69.

[0130] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence which has at least 80% sequence identity thereto.

[0131] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61.

[0132] In some embodiments, the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 61.

[0133] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence which has at least 80% sequence identity thereto.

[0134] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 71.

[0135] In some embodiments, the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 71.

[0136] In some embodiments, the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence which has at least 80% sequence identity thereto.

[0137] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63.

[0138] In some embodiments, the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 63.

[0139] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, and the IL-12 p35 subunit, or the functional fragment or derivative thereof, are connected by a linker.

[0140] In some embodiments, the linker is from about 5 amino acids to about 30 amino acids in length.

[0141] In some embodiments, the linker comprises the amino acid sequence (VPGVG)2 (SEQ ID NO: 17).

[0142] In some embodiments, the linker consists of the amino acid sequence (VPGVG) (SEQ ID NO: 17).

[0143] In some embodiments, the recombinant protein further comprises a signal peptide operably linked to the IL- 12 polypeptide.

[0144] In some embodiments, the signal peptide is operably linked to the IL-12 p40 subunit, or the functional fragment or derivative thereof, and / or the IL-12 p35 subunit, or the functional fragment or derivative thereof.

[0145] In some embodiments, the signal peptide is operably linked to the IL-12 p40 subunit, or the functional fragment or derivative thereof, to form an IL-12 p40 subunit precursor, or a functional fragment or derivative thereof.

[0146] In some embodiments, the signal peptide operably linked to the IL-12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence that has at least 80% sequence identity thereto.

[0147] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that has at least 80% sequence identity thereto.

[0148] In some embodiments, the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 27.

[0149] In some embodiments, the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 27.

[0150] In some embodiments, the IL-12 p40 subunit precursor, or a functional fragment or derivative thereof, comprises one or more amino acid changes at positions E81, F82, KI 06, or K217 of the amino acid sequence of SEQ ID NO: 27.

[0151] In some embodiments, the one or more amino acid changes comprises one or more alanine (A) substitutions.

[0152] In some embodiments, the signal peptide operably linked to the IL- 12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence that has at least 80% sequence identity thereto.

[0153] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that has at least 80% sequence identity thereto.

[0154] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 12.

[0155] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 12.

[0156] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises one or more amino acid changes at positions E81, F82, K106, or K217 of the amino acid sequence of SEQ ID NO: 12.

[0157] In some embodiments, the one or more amino acid changes comprises one or more alanine (A) substitutions.

[0158] In some embodiments, the signal peptide is operably linked to the IL-12 p35 subunit, or the functional fragment or derivative thereof, to form an IL-12 p35 subunit precursor, or a functional fragment or derivative thereof.

[0159] In some embodiments, the signal peptide operably linked to the IL-12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that has at least 80% sequence identity thereto.

[0160] In some embodiments, the signal peptide operably linked to the IL-12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence that has at least 80% sequence identity thereto.

[0161] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence that has at least 80% sequence identity thereto.

[0162] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 159.

[0163] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 159.

[0164] In some embodiments, the IL-12 polypeptide, or a functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 154, or an amino acid sequence that has at least 80% sequence identity thereto.

[0165] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 154.

[0166] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 154.

[0167] In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding, from the 5’ end to the 3’ end of said nucleotide sequence, a VSV N polypeptide, or a functional fragment or derivative thereof, a VSV P polypeptide, or a functional fragment or derivative thereof, a VSV M polypeptide, or a functional fragment or derivative thereof, a fusogenic polypeptide, or a functional fragment or derivative thereof, the recombinant protein, and a VSV L polypeptide, or a functional fragment or derivative thereof.

[0168] In some embodiments, the VSV M polypeptide is a wild-type VSV M polypeptide, or a functional fragment or derivative thereof.

[0169] In some embodiments, the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that has at least 80% sequence identity thereto.

[0170] In some embodiments, the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 3.

[0171] In some embodiments, the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 3.

[0172] In some embodiments, the VSV M polypeptide is a mutant VSV M polypeptide, or a functional fragment or derivative thereof.

[0173] In some embodiments, the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises an amino acid change at position M51 of the amino acid sequence of SEQ ID NO: 3.

[0174] In some embodiments, the amino acid change comprises a methionine (M) substitution.

[0175] In some embodiments, the methionine (M) substitution comprises a methionine (M) to an arginine (R) substitution (M51R).

[0176] In some embodiments, the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that has at least 80% sequence identity thereto.

[0177] In some embodiments, the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 5.

[0178] In some embodiments, the mutant VSV M polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 5.

[0179] In some embodiments, the fusogenic polypeptide is a VSV glycoprotein (G) polypeptide, or a functional fragment or derivative thereof.

[0180] In some embodiments, the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence that has at least 80% sequence identity thereto.

[0181] In some embodiments, the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 49.

[0182] In some embodiments, the recombinant rhabdovirus consists of the amino acid sequence of SEQ ID NO: 49.

[0183] In some embodiments, the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 113, or an amino acid sequence that has at least 80% sequence identity thereto.

[0184] In some embodiments, the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 113.

[0185] In some embodiments, the recombinant rhabdovirus consists of the amino acid sequence of SEQ ID NO: 113.

[0186] In some embodiments, the polynucleotide is DNA.

[0187] In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 50, or a nucleotide sequence which has at least 65% sequence identity thereto.

[0188] In some embodiments, the recombinant polynucleotide comprises the amino acid sequence of SEQ ID NO: 50.

[0189] In some embodiments, the recombinant polynucleotide consists of the amino acid sequence of SEQ ID NO: 50.

[0190] In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 112, or a nucleotide sequence which has at least 65% sequence identity thereto.

[0191] In some embodiments, the recombinant polynucleotide comprises the amino acid sequence of SEQ ID NO: 112.

[0192] In some embodiments, the recombinant polynucleotide consists of the amino acid sequence of SEQ ID NO: 112.

[0193] In some embodiments, the polynucleotide is RNA.

[0194] In another aspect, provided herein is a recombinant polynucleotide, comprising a nucleotide sequence encoding, from the 5’ end to the 3’ end of said nucleotide sequence, a VSV N polypeptide, or a functional fragment or derivative thereof, a VSV P polypeptide, or a functional fragment or derivative thereof, a VSV M polypeptide, or a functional fragment or derivative thereof, a fusogenic polypeptide, or a functional fragment or derivative thereof, a recombinant protein, and a VSV L polypeptide, or a functional fragment or derivative thereof, wherein: i. the VSV M polypeptide, or the functional fragment or derivative thereof, is a mutant VSV M polypeptide, or a functional fragment or derivative thereof, said mutant VSV M polypeptide comprising the amino acid sequence of SEQ ID NO: 5; ii. the fusogenic polypeptide, or the functional fragment thereof, is a VSV glycoprotein (G) polypeptide, or a functional fragment or derivative thereof; and iii. the recombinant protein comprises an IL-12 polypeptide, or a functional fragmentor derivative thereof, said IL-12 polypeptide comprising the amino acid sequence of SEQ ID NO: 159 or SEQ ID NO: 154.

[0195] In some embodiments, the IL-12 polypeptide comprises the amino acid sequence of SEQ ID NO: 159, and the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 49.

[0196] In some embodiments, the IL-12 polypeptide comprises the amino acid sequence of SEQ ID NO: 154, and the recombinant protein comprises the amino acid sequence of SEQ ID NO: 113.

[0197] In a further aspect, provided herein is a recombinant polynucleotide, wherein the recombinant polynucleotide is an RNA molecule comprising: i. a nucleotide sequence that is a template for a positive sense transcript encoding a VSV nucleoprotein (N) polypeptide, or a functional fragment or derivative thereof; ii. a nucleotide sequence that is a template for a positive sense transcript encoding a VSV phosphoprotein (P) polypeptide, or a functional fragment or derivative thereof; iii. a nucleotide sequence that is a template for a positive sense transcript encoding a VSV matrix (M) polypeptide, or a functional fragment or derivative thereof; iv. a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide, or a functional fragment or derivative thereof; v. a nucleotide sequence that is a template for a positive sense transcript encoding a VSV large protein (L) polypeptide, or a functional fragment or derivative thereof; and vi. a nucleotide sequence that is a template for a positive sense transcript encoding a recombinant protein comprising an IL-12 polypeptide, or a functional fragment or derivative thereof.

[0198] In some embodiments, the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein is positioned between the nucleotide sequence that is a template for a positive sense transcript encoding the fusogenic polypeptide, or the functional fragment or derivative thereof, and the nucleotide sequence that is a template for a positive sense transcript encoding the VSV L polypeptide, or a functional fragment or derivative thereof.

[0199] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises an IL-12 p40 subunit, or a functional fragment or derivative thereof.

[0200] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, further comprises an IL-12 p35 subunit, or a functional fragment or derivative thereof.

[0201] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises an IL-12 p35 subunit, or a functional fragment or derivative thereof.

[0202] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence which has at least 80% sequence identity thereto.

[0203] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 69.

[0204] In some embodiments, the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 69.

[0205] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence which has at least 80% sequence identity thereto.

[0206] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61.

[0207] In some embodiments, the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 61.

[0208] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence which has at least 80% sequence identity thereto.

[0209] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 71.

[0210] In some embodiments, the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 71.

[0211] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence which has at least 80% sequence identity thereto.

[0212] In some embodiments, the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63.

[0213] In some embodiments, the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 63.

[0214] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, and the IL-12 p35 subunit, or the functional fragment or derivative thereof, are connected by a linker.

[0215] In some embodiments, the linker is from about 5 amino acids to about 30 amino acids in length.

[0216] In some embodiments, the linker comprises the amino acid sequence (VPGVG)2 (SEQ ID NO: 17).

[0217] In some embodiments, the linker consists of the amino acid sequence (VPGVG)2 (SEQ ID NO: 17).

[0218] In some embodiments, the recombinant protein further comprises a signal peptide operably linked to the IL- 12 polypeptide.

[0219] In some embodiments, the signal peptide is operably linked to the IL-12 p40 subunit, or the functional fragment or derivative thereof, and / or the IL-12 p35 subunit, or the functional fragment or derivative thereof.

[0220] In some embodiments, the signal peptide is operably linked to the IL-12 p40 subunit, or the functional fragment or derivative thereof, to form an IL-12 p40 subunit precursor, or a functional fragment or derivative thereof.

[0221] In some embodiments, the signal peptide operably linked to the IL-12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence that has at least 80% sequence identity thereto.

[0222] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that has at least 80% sequence identity thereto.

[0223] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 27.

[0224] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 27.

[0225] In some embodiments, the IL-12 p40 subunit precursor, or a functional fragment or derivative thereof, comprises one or more amino acid changes at positions E81, F82, K106, or K217 of the amino acid sequence of SEQ ID NO: 27.

[0226] In some embodiments, the one or more amino acid changes comprises one or more alanine (A) substitutions.

[0227] In some embodiments, the signal peptide operably linked to the IL- 12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence that has at least 80% sequence identity thereto.

[0228] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that has at least 80% sequence identity thereto.

[0229] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 12.

[0230] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 12.

[0231] In some embodiments, the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises one or more amino acid changes at positions E81, F82, K106, or K217 of the amino acid sequence of SEQ ID NO: 12.

[0232] In some embodiments, the one or more amino acid changes comprises one or more alanine (A) substitutions.

[0233] In some embodiments, the signal peptide is operably linked to the IL-12 p35 subunit, or the functional fragment or derivative thereof, to form an IL- 12 p35 subunit precursor, or a functional fragment or derivative thereof.

[0234] In some embodiments, the signal peptide operably linked to the IL- 12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that has at least 80% sequence identity thereto.

[0235] In some embodiments, the signal peptide operably linked to the IL- 12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence that has at least 80% sequence identity thereto.

[0236] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence that has at least 80% sequence identity thereto.

[0237] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 159.

[0238] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 159.

[0239] In some embodiments, the IL- 12 polypeptide, or a functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 154, or an amino acid sequence that has at least 80% sequence identity thereto.

[0240] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 154.

[0241] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 154.

[0242] In some embodiments, the recombinant polynucleotide comprises, from the 3’ end to the 5’ end of said recombinant polynucleotide, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein, and a nucleotide sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, or a functional fragment or derivative thereof.

[0243] In some embodiments, the VSV M polypeptide is a wild-type VSV M polypeptide, or a functional fragment or derivative thereof.

[0244] In some embodiments, the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that has at least 80% sequence identity thereto.

[0245] In some embodiments, the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 3.

[0246] In some embodiments, the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 3.

[0247] In some embodiments, the VSV M polypeptide is a mutant VSV M polypeptide, or a functional fragment or derivative thereof.

[0248] In some embodiments, the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises an amino acid change at position M51 of the amino acid sequence of SEQ ID NO: 3.

[0249] In some embodiments, the amino acid change comprises a methionine (M) substitution.

[0250] In some embodiments, the methionine (M) substitution comprises a methionine (M) to an arginine (R) substitution (M51R).

[0251] In some embodiments, the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that has at least 80% sequence identity thereto.

[0252] In some embodiments, the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 5.

[0253] In some embodiments, the mutant VSV M polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 5.

[0254] In some embodiments, the fusogenic polypeptide is a VSV glycoprotein (G) polypeptide, or a functional fragment or derivative thereof.

[0255] In some embodiments, the recombinant protein comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence that has at least 80% sequence identity thereto.

[0256] In some embodiments, the recombinant protein comprises the amino acid sequence of SEQ ID NO: 49.

[0257] In some embodiments, the recombinant protein consists of the amino acid sequence of SEQ ID NO: 49.

[0258] In some embodiments, the recombinant protein comprises the amino acid sequence of SEQ ID NO: 113, or an amino acid sequence that has at least 80% sequence identity thereto.

[0259] In some embodiments, the recombinant protein comprises the amino acid sequence of SEQ ID NO: 113.

[0260] In some embodiments, the recombinant protein consists of the amino acid sequence of SEQ ID NO: 113.

[0261] In another aspect, provided herein is a recombinant polynucleotide, wherein the recombinant polynucleotide is an RNA molecule comprising, from the 3’ end to the 5’ end of said recombinant polynucleotide, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a recombinant protein, and a nucleotide sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, or a functional fragment or derivative thereof, wherein: i. the VSV M polypeptide, or the functional fragment or derivative thereof, is a mutant VSV M polypeptide, or a functional fragment or derivative thereof, said mutant VSV M polypeptide comprising the amino acid sequence of SEQ ID NO: 5; ii. the fusogenic polypeptide, or the functional fragment thereof, is a VSV glycoprotein (G) polypeptide, or a functional fragment or derivative thereof; and iii. the recombinant protein comprises an IL-12 polypeptide, or a functional fragment or derivative thereof, said IL-12 polypeptide comprising the amino acid sequence of SEQ ID NO: 159 or SEQ ID NO: 154.

[0262] In some embodiments, the IL-12 polypeptide comprises the amino acid sequence of SEQ ID NO: 159, and the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 49.

[0263] In some embodiments, the IL-12 polypeptide comprises the amino acid sequence of SEQ ID NO: 154, and the recombinant protein comprises the amino acid sequence of SEQ ID NO: 113.

[0264] In a further aspect, provided herein is a vector comprising any of the recombinant polynucleotides as described herein.

[0265] In another aspect, provided herein is a recombinant vesicular stomatitis virus (VSV) comprising any of the recombinant polynucleotides as described herein.

[0266] In a further aspect, provided herein is a composition comprising any of the recombinant rhabdoviruses as described herein, and a carrier and / or excipient.

[0267] In another aspect, provided herein is a composition comprising any of the recombinant polynucleotides as described herein, and a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

[0268] In a further aspect, provided herein is a product of manufacture comprising any of the recombinant rhabdoviruses as described herein or any of the compositions as described herein in a sterile vial, ampoule or syringe.

[0269] In another aspect, provided herein is a host cell comprising any of the recombinant rhabdoviruses as described herein.

[0270] In a further aspect, provided herein is a host cell comprising any of the recombinant polynucleotides as described herein.

[0271] In another aspect, provided herein is a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the recombinant rhabdoviruses as described herein or any of the compositions as described herein.

[0272] In some embodiments, the administration of the recombinant rhabdovirus or composition to the subject is under conditions wherein the recombinant rhabdovirus infects cancer cells of the subject to form infected cancer cells, wherein the infected cancer cells express the recombinant protein comprising the IL-12 polypeptide, or the functional fragment or derivative thereof, and wherein the number of cancer cells within the subject is reduced following the administration.

[0273] In some embodiments, the administration of the recombinant rhabdovirus or composition to the subject is under conditions wherein the recombinant rhabdovirus infects cancer cells of the subject to form infected cancer cells, wherein the infected cancer cells express the recombinant protein comprising the IL-12 polypeptide, or the functional fragment or derivative thereof, and wherein the number of cancer cells within the subject does not increase following the administration.

[0274] In a further aspect, provided herein is a method for inducing a cancer regression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the recombinant rhabdoviruses as described herein or any of the compositions as described herein.

[0275] In some embodiments, the cancer regression produces a reduced tumor size.

[0276] In some embodiments, the recombinant rhabdovirus or composition is administered via local delivery.

[0277] In some embodiments, the local delivery is intratumoral delivery.

[0278] In some embodiments, the recombinant rhabdovirus or composition is administered at a dose of from about lxlO6 / mL TCID50 / mLto about1x1011TCID50 / mL.

[0279] In some embodiments, the recombinant rhabdovirus or composition is administered at a dose of from about 1x107TCID50 / mL to about1x1010TCID50 / mL.

[0280] In some embodiments, the recombinant rhabdovirus or composition is administered at a dose of about 1x107TCID50 / mL, about 3xl07TCID50 / mL, about1x108TCID50 / mL, about 3xl08TCID50 / mL, about1x109TCID50 / mL, about 3xl09TCID50 / mL, or about 1x1010TCID50 / mL.

[0281] In some embodiments, the recombinant rhabdovirus or composition is administered at a dose of about1x1010TCID50 / mL.

[0282] In some embodiments, the recombinant rhabdovirus comprises a nucleotide sequence encoding a mutant VSV matrix (M) polypeptide, or a functional fragment or derivative thereof, said mutant VSV M polypeptide comprising a methionine (M) to an arginine (R) substitution at position M51 of the amino acid sequence of SEQ ID NO: 3, and the administration of the recombinant rhabdovirus to the subject produces a reduced serum interferon gamma (IFN-y) release in the subject as compared to a control recombinant rhabdovirus comprising a nucleotide sequence encoding a wild-type VSV M polypeptide, or a functional fragment or derivative thereof.

[0283] In some embodiments, the recombinant rhabdovirus comprises a nucleotide sequence encoding a mutant VSV matrix (M) polypeptide, or a functional fragment or derivative thereof, said mutant VSV M polypeptide comprising a methionine (M) to an arginine (R) substitution at position M51 of the amino acid sequence of SEQ ID NO: 3, and the administration of the recombinant rhabdovirus to the subject produces a maintained or increased body weight in the subject as compared to a control recombinant rhabdovirus comprising a nucleotide sequence encoding a wild-type VSV M polypeptide, or a functional fragment or derivative thereof.

[0284] In some embodiments, the administration of the recombinant rhabdovirus to the subject produces an enhanced tumor-specific CD8 T cell response in the subject as compared to a control recombinant rhabdovirus which does not comprise a nucleotide sequence encoding a recombinant protein comprising an IL-12 polypeptide, or a functional fragment or derivative thereof.

[0285] In some embodiments, the enhanced tumor-specific CD8 T cell response comprises an increased serum interferon gamma (IFN-y) release.

[0286] In some embodiments, the cancer is selected from head and neck cancer, colon cancer, lung cancer, prostate cancer, ovarian cancer, hepatocellular carcinoma, pancreatic cancer, kidney cancer, melanoma, brain cancer, lymphoma, myeloma, lymphocytic leukemia, myelogenous leukemia, and breast cancer.

[0287] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of one or more immune checkpoint inhibitors.

[0288] In some embodiments, the immune checkpoint inhibitors comprise one of more of an anti-PD-1 antibody, an anti-PDLl antibody, or an anti-CTLA-4 antibody.

[0289] In some embodiments, the immune checkpoint inhibitor comprises an anti-PD-1 antibody.

[0290] In some embodiments, the immune checkpoint inhibitor is administered via systemic delivery.

[0291] In some embodiments, the immune checkpoint inhibitor is administered via parenteral delivery.

[0292] In some embodiments, the parenteral delivery is subcutaneous, intraperitoneal, intradermal, intramuscular, or intravenous delivery.

[0293] In some embodiments, the parenteral delivery is intravenous delivery.

[0294] In some embodiments, the immune checkpoint inhibitor is administered at a dose from about 200 mg to about 400 mg.

[0295] In some embodiments, the immune checkpoint inhibitor is administered at a dose of about 350 mg.

[0296] In some embodiments, the subject is human.BRIEF DESCRIPTION OF THE DRAWINGS

[0297] Figure 1 is a schematic representation of a Vesicular Stomatitis Virus (VSV) encoding different versions of IL-12. Figure discloses “VPGVGx2” as SEQ ID NO: 17 and “G4Sx3” as SEQ ID NO: 18.

[0298] Figure 2 shows an in vivo experimental timeline for an experiment disclosed herein.

[0299] Figure 3 shows average body weight changes (% ± SEM [standard error of the mean]) in each treatment group at multiple post-virus infection time points, with treatment at day 0.

[0300] Figures 4A-4E show individual body weight changes after treatment at day 0. Figure 4A shows individual body weight changes in 5 mice after treatment with PBS. Figure 4B shows individual body weight changes in 5 mice after treatment with VSV-Mwt-mIL12p70. Figure 4C shows individual body weight changes in 5 mice after treatment with VSV-M51R-mIL12p70. Figure 4D shows individual body weight changes in 5 mice after treatment with VSV-M51R- mIL12p35_TM. Figure 4E shows individual body weight changes in 5 mice after treatment with VS V-M51 R-mIL 12p70_TM.

[0301] Figure 5 shows serum levels of interleukin- 12p70 (IL12p70) and interferon gamma (IFNy) 24 hours post infection (hpi) detected by enzyme-linked immunoassay (ELISA).

[0302] Figure 6 shows an in vivo experimental timeline for an experiment disclosed herein.

[0303] Figure 7 shows average tumor volumes (mm3± SEM) in each treatment group at multiple post-tumor implantation time points, with treatment days indicated by arrows.

[0304] Figure 8 shows individual tumor volumes at day 14 after treatment initiation.

[0305] Figure 9 shows Kaplan-Meier survival curves.

[0306] Figure 10 shows serum levels of IL12p70 and IFNy 24 hours post infection detected by ELISA.

[0307] Figure 11 shows an in vivo experimental timeline for an experiment disclosed herein.

[0308] Figure 12 shows average tumor volumes (mm3± SEM) in each treatment group at multiple post-tumor implantation time points, with treatment days indicated by arrows.

[0309] Figure 13 shows individual tumor volumes at day 11 (Dl l) after treatment initiation.

[0310] Figure 14 shows Kaplan-Meier survival curves.

[0311] Figure 15 shows individual body weight changes after treatment at day 0.

[0312] Figure 16 shows serum levels of IL12p70 and IFNy 24 hpi detected by ELISA.

[0313] Figures 17A-17K show examples of VSV-IL-12 constructs described herein. Figure 17A shows VSV-M51R-hIL12p70 (secreted IL12 p70). Figure 17B shows VSV-M51R- hIL12p40-hIL12p35 (secreted unlinked IL12 p40 and p35). Figure 17C shows VSV-M51R-L- hIL12p70 (secreted IL12 p70 downstream ofL). Figure 17D shows VSV-M51R-hIL12p70.P2A.L (secreted IL 12 p70 linked to L with P2A). Figure 17E shows VSV-M51R-hIL12p70TM (IL 12 p70 with transmembrane region). Figure 17F shows VSV-M51R-hIL12p70_hCD4 (IL12p70 linked with CD4). Figure 17G shows VSV-M51R-hIL12p70_hCD46 (IL12p70 linked with CD46). Figure 17H shows VSV-M5 !R-hIL12p70_G4Sx3_CBD (IL12p70 linked with VWF CBD XI). Figure 171 shows VSV-M51R-hIL12p70_(G4Sx3_CBD)x2 (IL12p70 linked with VWF CBD X2). Figure 17J shows VSV-M51R-hIL12p70_hIgGl (IL12p70 linked with IgGl [switch]). Figure 17K shows VSV-M51R-hIL12p70_hIgG4 (IL12p70 linked with IgG4 [switch]). Figures disclose “VPGVGx2” as SEQ ID NO: 17 and “G4Sx3” as SEQ ID NO: 18.

[0314] Figures 18A-18K show a secreted VSV-M51R-hIL12p70 (secreted human IL12 p70) construct nucleic acid sequence, SEQ ID NO: 112 (Figures 18A-18H) and amino acid sequence, SEQ ID NO: 113 (Figures 18I-18K). Figures disclose “VPGVGx2” as SEQ ID NO: 17.

[0315] Figures 19A-19K show a secreted VSV-M51R-hIL12p40-hIL12p35 (secreted unlinked IL12 p40 and p35, human) construct nucleic acid sequence, SEQ ID NO: 114 (Figures 19A-19H) and amino acid sequence, SEQ ID NO: 115 (Figures 19I-19K).

[0316] Figures 20A-20K show a secreted VSV-M51R-L-hIL12p70 (secreted human IL12 p70 downstream of L) construct nucleic acid sequence, SEQ ID NO: 116 (Figures 20A-20H) and corresponding amino acid sequence, SEQ ID NO: 117 (Figures 20I-20K). Figures disclose “VPGVGx2” as SEQ ID NO: 17.

[0317] Figures 21A-21K show a secreted VSV-M51R-hIL12p70.P2A.L (secreted human IL12 p70 linked to L with P2A) construct nucleic acid sequence, SEQ ID NO: 118 (Figures 21A-21H) and corresponding amino acid sequence, SEQ ID NO: 119 (Figures 21I-21K). Figures disclose “VPGVGx2” as SEQ ID NO: 17.

[0318] Figures 22A-22J show a human transmembrane VSV-M51R-hIL12p70TM (IL12 p70 with transmembrane region) construct nucleic acid sequence, SEQ ID NO: 120 (Figures 22A- 22H) and corresponding amino acid sequence, SEQ ID NO: 121 (Figures 22I-22J). Figures disclose “VPGVGx2” as SEQ ID NO: 17 and “GGGGSx3” as SEQ ID NO: 18.

[0319] Figures 23A-23L show a human transmembrane VSV-M51R-hIL12p70_hCD4 (IL12p70 linked with CD4) construct nucleic acid sequence, SEQ ID NO: 122 (Figures 23A-23I) and corresponding amino acid sequence, SEQ ID NO: 123 (Figures 23J-23L). Figures disclose “VPGVGx2” as SEQ ID NO: 17 and “GGGGSx3” as SEQ ID NO: 18.

[0320] Figures 24A-24K show a human transmembrane VSV-M51R-hIL12p70_hCD46 (IL12p70 linked with CD46) construct nucleic acid sequence, SEQ ID NO: 124 (Figures 24A- 24H) and corresponding amino acid sequence, SEQ ID NO: 125 (Figures 24I-24K). Figures disclose “VPGVGx2” as SEQ ID NO: 17 and “GGGGSx3” as SEQ ID NO: 18.

[0321] Figures 25A-25K show a human transmembrane VSV-M51R-hIL12p70_hIgGl (IL12p70 linked with hlgGl [switch]) construct nucleic acid sequence, SEQ ID NO: 132 (Figures 25A-25H) and corresponding amino acid sequence, SEQ ID NO: 133 (Figures 25I-25K). The human version of the construct comprises IgGl and the mouse version of the construct comprises IgG2a. Figures disclose “VPGVGx2” as SEQ ID NO: 17 and “GGGGSx3” as SEQ ID NO: 18.

[0322] Figures 26A-26K show a human transmembrane VSV-M51R-hIL12p70_hIgG4 (IL12p70 linked with hIgG4 [switch]) construct nucleic acid sequence, SEQ ID NO: 130 (Figures 26A-26H) and corresponding amino acid sequence, SEQ ID NO: 131 (Figures 26I-26K). The human version of the construct comprises IgG4 and the mouse version of the construct comprises IgGl. Figures disclose “VPGVGx2” as SEQ ID NO: 17 and “GGGGSx3” as SEQ ID NO: 18.

[0323] Figures 27A-27K show a secreted VSV-M51R-hIL12p70_CBDl (human IL12p70 linked with CBD1) construct nucleic acid sequence, SEQ ID NO: 126 (Figures 27A-27H) and corresponding amino acid sequence, SEQ ID NO: 127 (Figures 27I-27K). Figures disclose “VPGVGx2” as SEQ ID NO: 17 and “GGGGSx3” as SEQ ID NO: 18.

[0324] Figures 28A-28L show a secreted VSV-M51R-hIL12p70_CBD2 (human IL12p70 linked with CBD2) construct nucleic acid sequence, SEQ ID NO: 128 (Figures 28A-28I) and corresponding amino acid sequence, SEQ ID NO: 129 (Figures 28J-28L). Figures disclose “VPGVGx2” as SEQ ID NO: 17 and “GGGGSx3” as SEQ ID NO: 18.

[0325] Figures 29A-29J show a secreted VSV-M5 lR-mIL12p40-mIL12p35 (secreted unlinked IL12 p40 and p35, mouse) construct nucleic acid sequence, SEQ ID NO: 110 (Figures 29A-29H) and corresponding amino acid sequence, SEQ ID NO: 111 (Figures 29I-29J).

[0326] Figures 30A-30J show a mouse transmembrane VSV-M51R-mIL12p70TM construct nucleic acid sequence, SEQ ID NO: 56 (Figures 30A-30H) and corresponding amino acidsequence, SEQ ID NO: 55 (Figures 30I-30J). Figures disclose “VPGVGx2” as SEQ ID NO: 17 and “GGGGSx3” as SEQ ID NO: 18.

[0327] Figures 31A-31I show a mouse transmembrane VSV-M51R-mIL12p35TM construct nucleic acid sequence, SEQ ID NO: 58 (Figures 31A-31G) and corresponding amino acid sequence, SEQ ID NO: 57 (Figures 31H-31I). Figures disclose “GGGGSx3” as SEQ ID NO: 18.

[0328] Figures 32A-32J show a mouse secreted VSV-M5 !R-mIL12p70woSP construct nucleic acid sequence, SEQ ID NO: 60 (Figures 32A-32H) and corresponding amino acid sequence, SEQ ID NO: 59 (Figures 32I-32J) Figures disclose “VPGVGx2” as SEQ ID NO: 17.

[0329] Figures 33A-33J show a mouse secreted (mutein) VSV-M51R-mIL12p70_2xAla construct nucleic acid sequence, SEQ ID NO: 52 (Figures 33A-33H) and corresponding amino acid sequence, SEQ ID NO: 51 (Figures 33I-33J). Figures disclose “VPGVGx2” as SEQ ID NO: 17.

[0330] Figures 34A-34J show a mouse secreted (mutein) VSV-M51R-mIL12p70_3xAla construct nucleic acid sequence, SEQ ID NO: 54 (Figures 34A-34H) and corresponding amino acid sequence, SEQ ID NO: 53 (Figures 34I-34J). Figures disclose “VPGVGx2” as SEQ ID NO: 17.

[0331] Figures 35A-35J show a mouse secreted VSV-M51R-mIL12p70 construct nucleic acid sequence, SEQ ID NO: 50 (Figures 35A-35H) and corresponding amino acid sequence, SEQ ID NO: 49 (Figures 35I-35J). Figures disclose “VPGVGx2” as SEQ ID NO: 17.

[0332] Figures 36A-36K show a mouse transmembrane VSV-M51R-mIL12p70-mCD4-dl- 4TM construct nucleic acid sequence, SEQ ID NO: 134 (Figures 36A-36H) and corresponding amino acid sequence, SEQ ID NO: 135 (Figures 36I-36K). Figures disclose “VPGVGx2” as SEQ ID NO: 17 and “GGGGSx3” as SEQ ID NO: 18.

[0333] Figures 37A-37J show a mouse transmembrane VSV-M51R-mIL12p70-H4-2TM construct nucleic acid sequence, SEQ ID NO: 136 (Figures 37A-37H) and corresponding amino acid sequence, SEQ ID NO: 137 (Figures 37I-37J). Figures disclose “VPGVGx2” as SEQ ID NO: 17 and “A(EAAAK)4ALEA(EAAAK)4A” as SEQ ID NO: 73.

[0334] Figures 38A-38K show a mouse transmembrane VSV-M51R-mIL12p70- AEAAKEAAAKEAAAKATM construct nucleic acid sequence, SEQ ID NO: 138 (Figures 38A- 38H) and corresponding amino acid sequence, SEQ ID NO: 139 (Figures 38I-38K). Figures disclose “VPGVGx2” as SEQ ID NO: 17 and “A(EAAAK)3A” as SEQ ID NO: 74

[0335] Figures 39A-39K show a mouse transmembrane VSV-M51R-mIL12p70- D265A_mIgGlTM construct nucleic acid sequence, SEQ ID NO: 140 (Figures 39A-39H) and corresponding amino acid sequence, SEQ ID NO: 141 (Figures 39I-39K). Figures disclose “VPGVGx2” as SEQ ID NO: 17, “GGGGSx3” as SEQ ID NO: 18, and “GGGGSx4” as SEQ ID NO: 42.

[0336] Figures 40A-40K show a mouse transmembrane VSV-M51R-mIL12p70-mIgG2aTM construct nucleic acid sequence, SEQ ID NO: 142 (Figures 40A-40H) and corresponding amino acid sequence, SEQ ID NO: 143 (Figures 40I-40K). Figures disclose “VPGVGx2” as SEQ ID NO: 17 and “GGGGSx3” as SEQ ID NO: 18.

[0337] Figures 41A-41K show a mouse transmembrane VSV-mIL12p70TM-N-M51R-GFP construct nucleic acid sequence, SEQ ID NO: 144 (Figures 41A-41H) and corresponding amino acid sequence, SEQ ID NO: 145 (Figures 41I-41K). Figures disclose “VPGVGx2” as SEQ ID NO: 17 and “GGGGSx4” as SEQ ID NO: 42.

[0338] Figures 42A-42K show a mouse transmembrane VSV-N-mIL12p70TM-P-M51R-GFP construct nucleic acid sequence, SEQ ID NO: 146 (Figures 42A-42H) and corresponding amino acid sequence, SEQ ID NO: 147 (Figures 42I-42K). Figures disclose “VPGVGx2” as SEQ ID NO: 17 and “GGGGSx4” as SEQ ID NO: 42.

[0339] Figure 43 shows an example assay design that can be used for functional assessment of VSV-12p70TM (rigid linkers).

[0340] Figure 44 shows IL- 12 surface expression determined by flow cytometry -based measurement.

[0341] Figure 45 shows a splenocyte IFNy functional assay.

[0342] Figure 46 shows an example assay design for flow cytometry-based validation and ELISA for IL-12 (rigid linker payloads).

[0343] Figure 47 shows flow cytometry -based detection of membrane-bound IL-12p40.

[0344] Figure 48 shows median fluorescence intensity efluor450-IL-12p40 (18 hpi).

[0345] Figure 49 shows IL-12 levels in 5TGM1 supernatants (18 hpi).

[0346] Figure 50 shows average tumor volumes per group. Figure discloses “VPGVGx2” as SEQ ID NO: 17 and “AE A AKE A A AKE A A AKA” as SEQ ID NO: 74.

[0347] Figure 51 shows individual tumor growth curves per group.

[0348] Figure 52 shows Kaplan-Meier survival curves. Figure discloses “G4Sx3” as SEQ ID NO: 18 and “A(EAAAK)3A” as SEQ ID NO: 74.

[0349] Figure 53 shows body weight change after treatment on Day 0. Figure discloses “G4Sx3” as SEQ ID NO: 18 and “A(EAAAK)3A” as SEQ ID NO: 74.

[0350] Figure 54 shows serum IL-12p70 and IFNy levels 24 hpi. Figure discloses “G4Sx3” as SEQ ID NO: 18 and “A(EAAAK)3A” as SEQ ID NO: 74.

[0351] Figure 55 shows body weight change after treatment on Day 0. Figure discloses “G4Sx3” as SEQ ID NO: 18 and “A(EAAAK)3A” as SEQ ID NO: 74.

[0352] Figure 56 shows individual mice body weight per group. * represents mice either found dead or euthanized because of >20% body weight loss. Figure discloses “G4Sx3” as SEQ ID NO: 18 and “A(EAAAK)3A” as SEQ ID NO: 74.

[0353] Figure 57 shows Kaplan-Meier survival curves. Figure discloses “G4Sx3” as SEQ IDNO: 18 and “A(EAAAK)3A” as SEQ ID NO: 74.

[0354] Figure 58 shows serum IL-12p70 and IFNy levels at 24 hpi. Figure discloses “G4Sx3” as SEQ ID NO: 18 and “A(EAAAK)3A” as SEQ ID NO: 74.

[0355] Figure 59 shows the components of a VSV-mIFNb-NIS virus and a VSV-M51R-IL12p70 virus (soluble form).

[0356] Figure 60 shows the anti-tumor efficacy of a single-dose VSV encoding IL- 12 administered intratumorally (IT) to a mouse.

[0357] Figure 61 shows the anti-tumor efficacy of a single-dose VSV encoding IL- 12 administered IT to a mouse.

[0358] Figure 62 shows the anti-tumor efficacy of a single-dose VSV encoding IL-12 administered IT to a mouse.

[0359] Figure 63 shows the anti-tumor efficacy of a single-dose VSV encoding IL-12 administered IT to a mouse.

[0360] Figure 64 shows body weight changes in mice treated IT with VSV-M51R-mIL12p70, VSV-mIFNb-NIS, and VSV-Mwt-mIL12p70.

[0361] Figure 65 shows serum levels of IL-12 24 hpi.

[0362] Figure 66 shows VSV-M5 lR-mIL12p70 dosed IT compared to intravenously (IV) in the CMT64 therapeutic model.

[0363] Figure 67 shows VSV-M51R-mIL12p70 dosed IT compared to IV in the CMT64 therapeutic model.

[0364] Figure 68 shows VSV-M51R-mIL12p70dosed IT compared to IV in the CMT64 therapeutic model.

[0365] Figure 69 shows serum IL-12p70 and IFNy levels after 24 hpi.

[0366] Figure 70 shows that single-dose IT VSV-M5 lR-mZL12p70 had robust anti-tumor efficacy compared to VSV-mIFNb-NIS, on treated and contralateral non-treated tumors in MC38 and CMT64 tumors.

[0367] Figure 71 shows that single-dose IT VSV-M51R-mIL12p70 had robust anti-tumor efficacy compared to VSV-mIFNb-NIS, on treated and contralateral non-treated tumors in MC38 and CMT64 tumors.

[0368] Figure 72 shows that single-dose IT VSV-M51R-mIL12p70 had robust anti-tumor efficacy compared to VSV-mIFNb-NIS, on treated and contralateral non-treated tumors in MC38 and CMT64 tumors.

[0369] Figures 73A-73D show VSV-mIL12p70 transcript variants for reduction of IL-12 leakage from tumors, which can result in systemic exposure to IL- 12 and toxicity. Figure 73A displays an example of a toxicity mitigation strategy for VSV-mIL12p70 transcript variants. A schematic representation of the dosing regimen is shown in Figure 73B. Quantification of average tumor volumes (Figure 73C) and average body weights (% body weight change) (Figure 73D) across days post-infection are shown for VSV-M51R-GFP-L-mIL12p70 versus VSV-M51R- mIL12p70 groups. Phosphate buffered saline (PBS) was used as a control.

[0370] Figure 74 shows serum expression levels of IL12p70 (left panel) and IFNy (right panel) quantified at 24 hours post infection.

[0371] Figure 75 shows IL-12 expression across treatment groups.

[0372] Figures 76A-76B illustrate an IL-12 functional assay involving induction of IFNy from splenocytes. Figure 76A shows an example of an IL-12 functional assay design. Figure 76B shows quantification of IFNy in supernatants collected from 5TGM1 cells infected with IL- 12 transmembrane variant and soluble IL- 12 variant viruses.

[0373] Figures 77A-77B show tumor growth upon administration of VSV-M51R-mIL12 encoding murine single chain IL- 12 (soluble form) in combination with an anti -programmed cell death protein-1 (PD-1) antibody on both dosed (Figure 77A) and contralateral, non-dosed (Figure77B) tumors. Symbols on the graphs represent group means and error bars represent standard error of the mean (SEM). Error bars are present at Day 10 for all 4 dosing groups but were too small to discern for groups administered VSV-M51R-mIL12 or VSV-M51R-mIL12 + anti-PD-1.

[0374] Figure 78 shows survival upon administration of VSV-M51R-mIL12 encoding murine single chain IL- 12 (soluble form) in combination with anti-PD-1 antibody compared with administration of either reagent individually. Data are plotted as Kaplan-Meier curves.

[0375] Figures 79A-79LL show an example of a linear vector map of a VSV-M5 lR-hIL12p70 construct. The vector can be circular and comprise, e.g., 12,825 bp. Unique cutters are shown in bold text. The full-length VSV-M51R-hIL12p70 construct comprises a polynucleotide sequence, from 5’ to 3’, SEQ ID NO: 163. The VSV-M51R-hIL12p70 construct represented in the linear vector map comprises nucleotide sequences for, from 5’ to 3’, a VSV leader sequence (SEQ ID NO: 165), an N protein (SEQ ID NO: 2), a P protein (SEQ ID NO: 10), an M protein (SEQ ID NO: 6), a G signal peptide (GSP) protein (SEQ ID NO: 198), a G protein in frame with GSP protein (SEQ ID NO: 171), an intravirion tail-VSVG in frame with a GSP protein (SEQ ID NO: 174), an hIL12p70 protein (SEQ ID NO: 155) comprising an hIL12p40 protein (SEQ ID NO: 156), a linker (SEQ ID NO: 179), and an hIL12p35 protein (SEQ ID NO: 181), an L protein (SEQ ID NO: 183), and a VSV trailer sequence (SEQ ID NO: 185). The full-length VSV-M51R-hIL12p70 construct comprises a polynucleotide sequence, from 3’ to 5’, SEQ ID NO: 164. The VSV-M51R-hIL12p70 construct represented in the linear vector map comprises nucleotide sequences for, from 3’ to 5’, a VSV leader sequence (SEQ ID NO: 166), an N protein (SEQ ID NO: 167), a P protein (SEQ ID NO: 168), an M protein (SEQ ID NO: 169), a GSP protein (SEQ ID NO: 199), a G protein in frame with GSP protein (SEQ ID NO: 172), an intravirion tail-VSVG in frame with GSP protein (SEQ ID NO: 175), an hIL12p70 (SEQ ID NO: 177) comprising a hIL12p40 protein (SEQ ID NO: 178), a linker (SEQ ID NO: 180) and an hIL12p35 protein (SEQ ID NO: 182), an L protein (SEQ ID NO: 184), and a VSV trailer sequence (SEQ ID NO: 186). The VSV-M51R-hIL12p70 construct encodes for a full-length polypeptide sequence comprising, from N-terminus to C-terminus, SEQ ID NO: 113. The VSV-M51R-hIL12p70 construct encodes for a protein comprising an N protein (SEQ ID NO: 1), a P protein (SEQ ID NO: 9), an M protein (SEQ ID NO: 5), a GSP protein (SEQ ID NO: 197), a G protein in frame with a GSP protein (SEQ ID NO: 173), an intravirion tail- VSVG protein in frame with a GSP protein (SEQ ID NO: 176), hIL12p70 protein (SEQ ID NO:154) comprising an hTL12p40 protein (SEQ ID NO: 12), a linker protein (SEQ ID NO: 17) and an hIL12p35 protein (SEQ ID NO: 63), and an L protein (SEQ ID NO: 94).

[0376] Figures 80A-80C show C57BL / 6 mice treated intravenously with one dose of VSV- Mwt-GFP, VSV-Mwt-mIL12p70, or VSV-M51R-mIL12p70 at9TC1IxD1500. IL-12p70 (Figure 80A) and IFNy (Figure 80B) ELISA was performed on serum collected 24 hours post infection; Body weight (Figure 80C) was measured after treatment over time.

[0377] Figure 81 shows mice with established CMT64 subcutaneous tumors treated intratumorally (IT) with PBS, VSV-M51R-GFP, or VSV-M51R-mIL12p70 (5 x 108TCID50) and harvested for tumor-infiltrating lymphocyte (TIL) isolation seven days post infection. To detect antigen specific anti-tumor responses to previously published peptides, an IFNY ELISpot assay was performed (n=3). ***p<0.001; *p<0.05; Two-way ANOVA compared to PBS. The peptides along the x-axis are listed sequentially, from left to right (following a DMSO [Dimethyl Sulfoxide] negative control condition), as Aiml, Lyst, Rpp40, Naip2, Zhx2, Cepl92, Ndufsl, Arhgefl 1, Nes, Rabl3, Akap9, and ArhgeflO (followed by VSV-NP and PMA [Phorbol 12-myristate 13- acetate] / Ionomycin positive control conditions, respectively). For each of the aforementioned conditions, the three bars (per condition) represent the treatment groups, listed sequentially, from left to right, as PBS, VSV-M51R-GFP, and VSV-M51R-mIL12p70.

[0378] Figure 82 shows quantification of IL-12 levels from infected cell supernatants. Human tumor (SCC-15, SCC-25, HT1080, DLD-1, A375) and non-tumor (LaSt293) cell lines were infected with either VSV-M51R-hIL12p70 or VSV-P-hIL12p70-AM51 at varying multiplicities of infection (MOI: 0.01, 0.1, 1.0). SCC-25, a highly VSV-resistant line, was also tested at MOI 10. IL-12 levels (pg / mL) were quantified from culture supernatants 24 hours post-infection. Data represent mean ± SE from biological duplicates. Statistical significance was determined using two- way ANOVA test with Tukey’s multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0 0001, ns = not significant.

[0379] Figure 83 shows cell viability using a CCK-8 assay. Human tumor (SCC-15, SCC-25, HT1080, DLD-1, A375) and non-tumor (LaSt293) cell lines were infected with two recombinant VSVs: VSV-M51R-hIL12p70 and VSV-P-hIL12p70-AM51, at indicated multiplicities of infection (MOI: 0.01, 0.1, 1). Cell viability was assessed 24 hours post-infection and is expressed as a percentage relative to uninfected controls. Error bars represent standard errors of biological replicates.

[0380] Figures 84A-84B show virus growth over time in LaSt293 and HT1080 cells. Human HT1080 (Figure 84A) cells were seeded at 500,000 cells / well in 6-well plates in DMEM with 10% FBS and incubated 24 hours. Prior to infection, the media was replaced with Opti-MEM. Suspension Last293 (Figure 84B) cells were seeded at a density of 2e6 cells / mL in 25mL culture volume and infected immediately. Viruses diluted in Opti-MEM were added to cells to achieve final MOI of 0.1, for each cell line. At 8, 16, 24, 32, and 48 hours, samples were collected. One entire well from the HT1080 6-well plates was harvested, and 500uL was collected from the LaSt293 flasks at each timepoint. Viral titer was quantified by TCID50 assay.

[0381] Figures 85A-85B show kinetics of human IL12p70 produced over time in LaSt293 and HT1080 cells. Human HT1080 (Figure 85A) cells were seeded at 500,000 cells / well in 6-well plates in DMEM with 10% FBS and incubated 24 hours. Prior to infection, the media was replaced with Opti-MEM. Suspension LaSt293 (Figure 85B) cells were seeded at a density of 2e6 cells / mL in 25 m culture volume and infected immediately. Viruses diluted in Opti-MEM were added to cells to achieve final MOI of 0.1, for each cell line. At 8, 16, 24, 32, and 48 hours, samples were collected. One entire well from the HT10806-well plates was harvested, and 500 pL was collected from the LaSt293 flasks at each timepoint. Samples were assayed for hIL12p70 concentration by ELISA.DETAILED DESCRIPTION

[0382] Oncolytic virus therapy is an anti-cancer approach that uses viruses to selectively replicate and kill cancer cells without harming healthy cells. Such viruses may be used as delivery vehicles to express heterologous genes inserted into the viral genome, which may help to selectively hone the systemic anti-tumor immune response in infected cells. Vesicular stomatitis virus (VSV), a member of the Rhabdoviridae family, can be a potential oncolytic virus.

[0383] Interleukin-12 (IL-12), an IL-12 family cytokine, can be a promising target for cancer immunotherapy. However, the clinical success of therapeutic approaches involving cytokines has been limited due to pleiotropy and off-target toxicity. In particular, systemic administration of IL- 12 may lead to toxicity due to innate lymphocyte-mediated lENy production. Cytokine engineering is a potential strategy to develop cytokines with desired activities but lacking toxic side effects.

[0384] Systemic accumulation of IL-12 after delivery by oncolytic viruses remains potentially toxic to patients. There is a need for approaches to improve properties of IL-12 for use in cancer immunotherapy, for example, oncolytic virus therapy.

[0385] The present disclosure provides compositions and methods relating to a recombinant virus comprising a rhabdovirus genome, for example, a vesicular stomatitis virus (VSV) genome, and a nucleotide sequence encoding a recombinant protein comprising, e.g., an IL-12 polypeptide, or a functional fragment or derivative thereof. In various aspects, the recombinant protein can comprise a transmembrane domain and can, e.g., be membrane bound. In various other aspects, the recombinant protein may not comprise a transmembrane domain and may not be membrane bound. When the recombinant protein is not membrane bound, the recombinant protein may be a soluble and / or secreted protein. Recombinant polynucleotides and recombinant vectors encoding virus polypeptides and nucleotide sequence(s) encoding an IL- 12 polypeptide(s), or a functional fragment(s) or derivative(s) thereof, as well as host cells and methods for preparing host cells that express the polypeptides are disclosed. The disclosure further provides pharmaceutical compositions comprising the recombinant virus, and methods for treating cancer, e.g., a tumor, and for inducing cancer regression, in a subject in need thereof, using the recombinant virus.Definitions

[0386] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0387] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0388] The term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.

[0389] The term “antigen” refers to any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portions thereof, or combinations thereof) that, when introduced into a host, animal or human, having an immune system (directly or upon expression as in, e.g., DNA vaccines), is recognized by the immune system of the host and is capable of eliciting, or does elicit, an immune response.

[0390] The terms “viral element” and “viral component” are used herein to refer to viral genes (e.g., genes encoding polymerase or structural proteins) or other elements of the viral genome (e.g., packaging signals, regulatory elements, LTRs, ITRs, etc.).

[0391] The term “oncolytic virus” is used herein to refer to a virus that is capable of infecting and replicating in a tumor cell such that the tumor cell may be killed. The oncolytic virus may be replication competent. As a non-limiting example, the oncolytic virus may comprise a rhabdovirus, i.e., any of a group of viruses comprising the family Rhabdoviridae, e.g., a vesicular stomatitis virus (VSV).

[0392] As used herein, the term “vesiculovirus” refers to any virus in the Vesiculovirus genus. Non-limiting examples of vesiculoviruses include Vesicular Stomatitis Virus (VSV) (e.g., VSV- New Jersey, VSV-Indiana), Alagoas vesiculovirus, Cocal vesiculovirus, Jurona vesiculovirus, Carajas vesiculovirus, Maraba vesiculovirus, Piry vesiculovirus, Calchaqui vesiculovirus, Yug Bogdanovac vesiculovirus, Isfahan vesiculovirus, Chandipura vesiculovirus, Perinct vesiculovirus, and Porton-S vesiculovirus. Vesicular Stomatitis Virus (VSV), in the Vesiculovirus genus, is a prototypic rhabdovirus. While VSV is used as an example in the present disclosure, this disclosure can also be used for other vesiculoviruses and other rhabdoviruses. There are two major serotypes of VSV, New Jersey and Indiana, both of which can infect insects and mammals, causing disease in cattle, equines, and swine. The VSV genome is composed of single-stranded, negative-sense RNA of 11-12 kb, which encodes five viral proteins: the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G) and the viral polymerase (also known as large protein) (L). G monomers associate to form trimeric spikes anchored in the viral membrane.

[0393] The terms “vector”, “expression vector”, and “cloning vector” refer to any vehicle by which a nucleotide sequence, e.g., an RNA sequence or a DNA sequence, encoding for example, a foreign gene, may be introduced into a cell (e.g., a host cell) to genetically modify the cell and promote expression (e.g., transcription and translation) of said introduced nucleotidesequence. Non-limiting examples of vectors include synthesized RNA and DNA molecules plasmids, viruses, phages, and the like. In some embodiments, the vector may be a viral vector including, without limitation, a baculoviral vector, a herpes virus vector, a lentiviral vector, a retroviral vector, a vaccinia virus vector, an adeno-associated virus vector, an adenoviral vector, and an alphaviral vector.

[0394] The term “fusogen” or “fusogenic molecule” is used herein to refer to any molecule that can trigger membrane fusion when present on the surface of a virus. A fusogen can be, for example, a protein (e.g., a viral glycoprotein) or a fragment or derivative thereof.

[0395] The term “replication-competent” is used herein to refer to viruses (including wild-type and recombinant viruses) that are capable of infecting and propagating within a cell.

[0396] The term “encoding” can refer to encoding from either the (+) or (-) sense strand of a polynucleotide, for example, for expression in the virus particle.

[0397] The terms “antibody” and “antibodies” refer to monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, minibodies, diabodies and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antigen specific TCR), and epitope-binding fragments of any of the above. The terms “antibody” and “antibodies” also refer to covalent diabodies such as those disclosed in U.S. Pat. Appl. Pub. 2007 / 0004909 and Ig-DARTS such as those disclosed in U.S. Pat. Appl. Pub. 2009 / 0060910. Antibodies useful in the present disclosure include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.

[0398] The term “operably linked”, or the like, refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. For example, a control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. “Operably linked” sequences include both expression control sequences that are contiguous with a gene of interest and expression control sequences that act in trans or at a distance to control a gene of interest (or sequence of interest). The term “expression control sequence” includes polynucleotide sequences, which are necessary to affect the expression and processing of codingsequences to which they are ligated. “Expression control sequences” include: appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance polypeptide stability; and when desired, sequences that enhance polypeptide secretion. The nature of such control sequences differs depending upon the host organism. For example, in prokaryotes, such control sequences generally include promoter, ribosomal binding site and transcription termination sequence, while in eukaryotes typically such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include components whose presence is essential for expression and processing and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0399] The term “host cell” refers to any cell that comprises a heterologous nucleic acid. By way of a non-limiting example, the heterologous nucleic acid may be a vector. A host cell, for example, without limitation, may be a cell from any organism that is used, manipulated, modified, selected, transformed, or grown, for the production of a substance by the cell, e.g., the expression by the cell of, a RNA or DNA sequence, a gene, a protein, or an enzyme.

[0400] An “individual” or “subject” or “animal” refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models (e.g., mice, rats), e.g., of diseases. In a preferred embodiment, the subject is a human.

[0401] The terms “nucleic acid,” “polynucleotide,” and “nucleotide” used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides (RNA), deoxyribonucleotides (DNA), or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases. A single-stranded nucleic acid can be the sense strand or the antisense strand.

[0402] Nucleic acids are said to have a “5’ end” and a “3’ end” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5’ phosphate of one mononucleotide pentose ring is attached to the 3’ oxygen of its neighbor in one direction via a phosphodiester linkage. An end of an oligonucleotide is referred to as the “5’ end” if its 5’ phosphate is not linkedto the 3’ oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the “3’ end” if its 3’ oxygen is not linked to a 5’ phosphate of another mononucleotide pentose ring. A nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5’ and 3’ ends. In either a linear or circular DNA molecule, discrete elements are referred to as being “upstream” or 5’ of the “downstream” or 3’ elements.

[0403] The term “fragment” when referring to a protein means a protein that is shorter or has fewer amino acids than the full-length protein. A fragment can be, for example, an N-terminal fragment (i.e., removal of a portion of the C-terminal end of the protein), a C-terminal fragment (i.e., removal of a portion of the N-terminal end of the protein), or an internal fragment. The term “fragment” when referring to a nucleic acid means a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. A fragment can be, for example, a 5’ fragment (i.e., removal of a portion of the 3’ end of the nucleic acid), a 3’ fragment (i.e., removal of a portion of the 5’ end of the protein), or an internal fragment.

[0404] The term “derivative” as used herein refers to a nucleic acid, or protein, or a variant, or an analog thereof comprising one or more mutations and / or chemical modifications as compared to a 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 a combination thereof.

[0405] “Sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score ofzero, a conservative substitution is given a score between zero and 1 . The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC / GENE.

[0406] “Percentage of sequence identity” includes the value determined by comparing two optimally aligned sequences (greatest number of perfectly matched residues) over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise specified (e g., the shorter sequence includes a linked heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.

[0407] The terms “treat” or “treatment” of a state, disorder, disease, or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder, disease, or condition developing in a subject that may be afflicted with or predisposed to the state, disorder, disease, or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder, disease, or condition; or (2) inhibiting the state, disorder, disease, or condition, i.e., arresting, reducing, or delaying the development of the disease, or a relapse thereof, or at least one clinical or sub-clinical symptom thereof; or (3) relieving the state, disorder, disease, or condition, i.e., causing regression of the state, disorder, disease or condition or at least one of the clinical or sub-clinical symptoms of the state, disorder, disease or condition. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0408] The term “effective” as applied to a dose or an amount refers to the quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of thesubject, the severity of the condition being treated, the particular drug or drugs employed, the mode of administration, and the like.

[0409] The phrase “pharmaceutically acceptable”, as used in connection with compositions described herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0410] In accordance with the disclosure herein, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, NY : Cold Spring Harbor Laboratory Press, 1989 (herein “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins eds. (1985)]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel, F.M. 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), U. S. Patent No. 5,071, 743, Fukuoka et al., Biochem. Biophys. Res. Commun. 263: 357-360 (1999); Kim and Maas, BioTech. 28: 196- 198 (2000); Parikh and Guengerich, BioTech. 24: 4 28-431 (1998); Ray and Nickoloff, BioTech. 13: 342-346 (1992); Wang et al., BioTech. 19: 556-559 (1995); Wang and Malcolm, BioTech. 26: 680-682 (1999); Xu and Gong, BioTech. 26: 639-641 (1999), U.S. Patents Nos. 5,789, 166 and 5,932, 419, Hogrefe, Strategies 14. 3: 74-75 (2001), U. S. Patents 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.Recombinant Viruses

[0411] In one aspect, the present disclosure provides a recombinant virus comprising a virus genome, and further comprising a nucleotide sequence encoding, for example, a recombinant protein comprising, e.g., an IL-12 polypeptide, or a functional fragment or derivative thereof.

[0412] In some embodiments, the recombinant virus may be derived from a rhabdovirus. In some embodiments, the recombinant virus may comprise a rhabdovirus genome. The Rhabdoviridae family is mainly composed of a cage, bullet-shaped or bacilliform virus and has a negative-sense single-stranded RNA genome that infects vertebrates, invertebrates, or plants. Nonlimiting examples of rhabdoviruses that can be used in the present disclosure include rabies, cytolabudoviruses, dicholabdoviruses, ephemeraviruses, lyssaviruses, nobilabdoviruses, and vesiculoviruses. In some embodiments, a recombinant virus described herein is a vesiculovirus including, without limitation, a vesicular stomatitis virus (VSV).

[0413] In some embodiments, a recombinant virus (e.g., a recombinant rhabdovirus) described herein can be replication deficient, i.e., the recombinant virus can be non-replicative, and may only contain an incomplete genome of the virus from which it is derived. In some embodiments, the recombinant virus can be replication-competent. In certain aspects, a recombinant virus according to the disclosure (e.g., a recombinant rhabdovirus described herein) may be provided as an oncolytic virus.Oncolytic Viruses

[0414] Oncolytic virus therapy is an anti-cancer approach which uses viruses to selectively replicate and kill cancer cells without harming healthy cells. Such viruses are a powerful platform for oncolytic immunotherapy, which involves both direct tumor cell lysis and the induction of immune responses against tumor antigens. In general, viruses can enter cells at high efficiency. Viral genes are subsequently expressed, and the virus replicates. Viruses may also be used as delivery vehicles to express heterologous genes inserted into the viral genome in infected cells, and thus viruses such as oncolytic viruses can deliver encoded molecules which may help, for example, to selectively hone the systemic anti-tumor immune response.

[0415] In certain aspects, the recombinant virus according to the disclosure may be provided as an oncolytic virus. An oncolytic virus can be capable of infecting and replicating within tumor cells, such that the tumor cells are broken down, e.g., lysed, and killed. Therefore, an oncolytic virus of the disclosure may be replication competent. In some embodiments, the virus may beselectively replication competent in a specific tissue, for example, a tumor tissue. By way of a nonlimiting example, a virus is selectively replication competent in a tumor tissue if it replicates more effectively in a tumor tissue than in a non-tumor tissue. The ability of a virus to replicate in different tissue types may be determined using, for example, without limitation, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), electrophoresis, chromatography, flow cytometry, tunable resistive pulse sensing (TRPS), microscopy techniques (e.g., fluorescence microscopy, electron microscopy, including transmission electron microscopy), fluorescence in situ hybridization (FISH), infectivity assays, enzyme-linked immunosorbent assays (ELISAs), fluorescent-tagged antibody assays, and precipitation assays, loop-mediated amplification (LAMP) assay, plaque assay, focus forming assays (FFAs), 50% tissue culture infective dose (TCID50) assay, virus neutralization assays, homogenous virus assays, e.g., fluorescence polarization-based assays, AlphaScreen® / AlphaLISA® technologies, proximitybased assays, e.g. FRET, BRET, TR-FRET, time-resolved fluorescence (TRF), RNA dependent RNA polymerase assays, fluorescence in vitro and high throughput assays, cell-based microplate assays, protein assays (e.g., bicinchoninic acid assay, single radial immunodiffusion assay), and the like.

[0416] In some embodiments, oncolytic effects of the oncolytic virus disclosed herein may rely on the virus replicating in and killing infected cells (e.g., cancer cells including tumor cells). Progeny virions may then proceed to infect and kill other tumor cells, thereby spreading within the tumor. Hence, the ability of the oncolytic virus to effectively kill tumor cells and spread within tumors results in optimal anti-tumor effects. Efficient spread and virus replication can be associated with lysis of tumor cells, a process which may also maximize the quantity of tumor antigen released and therefore maximize the potency of the induced anti-tumor immune response.

[0417] An oncolytic virus (e.g., a recombinant rhabdovirus) disclosed herein may be unaltered from the parental virus species (i.e., wild-type), or with gene modifications, e.g., gene additions (i.e., mutant).

[0418] A rhabdovirus is member of the Rhabdoviridae family of viruses in the order Mononegavirales, encompassing more than 150 viruses of vertebrates, invertebrates, and plants. Examples of rhabdoviruses include rabies virus (RABV) from the Lyssavirus genus, vesiculoviruses from Vesiculovirus genus, the viral hemorrhagic septicemia virus (VHSV), and infectious hematopoietic necrosis virus, both from the Novirhabdovirus genus. Members of thegenus Lyssavirus may cause lethal meningoencephalitis in humans and animals while VSV (genus Vesiculovirus) may cause symptoms clinically identical to those of foot-and-mouth disease in cattle and occasional, limited infections in humans. Dimarhabdoviruses are the supergroup of rhabdoviruses that infect mammals and mosquitoes. Rhabdoviruses are bullet-shaped enveloped viruses with negative- sense single-stranded RNA genome 11-15 kb in length. The genome of rhabdoviruses can contain up to ten genes among which only five are common to all members of the family. These five common rhabdoviruses genes encode the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G), and the viral polymerase (also known as large protein) (L). The rhabdoviruses genome associates with N, L, and P to form the nucleocapsid, which is condensed by the M protein into a tightly coiled helical structure. The condensed nucleocapsid is surrounded by a lipid bilayer containing the viral glycoprotein G that constitutes the spikes that protrude from the viral surface. Rhabdoviruses enter a host cell via the endocytic pathway and subsequently fuse with the cellular membrane within the acidic environment of the endosome. Both receptor recognition and membrane fusion are mediated by a single transmembrane viral glycoprotein (G). Fusion between the viral envelope and the endosomal membrane is triggered via a low-pH induced (in the endosome) structural rearrangement of the G resulting in the release the viral genome and associated proteins into the cytoplasm of target cells.

[0419] In some embodiments, vesicular stomatitis virus (VSV), a member of the Rhabdoviridae family, can be a potential oncolytic virus. The advantages of VSV may include, for example: (i) an efficient life cycle, allowing for production of mature progeny virus in approximately 6 hours; (ii) multiple mechanisms of viral spread thereby ensuring rapid and efficient propagation of the virus; (iii) a large viral genome, allowing incorporation of substantial foreign DNA inserts; (iv) a lack of disease promotion in healthy humans; and, (v) use as a smallpox vaccine contributing to a considerable clinical knowledge base.

[0420] One aspect of the disclosure provides recombinant vesiculoviruses. Examples of vesiculoviruses that can be used in the present disclosure are listed in Table 1.Table 1. Examples of Vesiculoviruses

[0421] In some embodiments, an oncolytic virus, e.g., a recombinant rhabdovirus, disclosed herein can be a vesiculovirus such as, but not limited to, a vesicular stomatitis virus (VSV). In some embodiments, the VSV is a replication-competent VSV. In some embodiments, the VSV is non-replicative.

[0422] The genome of vesicular stomatitis virus (VSV), considered a prototypic rhabdovirus, comprises 11,161 nucleotides of negative-sense RNA. This RNA is tightly encapsidated by the viral nucleocapsid (N) polypeptide, and forms the template for the RNA-dependent RNA polymerase (RdRP), the viral components being a 241-kDa large protein (L) polypeptide and a phosphoprotein (P) polypeptide. The RdRP uses the RNA (viral) genome as a template for two reactions: (i) transcription of a short leader RNA (Le+) and 5 mRNAs that encode nucleoprotein (N) polypeptide, phosphoprotein (P) polypeptide, matrix (M) polypeptide, glycoprotein (G) polypeptide, and large protein (L) polypeptide; and (ii) replication to yield full-length antigenomic and then genomic strands.

[0423] In certain aspects, a recombinant rhabdovirus of the present disclosure may comprise a vesiculovirus genome, for example, a VSV genome, or any portion thereof. As a non-limiting example, a recombinant rhabdovirus genome may comprise genes which may encode, e.g., a VSV nucleoprotein (N) polypeptide, a VSV phosphoprotein (P) polypeptide, a VSV matrix (M) polypeptide (e.g., a wild-type VSV M polypeptide or a mutant VSV M polypeptide), a fusogenic polypeptide (e.g., a VSV glycoprotein (G) polypeptide), and / or a VSV large protein (L) polypeptide, or functional fragments or derivatives thereof.

[0424] In some embodiments, a recombinant rhabdovirus genome (e.g., a VSV genome) described herein comprises one or more genes encoding a VSV nucleoprotein (N) polypeptide, or a functional fragment or derivative thereof, a VSV phosphoprotein (P) polypeptide, or a functional fragment or derivative thereof, a VSV matrix (M) polypeptide, or a functional fragment or derivative thereof, a fusogenic polypeptide, or a functional fragment or derivative thereof, and / or a VSV large protein (L) polypeptide, or a functional fragment or derivative thereof. In some embodiments, the VSV matrix (M) polypeptide, or a functional fragment or derivative thereof, is a wild-type VSV M polypeptide. In some embodiments, the VSV matrix (M) polypeptide, or a functional fragment or derivative thereof, is a mutant VSV M polypeptide. In some embodiments, the fusogenic polypeptide is a VSV glycoprotein (G) polypeptide. In some embodiments, the fusogenic polypeptide may comprise, e.g., a combination of F and H polypeptides of a paramyxovirus.

[0425] In some embodiments, a VSV nucleoprotein (N) polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 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 the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the nucleotide sequence that encodes the VSV nucleoprotein (N) polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 1, 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 the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the nucleotide sequence that encodes the VSV nucleoprotein (N) polypeptide comprises the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 167, 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 the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO: 167. In certain embodiments, the VSV nucleoprotein (N) polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the nucleotide sequence that encodes the VSV nucleoprotein (N) polypeptide comprises the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 167.

[0426] In some embodiments, the VSV nucleoprotein (N) polypeptide comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0427] In some embodiments, a VSV phosphoprotein (P) polypeptide comprises the amino acid sequence of SEQ ID NO: 9, 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 the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the nucleotide sequence that encodes the VSV phosphoprotein (P) polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 9, 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 the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the nucleotide sequence that encodes the VSV phosphoprotein (P) polypeptide comprises the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 168, 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 the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 168. In certain embodiments, the VSV phosphoprotein (P) polypeptide comprises the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the nucleotide sequence that encodes the VSV phosphoprotein (P) polypeptide comprises the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 168.

[0428] In some embodiments, the VSV phosphoprotein (P) polypeptide comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0429] In some embodiments, a wild-type VSV matrix (M) polypeptide comprises the amino acid sequence of SEQ ID NO: 3, 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 the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the nucleotide sequence that encodes the wild-type VSV matrix (M) polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 3, 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 the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the nucleotide sequence that encodes the wild-type VSV matrix (M) polypeptide comprises the nucleotide sequence of SEQ ID NO: 4, 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 about80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about98% or at least about 99%, sequence identity with the nucleotide sequence of SEQ ID NO: 4. In certain embodiments, the wild-type VSV matrix (M) polypeptide comprises the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the nucleotide sequence that encodes the wild-type VSV matrix (M) polypeptide comprises the nucleotide sequence of SEQ ID NO: 4.

[0430] In some embodiments, the VSV M polypeptide comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0431] In some embodiments, a VSV glycoprotein (G) polypeptide comprises the amino acid sequence of SEQ ID NO: 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%, atleast 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 the amino acid sequence of SEQ ID NO: 92. In certain embodiments, the nucleotide sequence that encodes the VSV glycoprotein (G) polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 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 the amino acid sequence of SEQ ID NO: 92. In certain embodiments, the nucleotide sequence that encodes the VSV glycoprotein (G) polypeptide comprises the nucleotide sequence of SEQ ID NO: 93 or SEQ ID NO: 170, 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 the nucleotide sequence of SEQ ID NO: 93 or SEQ ID NO: 170. In certain embodiments, the VSV glycoprotein (G) polypeptide comprises the amino acid sequence of SEQ ID NO: 92. In certain embodiments, the nucleotide sequence that encodes the VSV glycoprotein (G) polypeptide comprises the nucleotide sequence of SEQ ID NO: 93 or SEQ ID NO: 170

[0432] In some embodiments, the VSV glycoprotein (G) polypeptide comprises the amino acid sequence of SEQ ID NO: 92, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 92.

[0433] In some embodiments, a VSV glycoprotein (G) polypeptide comprises the amino acid sequence of SEQ ID NO: 173, 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 the amino acid sequence of SEQ ID NO: 173. In certain embodiments, the nucleotide sequence that encodes the VSV glycoprotein (G) polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 173, ora 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 the amino acid sequence of SEQ ID NO: 173. In certain embodiments, the nucleotide sequence that encodes the VSV glycoprotein (G) polypeptide comprises the nucleotide sequence of SEQ ID NO: 171 or SEQ ID NO: 172, 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 the nucleotide sequence of SEQ ID NO: 171 or SEQ ID NO: 172. In certain embodiments, the VSV glycoprotein (G) polypeptide comprises the amino acid sequence of SEQ ID NO: 173. In certain embodiments, the nucleotide sequence that encodes the VSV glycoprotein (G) polypeptide comprises the nucleotide sequence of SEQ ID NO: 171 or SEQ ID NO: 172.

[0434] In some embodiments, the VSV glycoprotein (G) polypeptide comprises the amino acid sequence of SEQ ID NO: 173, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 173.

[0435] In some embodiments, an intravirion tail-VSVG polypeptide comprises the amino acid sequence of SEQ ID NO: 176, 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 the amino acid sequence of SEQ ID NO: 176. In certain embodiments, the nucleotide sequence that encodes the intravirion tail-VSVG polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 176, 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 the amino acid sequence of SEQ ID NO: 176. In certain embodiments, the nucleotidesequence that encodes the intravirion tail-VSVG polypeptide comprises the nucleotide sequence of SEQ ID NO: 174 or 175, 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 about80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about98% or at least about 99%, sequence identity with the nucleotide sequence of SEQ ID NO: 174 or175. In certain embodiments, the intravirion tail-VSVG polypeptide comprises the amino acid sequence of SEQ ID NO: 176. In certain embodiments, the nucleotide sequence that encodes the intravirion tail-VSVG polypeptide comprises the nucleotide sequence of SEQ ID NO: 174 or 175.

[0436] In some embodiments, the intravirion tail-VSVG polypeptide comprises the amino acid sequence of SEQ ID NO: 176, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 176.

[0437] In some embodiments, an hIL12p70 polypeptide comprises the amino acid sequence of SEQ ID NO: 154, 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 the amino acid sequence of SEQ ID NO: 154. In certain embodiments, the nucleotide sequence that encodes the hIL12p70 polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 154, 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 the amino acid sequence of SEQ ID NO: 154. In certain embodiments, the nucleotide sequence that encodes the hIL12p70 polypeptide comprises the nucleotide sequence of SEQ ID NO: 155 or 177, 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 the nucleotide sequence of SEQ ID NO: 155 or 177. In certainembodiments, the hlLl 2p70 polypeptide comprises the amino acid sequence of SEQ ID NO: 1 4. In certain embodiments, the nucleotide sequence that encodes the hIL12p70 polypeptide comprises the nucleotide sequence of SEQ ID NO: 155 or 177.

[0438] In some embodiments, the hIL12p70 polypeptide comprises the amino acid sequence of SEQ ID NO: 154, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 154.

[0439] In some embodiments, an linker polypeptide comprises the amino acid sequence of SEQ ID NO: 17, 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 the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the nucleotide sequence that encodes the linker polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 17, 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 the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the nucleotide sequence that encodes the linker polypeptide comprises the nucleotide sequence of SEQ ID NO: 179 or 180, 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 the nucleotide sequence of SEQ ID NO: 179 or 180. In certain embodiments, the linker polypeptide comprises the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the nucleotide sequence that encodes the linker polypeptide comprises the nucleotide sequence of SEQ ID NO: 179 or 180.

[0440] In some embodiments, the linker polypeptide comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 17.

[0441] In some embodiments, a VSV large protein (L) polypeptide comprises the amino acid sequence of SEQ ID NO: 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 the amino acid sequence of SEQ ID NO: 94. In certain embodiments, the nucleotide sequence that encodes the VSV large protein (L) polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 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 the amino acid sequence of SEQ ID NO: 94. In certain embodiments, the nucleotide sequence that encodes the VSV large protein (L) polypeptide comprises the nucleotide sequence of SEQ ID NO: 95, 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 the nucleotide sequence of SEQ ID NO: 95. In certain embodiments, the nucleotide sequence that encodes the VSV large protein (L) polypeptide comprises the nucleotide sequence of SEQ ID NO: 183 or 184, 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 nucleotide sequence of SEQ ID NO: 183 or 184. In certain embodiments, the VSV large protein (L) polypeptide comprises the amino acid sequence of SEQ ID NO: 94. In certain embodiments, the nucleotide sequence that encodes the VSV large protein (L) polypeptide comprises the nucleotide sequence of SEQ ID NO: 95. In certain embodiments, the nucleotide sequence that encodes the VSV large protein (L) polypeptide comprises the nucleotide sequence of SEQ ID NO: 183 or 184.

[0442] In some embodiments, the VSV large protein (L) polypeptide comprises the amino acid sequence of SEQ ID NO: 94, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 94.

[0443] In some embodiments, a VSV-M51R-hIL12p70 polypeptide comprises the amino acid sequence of SEQ ID NO: 113, 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 the amino acid sequence of SEQ ID NO: 113. In certain embodiments, the nucleotide sequence that encodes the VSV-M51R-hIL12p70 polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 113, 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 the amino acid sequence of SEQ ID NO: 113. In certain embodiments, the VSV- M51R-hIL12p70 polypeptide comprises the amino acid sequence of SEQ ID NO: 113.

[0444] In some embodiments, the VSV-M51R-hIL12p70 polypeptide comprises the amino acid sequence of SEQ ID NO: 113, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 113.

[0445] A rhabdovirus genome, e.g., a VSV genome, described herein can be genetically modified to include one or more mutations or “mutation classes” in the genome. “Mutation class”, “mutation classes” or “classes of mutation” are used interchangeably herein. Example mutation classes include, but are not limited to, a VSV temperature-sensitive N gene mutation, a temperature-sensitive L gene mutation, a point mutation, a G-stem mutation, a non-cytopathic M gene mutation, a gene shuffling or rearrangement mutation, a truncated G gene mutation, an ambisense RNA mutation, a G gene insertion mutation, a gene deletion mutation and the like. Mutations can be insertions, deletions, substitutions, gene rearrangement, or shuffling modifications.

[0446] Some mutations may attenuate the infectivity, virulence or pathogenic effects of the VSV. The attenuation can be additive or synergistic. With synergistic attenuation, the level ofVSV attenuation is greater than additive. Synergistic attenuation of VSV can arise from combining at least two classes of mutation in the same VSV genome, thereby resulting in a reduction of VSV pathogenicity much greater than an additive attenuation level observed for each VSV mutation class alone. A synergistic attenuation of VSV can provide for an LD50 at least greater than the additive attenuation level observed for each mutation class alone (i.e., the sum of the two mutation classes), where attenuation levels (i.e., the LD50) are determined in a small animal neurovirulence model.

[0447] The matrix (M) polypeptide of rhabdoviruses, such as VSVs, are small (approximately 20-25 kDa) multifunctional proteins that play a role in virus assembly, maturation, and budding (replication). The M protein also modulates the production of host and virus proteins, e.g., promoting viral egress from the host cell and cell death. From a structural standpoint, the M protein forms a layer between the glycoprotein- (G-) containing outer membrane and the nucleocapsid core which includes the virus nucleoprotein (N), large (L), phosphoprotein (P) and RNA (viral) genome.

[0448] The VSV M gene encodes the virus matrix (M) protein, and two smaller in-frame polypeptides (M2 and M3). The M2 and M3 polypeptides can be translated from the same open reading frame (ORF) as the M protein and lack the first 33 and 51 amino acids, respectively. A recombinant VSV vector comprising non-cytopathic M gene mutations (i.e., VSV vectors that also do not express M2 and M3 proteins) can be generated, and can further comprise one or more additional mutation(s) thereby resulting in a VSV vector that was highly attenuated in cell culture and in animals.

[0449] In certain embodiments, the recombinant rhabdovirus, e.g., recombinant VSV, described herein may comprise a non-cytopathic mutation in the M gene. The VSV (Indiana serotype) M gene encodes a 229 amino acid M (matrix) protein in which the first thirty amino acids of the NH2 -terminus comprise a proline-rich PPPY (SEQ ID NO: 187) (PY) motif. M is both a structural protein and inhibits nuclear export of host cell mRNAs. The PY motif of VSV M protein is located at amino acid positions 24-27 in both VSV Indiana (Genbank Accession Number X04452) and New Jersey (Genbank Accession Number M14553) serotypes. The VSV may comprise mutations in the PY motif (e.g., APPY (SEQ ID NO: 188), AAPY (SEQ ID NO: 189), PPAY (SEQ ID NO: 190), APPA (SEQ ID NO: 191), AAPA (SEQ ID NO: 192) and PPPA (SEQ ID NO: 193)). The VSV can comprise any of various amino acid mutations (e.g., deletions,substitutions, insertions, etc.) into the M protein PSAP (SEQ ID NO: 194) (PS) motif. These and other mutations in the PY motif may be effective to reduce virus yield by blocking a late stage in virus budding. A deletion mutant could be more disruptive to viral structure / function than a substitution mutant. Such disrupted viral structure / function could impact, for example, : a) replication kinetics, and b) other measures of viral fitness, i.e., the capacity of a virus to produce infectious progeny in a given environment.

[0450] The recombinant VSV described herein may comprise one or more M gene mutations. Non-limiting examples of M protein mutations include, e.g., a glycine changed to a glutamic acid at position (21), a leucine changed to a phenylalanine at position (111), a methionine changed to an arginine at position (51), a glycine changed to a glutamic acid at position (22), a methionine changed to an arginine at position (48), a leucine changed to a phenylalanine at position (110), a methionine changed to an alanine at position (51), and a methionine changed to an alanine at position (33). In various embodiments of the methods described herein, the genome of the recombinant VSV encodes a mutant VSV matrix M protein comprising the M51R variant M protein. The M51R mutation can eliminate the ability of the M protein to block cellular nucleo- cytoplasmic transport, and thus can substantially attenuate VSV infectivity.

[0451] In some embodiments, the VSV matrix (M) polypeptide is a mutant VSV M polypeptide. In some embodiments, the mutant VSV M polypeptide may comprise a mutation at methionine (M) 51 (M51R). In some embodiments, the mutation may comprise a substitution from methionine (M) to arginine I. In some embodiments, the mutant VSV M polypeptide may comprise a deletion at methionine (M) 51 (AM51). AM51 may also be referred to herein as deltaM51, dM51, A5 IM, or d5 IM, or the like.

[0452] In some embodiments, the mutant VSV matrix (M) polypeptide M51R comprises the amino acid sequence of SEQ ID NO: 5, 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 the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the nucleotide sequence that encodes the mutant VSV matrix (M) polypeptide M51R comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 5, or a variant thereof having at least about 50%, at least about 55%, at least about60%, 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 the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the nucleotide sequence that encodes the mutant VSV matrix (M) polypeptide M51R comprises the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 169, 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 the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 169. In certain embodiments, the mutant VSV matrix (M) polypeptide M51R comprises the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the nucleotide sequence that encodes the mutant VSV matrix (M) polypeptide M51R comprises the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 169.

[0453] In some embodiments, the VSV M polypeptide comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 5.

[0454] In some embodiments, the mutant VSV matrix (M) polypeptide AM51 comprises the amino acid sequence of SEQ ID NO: 7, 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 the amino acid sequence of SEQ ID NO:7. In certain embodiments, the nucleotide sequence that encodes the mutant VSV matrix (M) polypeptide AM51 comprises the nucleotide sequence that encodes the amino acid sequence of the amino acid sequence of SEQ ID NO: 7, 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 the amino acid sequence of SEQ ID NO:7. In certain embodiments, the nucleotide sequence that encodes the mutant VSV matrix (M)polypeptide AM51 comprises the nucleotide sequence of SEQ ID NO: 8, 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 the nucleotide sequence of SEQ ID NO: 8. In certain embodiments, the mutant VSV matrix (M) polypeptide AM51 comprises the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the nucleotide sequence that encodes the mutant VSV matrix (M) polypeptide AM51 comprises the nucleotide sequence of SEQ ID NO: 8.

[0455] In some embodiments, the VSV M polypeptide comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0456] In some embodiments, the VSV genome, provided herein that encodes a VSV N polypeptide, a VSV P polypeptide, a VSV M polypeptide, a VSV G polypeptide, and / or a VSV L polypeptide can be from a VSV Indiana strain as set forth in GenBank® Accession Nos. NC_001560 (GI No. 9627229) or can be from a VSV New Jersey strain.

[0457] In some embodiments, a VSV construct may comprise a hIL-12 gene inserted between a VSV M gene (e.g., a VSV M gene encoding a VSVAM51 protein) and a VSV P gene to generate a VSV-P-hIL12p70-AM51 construct.

[0458] In some embodiments, a VSV-M51R-hIL12p70 construct described herein exhibits increased efficacy compared to a similar construct having IL- 12 in a different position within the construct. In some embodiments, the VSV-M51R-hIL12p70 construct exhibits increased killing efficacy of cancer cells compared to the similar construct having IL-12 in a different position within the construct. In certain embodiments, the VSV-M5 !R-hIL12p70 construct exhibits at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, 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 95%, or at least about 100%, increased killing efficacy of cancer cells compared to the similar construct having IL-12 in a different position within the construct. In some embodiments, the VSV-M5 !R-hIL12p70 construct exhibits an enhanced replication rate compared to the similar construct having IL-12 in a differentposition within the construct. In certain embodiments, the VSV-M51R-hIL12p70 construct exhibits at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, 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 95%, or at least about 100%, enhanced replication rate compared to the similar construct having IL-12 in a different position within the construct.

[0459] In some embodiments, the VSV-M51R-hIL12p70 construct exhibits decreased toxicity compared to the similar construct having IL- 12 in a different position within the construct. In certain embodiments, the VSV-M5 !R-hIL12p70 construct exhibits at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, or at least about 100%, decreased toxicity compared to the similar construct having IL-12 in a different position within the construct.

[0460] In some embodiments, the VSV-M51R-hIL12p70 construct exhibits less IL-12 production compared to the similar construct having IL- 12 in a different position within the construct. In certain embodiments, the VSV-M51R-hIL12p70 construct exhibits at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%, less IL-12 production compared to the similar construct having IL- 12 in a different position within the construct. As a non-limiting example, the similar construct having IL- 12 in a different position within the construct may have IL-12 positioned between a polynucleotide encoding a VSV P protein and VSV M protein. In some embodiments, the similar construct having IL-12 in a different position within the construct can comprise a polynucleotide encoding a mutant M protein comprising, for example, a deletion at methionine (M) 51 (AM51).

[0461] In some embodiments, high levels of IL- 12 may be associated with, e.g., lymphopenia, which may be characterized by low white blood cell counts and / or low lymphocyte counts. In some embodiments, high levels of IL-12 may be associated with anemia, which may be characterized by low red blood cell counts and / or low hemoglobin..

[0462] In some embodiments, a VSV-M51R-hTL12p70 construct described herein exhibits increased efficacy compared to a similar construct having AM51 within the construct. In some embodiments, the VSV-M51R-hIL12p70 construct exhibits increased killing efficacy of cancer cells compared to the similar construct having AM51 within the construct. In certain embodiments, the VSV-M51R-hIL12p70 construct exhibits at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 100%, increased killing efficacy of cancer cells compared to the similar construct having AM51 within the construct. In some embodiments, the VSV-M51R- hIL12p70 construct exhibits an enhanced replication rate compared to the similar construct having AM51 within the construct. In certain embodiments, the VSV-M51R-hIL12p70 construct exhibits at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, 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 95%, or at least about 100%, enhanced replication rate compared to the similar construct having AM51 within the construct.

[0463] In some embodiments, the VSV-M51R-hIL12p70 construct exhibits decreased toxicity compared to the similar construct having AM51 within the construct. In certain embodiments, the VSV-M5 !R-hIL12p70 construct exhibits at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, 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 95%, or at least about 100%, decreased toxicity compared to the similar construct having AM51 within the construct.

[0464] In some embodiments, the VSV-M51R-hIL12p70 construct exhibits less IL-12 production compared to the similar construct having AM51 within the construct. In certain embodiments, the VSV-M51R-hIL12p70 construct exhibits at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%, less IL-12 production compared tothe similar construct having AM51 within the construct. Tn some embodiments, the similar construct having AM51 within the construct may have IL-12 positioned in a different position within the construct. In some embodiments, the similar construct having AM51 within the construct may have IL- 12 positioned between a polynucleotide encoding a VSV P protein and VSV M protein.

[0465] Sequences disclosed herein with respect to recombinant rhabdoviruses, e.g., recombinant VSVs, disclosed herein, can be incorporated into a plasmid coding for a positive sense cDNA of the viral genome allowing generation of, for example, a negative sense genome of the recombinant rhabdoviruses, e.g., recombinant VSVs. Thus, a nucleic acid sequence that encodes a VSV polypeptide, for example, can refer to a nucleotide sequence, e.g., an RNA sequence, that is the template for the positive sense transcript that encodes (e.g., via direct translation) that polypeptide.

[0466] In certain aspects, the present disclosure provides a recombinant rhabdovirus comprising an RNA molecule. In some embodiments, the RNA molecule may comprise, or consist essentially of, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV nucleoprotein (N) polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV phosphoprotein (P) polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV matrix (M) polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide (e.g., a VSV glycoprotein (G) polypeptide, or a combination of F and H polypeptides of a paramyxovirus), or a functional fragment or derivative thereof, and / or a nucleotide sequence that is a template for a positive sense transcript encoding a VSV large protein (L) polypeptide (polymerase), or a functional fragment or derivative thereof, or any combination thereof. In some embodiments, the RNA molecule may comprise a nucleotide sequence that is a template for a positive sense transcript encoding a recombinant protein, e.g., a recombinant protein comprising an IL-12 polypeptide, or the functional fragment or derivative thereof. In various embodiments, the recombinant protein may comprise a transmembrane domain (e.g., a transmembrane domain derived from a B7.1 antigen) described herein. In various embodiments, the recombinant protein may comprise a signal peptide which may be operably linked to the IL- 12 polypeptide. In some embodiments, the recombinant protein may further comprise one or more an additional polypeptide(s) such as, but not limited, to a cluster of differentiation 4 (CD4)polypeptide, or a functional fragment or derivative thereof, a cluster of differentiation 46 (CD46) polypeptide, or a functional fragment or derivative thereof, a collagen-binding domain (CBD) derived from von Willebrand factor (VWF), or a functional fragment or derivative thereof, an IgG2a polypeptide, or a functional fragment or derivative thereof, an IgGl polypeptide, or a functional fragment or derivative thereof, and / or an IgG4 polypeptide, or a functional fragment or derivative thereof. In some embodiments, the recombinant protein can be membrane bound. In some embodiments, the recombinant protein may not comprise a transmembrane domain and is not membrane bound. When the recombinant protein is not membrane bound, the recombinant protein may be a soluble and / or secreted protein.

[0467] In various embodiments, the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein is positioned in one of the following positions: between the nucleotide sequence that is a template for a positive sense transcript encoding the fusogenic polypeptide (e.g., a VSV glycoprotein (G) polypeptide), or the functional fragment or derivative thereof, and the nucleotide sequence that is a template for a positive sense transcript encoding the VSV L polypeptide, or a functional fragment or derivative thereof; at the 5’ end of the nucleotide sequence that is a template for a positive sense transcript encoding the VSV L polypeptide, or the functional fragment or derivative thereof; at the 3’ end of the nucleotide sequence that is a template for a positive sense transcript encoding the VSV N polypeptide, or the functional fragment or derivative thereof; between the nucleotide sequence that is a template for a positive sense transcript encoding the VSVN polypeptide, or the functional fragment or derivative thereof, and the nucleotide sequence that is a template for a positive sense transcript encoding the VSV P polypeptide, or the functional fragment or derivative thereof; or between the nucleotide sequence that is a template for a positive sense transcript encoding the VSV P polypeptide, or the functional fragment or derivative thereof, and the nucleotide sequence that is a template for a positive sense transcript encoding the VSV M polypeptide, or the functional fragment or derivative thereof.

[0468] In some embodiments, the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein is positioned between the nucleotide sequence that is a template for a positive sense transcript encoding the fusogenic polypeptide (e.g., a VSV glycoprotein (G) polypeptide), or the functional fragment or derivative thereof, and the nucleotidesequence that is a template for a positive sense transcript encoding the VSV L polypeptide, or a functional fragment or derivative thereof.

[0469] In some embodiments, the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein is positioned at the 5’ end of the nucleotide sequence that is a template for a positive sense transcript encoding the VSV L polypeptide, or the functional fragment or derivative thereof.

[0470] In some embodiments, the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein is positioned at the 3’ end of the nucleotide sequence that is a template for a positive sense transcript encoding the VSV N polypeptide, or the functional fragment or derivative thereof.

[0471] In some embodiments, the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein is positioned between the nucleotide sequence that is a template for a positive sense transcript encoding the VSV N polypeptide, or the functional fragment or derivative thereof, and the nucleotide sequence that is a template for a positive sense transcript encoding the VSV P polypeptide, or the functional fragment or derivative thereof.

[0472] In some embodiments, the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein is positioned between the nucleotide sequence that is a template for a positive sense transcript encoding the VSV P polypeptide, or the functional fragment or derivative thereof, and the nucleotide sequence that is a template for a positive sense transcript encoding the VSV M polypeptide, or the functional fragment or derivative thereof.

[0473] In some embodiments, the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein comprises, from the 3’ end to the 5’ end of said nucleotide sequence: a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide (e.g., a VSV glycoprotein (G) polypeptide), or a functional fragment or derivative thereof, the nucleotide sequence that is a template for the positive sense transcript encoding the recombinant protein, and a nucleotide sequence that is a template for a positive sense transcriptencoding VSV L polypeptide, or a functional fragment or derivative thereof; a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide (e.g., a VSV glycoprotein (G) polypeptide), or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, or a functional fragment or derivative thereof, and the nucleotide sequence that is a template for the positive sense transcript encoding the recombinant protein; the nucleotide sequence that is a template for the positive sense transcript encoding the recombinant protein, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide (e.g., a VSV glycoprotein (G) polypeptide), or a functional fragment or derivative thereof, and a nucleotide sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, or a functional fragment or derivative thereof; a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, the nucleotide sequence that is a template for the positive sense transcript encoding the recombinant protein, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide (e.g., a VSV glycoprotein (G) polypeptide), or a functional fragment or derivative thereof, and a nucleotide sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, or a functional fragment or derivative thereof; or a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, a nucleotidesequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, the nucleotide sequence that is a template for the positive sense transcript encoding the recombinant protein, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide (e.g., a VSV glycoprotein (G) polypeptide), or a functional fragment or derivative thereof, and a nucleotide sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, or a functional fragment or derivative thereof.

[0474] In some embodiments, the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein comprises, from the 3’ end to the 5’ end of said nucleotide sequence, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide (e.g., a VSV glycoprotein (G) polypeptide), or a functional fragment or derivative thereof, a nucleotide sequence that is the template for a positive sense transcript encoding the recombinant protein, and a VSV L polypeptide, or a functional fragment or derivative thereof.

[0475] In some embodiments, the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein comprises, from the 3’ end to the 5’ end of said nucleotide sequence, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide (e.g., a VSV glycoprotein (G) polypeptide), or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, or a functional fragment or derivative thereof, and the nucleotide sequence that is a template for the positive sense transcript encoding the recombinant protein.

[0476] In some embodiments, the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein comprises, from the 3’ end to the 5’ end of said nucleotide sequence, a nucleotide sequence that is the template for a positive sense transcript encoding the recombinant protein, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide (e.g., a VSV glycoprotein (G) polypeptide), or a functional fragment or derivative thereof, and a nucleotide sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, or a functional fragment or derivative thereof

[0477] In some embodiments, the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein comprises, from the 3’ end to the 5’ end, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, the nucleotide sequence that is a template for the positive sense transcript encoding the recombinant protein, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide (e.g., a VSV glycoprotein (G) polypeptide), or a functional fragment or derivative thereof, and a nucleotide sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, or a functional fragment or derivative thereof.

[0478] In some embodiments, the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein comprises, from the 3’ end to the 5’ end of said nucleotide sequence, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, the nucleotide sequence that is a template for the positive sense transcript encoding the recombinant protein, a nucleotide sequence that is a template for apositive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide (e.g., a VSV glycoprotein (G) polypeptide), or a functional fragment or derivative thereof, and a nucleotide sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, or a functional fragment or derivative thereof.

[0479] In certain embodiments, recombinant viruses (e.g., recombinant VSVs) of the present disclosure may provide one or more of any viral genes described herein (e.g., genes encoding a VSV N polypeptide, a VSV P polypeptide, a VSV M polypeptide, a VSV G polypeptide, and / or a VSV L polypeptide, or functional fragments or derivatives thereof), and / or one or more of any nucleotides described herein (e.g., nucleotides encoding recombinant proteins comprising an IL- 12 polypeptide, or a functional fragments or derivatives thereof, or additional polypeptides) in trans, and thus the one or more viral genes and / or nucleotides may be provided in a separate virus from any of the other viral genes and / or nucleotides.

[0480] Any appropriate method may be used to insert a nucleic acid disclosed herein (e.g., nucleotide sequence that is a template for a positive sense transcript encoding a polypeptide such as, but not limited to, a virus polypeptide, and / or a nucleotide sequence that is a template for a positive sense transcript encoding a virus polypeptide) into the genome of a recombinant virus of the present disclosure. For example, any of various nucleic acid generation and / or amplification techniques via, e g., polymerase chain reaction (PCR) and the like, including from a plasmid, gene excision, reverse genetics, cloning, and / or subcloning steps, virus preparation, growth, propagation, and / or recovery, and / or cell culture approaches comprising, e.g., infection, transduction and / or transfection, or combinations thereof, may be used to insert nucleic acid into the genome of a recombinant virus. Any appropriate method may be used to identify recombinant virus-containing a nucleic acid molecule disclosed herein. Non-limiting examples of such methods include and nucleic acid hybridization techniques including Northern and Southern analysis, and polymerase chain reaction (PCR). In some embodiments, biochemical techniques and / or immunohistochemistry may be used to determine if a recombinant virus contains a specific nucleic acid molecule by detecting the expression of a polypeptide encoded by that specific nucleic acid molecule.

[0481] In some embodiments, upon infection of a cell with a recombinant virus, e.g., a recombinant rhabdovirus, described herein, the cell can express a recombinant protein comprisingthe IL-12 polypeptide, or a functional fragment or derivative thereof, described herein. The cell can be a cancer cell (e.g., a tumor cell). The cancer cell can be a human cell.Fusogenic Molecules

[0482] In some embodiments, the recombinant virus disclosed herein may comprise a rhabdovirus genome, e.g., a vesiculovirus genome such as, but not limited to, a VSV genome, which may comprise fusogenic molecule, or fusogen, e.g., a fusogenic polypeptide. A “fusogen” or “fusogenic molecule” may refer to any molecule that can trigger membrane fusion when present on the surface of a virus. In some embodiments, a fusogen may act on the cell membrane to prevent spontaneous membrane fusion and promote fusion that may occur in a controlled and / or regulated manner. Upon activation, a fusogen may extend trimers anchored at one end by their transmembrane domains and expose an amphiphilic loop or hydrophobic fusion peptide that inserts into the target membrane. At this time, the two interacting domains are positioned in different membranes. Regulated refolding of the fusogenic complex into a hairpin-like structure then brings the fusion peptide and transmembrane domains to the same end of the molecule, which generates a pulling force that brings the two membranes into close (approximately 1 nm) apposition. The accumulated energy from this event is thought to drive fusion through the formation of a hemifusion stalk-like connection, where only the contacting proximal leaflets of the membranes are fused while the inner leaflets remain intact. Expansion of the hemifusion stalk, and the subsequent fusion of the distal leaflets, completes the reaction by opening a fusion pore that permits the contents of the two compartments to mix. Fusion pore expansion is considered a final energy barrier before membrane fusion becomes permanent. Without wishing to be bound by theory, there is evidence that viral fusogens mediate fusion via hemifusion. Many different protein and non-protein fusogenic molecules may be used herein. In some embodiments, the fusogenic molecule is a fusogenic protein or polypeptide.

[0483] In some embodiments, the fusogenic molecule is a viral fusogenic molecule. Nonlimiting examples of viral fusogenic molecules include, e.g., vesiculovirus fusogens (e.g., vesicular stomatitis virus G glycoprotein, alphavirus fusogens (e.g., a Sindbis virus glycoprotein), orthomyxovirus fusogens (e.g., influenza HA protein), paramyxovirus fusogens (e.g., a Nipah virus F protein or a measles virus F protein), as well as fusogens from Dengue virus (DV), Lassa fever virus, tick-borne encephalitis virus, Dengue virus, Hepatitis B virus, Rabies virus, SemlikiForest virus, Ross River virus, Aura virus, Boma disease virus, Hantaan virus, SARS-CoV virus, and various fragments, mutants, and derivatives thereof.

[0484] In some embodiments, the fusogenic molecule is heterologous to the virus from which the virus is derived. In some embodiments, the fusogenic molecule is a mutated protein which does not bind the fusogenic molecule’s natural ligand(s).

[0485] The recombinant virus disclosed herein may comprise multiple copies of the fusogenic polypeptide-encoding gene, preferably 1 or 2 copies. The virus may comprise two or more different fusogenic polypeptides, including, without limitation, any of the fusogenic polypeptides listed above. The fusogenic polypeptide or polypeptides expressed by a virus disclosed herein may be identical to a naturally occurring polypeptide, or may be a modified polypeptide. The fusogenic polypeptide-encoding gene (fusogenic gene) may have a naturally occurring nucleic acid sequence or a modified sequence. The sequence of the fusogenic gene may, for example, be modified to increase the fusogenic properties of the encoded polypeptide, or to provide codon optimization, and therefore increase the efficiency of expression of the encoded polypeptide.

[0486] In some embodiments, mutant fusogens are used which maintain their fusogenic ability but have a decreased or eliminated binding ability or specificity. Functional properties of mutant fusogens can be tested, e.g., in cell culture or by determining their ability to stimulate an immune response without causing undesired side effects in vivo.

[0487] To select the most effective and non-toxic combinations of fusogens (either wild-type or mutant), viruses bearing such molecules can be tested for selectivity and / or ability to facilitate penetration of the target cell membrane. Viruses that display wild-type fusogens can be used as controls for examining titer effects in mutants. For example, cells can be transduced by the viruses using a standard infection assay. After a specified time, for example, 48 hours post-transduction, cells can be collected and the percentage of transduced cells can be determined by, for example, monitoring reporter gene expression (e.g., using FACS analysis). The selectivity can be scored by calculating the percentage of cells infected by the viruses. Similarly, the effect of mutations on viral titer can be quantified by dividing the percentage of cells infected by viruses comprising a mutant targeting molecule by the percentage of cells infected by virus comprising the corresponding wild type targeting molecule. The titers of viruses can be determined, e.g., by limited dilution of the stock solution and transduction of cells expressing a polypeptide of interest.

[0488] To investigate whether fusogen-mediated cell penetration is dependent upon pH, and to select fusogens with the desired pH dependence, NH4CI or another compound that alters pH can be added at the infection step (NH4CI will neutralize the acidic compartments of endosomes). In the case of NH4CI, the disappearance of cells expressing the reporter will indicate that penetration of viruses is acidic pH-dependent. In addition, to confirm that the activity is pH-dependent, lysosomotropic agents, such as ammonium chloride, chloroquine, concanamycin, bafilomycin Al, monensin, nigericin, etc., may be added into the incubation buffer. These agents can elevate the pH within the endosomal compartments. The inhibitory effect of these agents can reveal the role of pH for viral fusion and entry. The different entry kinetics between viruses displaying different fusogenic molecules may be compared and the most suitable selected for a particular application.

[0489] PCR-based viral entry assays may be utilized to measure kinetics of viral DNA synthesis as an indication of the kinetics of viral entry. For example, viruses comprising a particular TCR-binding molecule and fusogen can be incubated with target cells, unbound viruses can be then removed, and aliquots of the cells can be analyzed by extracting DNA and performing semi-quantitative PCR. The appearance of virus-specific DNA products will indicate the success of viral entry and uncoating.

[0490] The fusogen preferably exhibits fast enough kinetics such that the virus contents can empty into the cytosol before the degradation of the virus. In addition, the fusogen can be modified to reduce or eliminate any binding activity and thus reduce or eliminate any non-specific binding. Recombinant Proteins

[0491] In various aspects, a recombinant virus, e.g., a recombinant rhabdovirus, of the present disclosure, comprises a nucleotide sequence encoding a recombinant protein. In some embodiments, the recombinant protein comprises an interleukin- 12 (IL-12) polypeptide, or a functional fragment or derivative thereof. In some embodiments, the recombinant protein comprises an IL-12 polypeptide and a transmembrane domain. In some embodiments, the recombinant protein does not comprise a transmembrane domain. In some embodiments, the recombinant protein is membrane bound. In some embodiments, the recombinant protein is not membrane bound. When the recombinant protein is not membrane bound, the recombinant protein may be a soluble and / or secreted protein.

[0492] In some embodiments, the nucleotide sequence encoding the recombinant protein described herein is a template for a positive sense transcript encoding the recombinant protein.

[0493] In some embodiments, the nucleotide sequence encoding the IL-12 polypeptide, or a functional fragment or derivative thereof is a template for a positive sense transcript encoding the IL- 12 polypeptide, or the functional fragment or derivative thereof.

[0494] In some embodiments, when the recombinant virus infects a cell, e.g., a host cell such as, but not limited to, a mammalian host cell, the cell expresses the IL- 12 polypeptide, or functional fragment or derivative thereof. In some embodiments, the cell may be a human cell. In some embodiments, the cell may be a cancer cell, e.g., a tumor cell.IL- 12 Polypeptides

[0495] Interleukin- 12 (IL-12) is a heterodimeric molecule composed of an alpha chain (the p35 subunit) and a beta chain (the p40 subunit) which are covalently linked via a disulfide bridge to form a biologically active 70 kDa dimer. Without wishing to be bound by theory, IL-12 is an inflammatory cytokine that may be produced, for example, in response to infection by various immune cells including, but not limited to, B cells, phagocytic cells, and dendritic cells. IL-12 plays a key role in modulating the interaction between the adaptive and innate divisions of the immune system. As an example, IL-12 has been shown to act on T-cells and natural killer (NK) cells, enhance proliferation and activity of cytotoxic lymphocytes, as well as participate in the production of other inflammatory cytokines, in particular, IFNy. IL-12 has been tested in human clinical trials as an immunotherapeutic agent for the treatment of a number of cancers such as, but not limited to, ovarian cancer, colon cancer, renal cancer, melanoma, and T-cell lymphoma. Further, IL-12 has been tested as an adjuvant for cancer vaccines. IL-12 gene therapy approaches may allow cytokine production at the tumor site to achieve high local levels of IL-12 with low serum concentration, thereby maximizing the anti-tumoral effect of IL-12 while minimizing the systemic toxicity of IL- 12.

[0496] A polypeptide (also referred to as a protein, or peptide herein) of the present disclosure (e.g., an IL-12 polypeptide) may include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. A polypeptide may also include polymers that have been modified, such as polypeptides having modified peptide backbones. A polypeptide typically has an N-terminus and a C-terminus. The N-terminus is the start of an amino acid chain of a protein, terminated by an amino acid with a free amine group (-NH2). The C-terminus is the end of an amino acid chain of a protein, terminated by a free carboxyl group (-COOH).

[0497] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, described herein may function as an immune stimulatory molecule.

[0498] In some embodiments, the IL-12 polypeptide of the present disclosure, or the functional fragment or derivative thereof, comprises an IL- 12 p40 subunit, or a functional fragment or derivative thereof. In some embodiments, the IL- 12 polypeptide, or functional fragment of derivative thereof, further comprises an IL-12 p35 subunit, or a functional fragment or derivative thereof. In some embodiments, the IL- 12 polypeptide, or the functional fragment or derivative thereof, comprises an IL-12 p35 subunit, or a functional fragment or derivative thereof.

[0499] In some embodiments, the IL- 12 polypeptide, or functional fragment or derivative thereof, may be derived from a rodent, e.g., a mouse. In some embodiments, the IL- 12 polypeptide, or functional fragment or derivative thereof, may be derived from a human. In some embodiments, the IL-12 p40 subunit, or functional fragment or derivative thereof, may be derived from a rodent, e.g., a mouse. In some embodiments, the IL-12 p40 subunit, or functional fragment or derivative thereof, may be derived from a human. In some embodiments, the IL-12 p35 subunit, or functional fragment or derivative thereof, may be derived from a rodent, e.g., a mouse. In some embodiments, the IL-12 p35 subunit, or functional fragment or derivative thereof, may be derived from a human.

[0500] In some embodiments, the IL- 12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 69, 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 the amino acid sequence of SEQ ID NO: 69. In certain embodiments, the nucleotide sequence that encodes IL-12 p40 subunit protein comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 69, 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 the amino acid sequence of SEQ ID NO: 69. In certain embodiments, the nucleotide sequence that encodes the IL- 12 p40 subunit protein comprises the nucleotide sequence of SEQ ID NO: 70, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about70%, 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 the nucleotide sequence of SEQ ID NO: 70. In certain embodiments, the IL-12 p40 subunit protein comprises the amino acid sequence of SEQ ID NO: 69. In certain embodiments, the nucleotide sequence that encodes the IL- 12 p40 subunit protein comprises the nucleotide sequence of SEQ ID NO: 70.

[0501] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the IL-12 p40 subunit, or the functional fragment or derivative thereof comprises the amino acid sequence of SEQ ID NO: 69. In some embodiments, the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 69.

[0502] In some embodiments, the IL-12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 61, 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 the amino acid sequence of SEQ ID NO: 61. In certain embodiments, the nucleotide sequence that encodes IL-12 p40 subunit protein comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 61, 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 about75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98% or at least about 99%, sequence identity with the amino acid sequence ofSEQ ID NO: 61. In certain embodiments, the nucleotide sequence that encodes the IL-12 p40 subunit protein comprises the nucleotide sequence of SEQ ID NO: 62, 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 the nucleotide sequence of SEQ ID NO: 62. In certain embodiments, the IL- 12 p40 subunit proteincomprises the amino acid sequence of SEQ ID NO: 61. In certain embodiments, the nucleotide sequence that encodes the IL-12 p40 subunit protein comprises the nucleotide sequence of SEQ ID NO: 62.

[0503] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 61. In some embodiments, the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61. In some embodiments, the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 61.

[0504] In some embodiments, the IL- 12 p40 subunit, or functional fragment or derivative thereof, may comprise one or more amino acid substitutions and / or insertions and / or deletions at one or more locations in the amino acid sequence, e.g., as compared to an amino acid sequence of a reference IL-12 p40 subunit. For example, the IL-12 p40 subunit may include a substitution(s) of one or more amino acids in the amino acid sequence of a parent IL-12 p40 subunit with a similar or homologous amino acid(s) or a dissimilar amino acid(s).

[0505] In some embodiments, the amino acid positions of one or more amino acid substitutions and / or insertions and / or deletions at one or more locations in the amino acid sequence of an IL-12 p40 subunit, or a functional fragment or derivative thereof described herein, may be described in reference to the amino acid positions of an IL- 12 p40 subunit reference sequence. In some embodiments, the IL-12 p40 subunit reference sequence may comprise a precursor form of an IL- 12 p40 subunit described herein (i.e., an IL-12 p40 subunit precursor form). In some embodiments, the precursor form of the IL-12 p40 subunit may comprise a signal peptide described herein such as, but not limited to, the amino acid sequence MCPQKLTISWFAIVLLVSPLMA (SEQ ID NO: 31), or a fragment or derivative thereof. In some embodiments, the precursor form of the IL-12 p40 subunit may comprise a signal peptide described herein such as, but not limited to, the amino acid sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO: 65), or a fragment or derivative thereof.

[0506] In some embodiments, the IL-12 p40 subunit reference sequence can comprise a precursor form of an IL-12 p40 subunit disclosed herein, for example, the sequence of SEQ ID NO: 27. In some embodiments, the amino acid positions of the one or more amino acid substitutions and / or insertions and / or deletions at one or more locations in the amino acid sequenceof the IL-12 p40 subunit, or functional fragment or derivative thereof, may be described in reference to the amino acid positions in the amino acid sequence of SEQ ID NO: 27.

[0507] In some embodiments, the IL-12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 27, 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 the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the nucleotide sequence that encodes IL- 12 p40 subunit comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 27, 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 about75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98% or at least about 99%, sequence identity with the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the nucleotide sequence that encodes the IL- 12 p40 subunit comprises the nucleotide sequence of SEQ ID NO: 28, 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 the nucleotide sequence of SEQ ID NO: 28. In certain embodiments, the IL-12 p40 comprises the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the nucleotide sequence that encodes the IL- 12 p40 subunit comprises the nucleotide sequence of SEQ ID NO: 28.

[0508] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 27.

[0509] In some embodiments, the IL-12 p40 subunit reference sequence can comprise a precursor form of an IL-12 p40 subunit disclosed herein, for example, the sequence of SEQ ID NO: 12. In some embodiments, the amino acid positions of the one or more amino acidsubstitutions and / or insertions and / or deletions at one or more locations in the amino acid sequence of the IL-12 p40 subunit, or functional fragment or derivative thereof, may be described in reference to the amino acid positions in the amino acid sequence of SEQ ID NO: 12.

[0510] In some embodiments, the IL- 12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 12, 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 the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the nucleotide sequence that encodes IL-12 p40 subunit comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 12, 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 the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the nucleotide sequence that encodes the IL-12 p40 subunit comprises the nucleotide sequence of SEQ ID NO: 13 or 14, 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 the nucleotide sequence of SEQ ID NO: 13 or 14. In certain embodiments, the nucleotide sequence that encodes the IL-12 p40 subunit comprises the nucleotide sequence of SEQ ID NO: 156 or 178, 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 the nucleotide sequence of SEQ ID NO: 156 or 178. In certain embodiments, the IL-12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the nucleotide sequence that encodes the IL-12 p40 subunit comprises thenucleotide sequence of SEQ ID NO: 13 or 14. In certain embodiments, the nucleotide sequence that encodes the IL-12 p40 subunit comprises the nucleotide sequence of SEQ ID NO: 156 or 178.

[0511] In some embodiments, the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the IL-12 p40 subunit, or the functional fragment or derivative thereof comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 12.

[0512] In some embodiments, an IL- 12 p40 subunit reference sequence may comprise a mature form of an IL-12 p40 subunit described herein (i.e., an IL-12 p40 subunit mature form). In some embodiments, the sequence of the mature form of the IL- 12 p40 subunit may be shorter in length than the sequence of the precursor form of the IL- 12 p40 subunit described herein owing, at least in part, to the absence of a signal peptide from the mature form which may be present in the precursor form. In some embodiments, the IL-12 p40 subunit reference sequence can comprise a mature form an IL-12 p40 subunit, for example, the sequence of SEQ ID NO: 69. In some embodiments, the amino acid positions of the one or more amino acid substitutions and / or insertions and / or deletions at one or more locations in the amino acid sequence of the IL-12 p40 subunit, or functional fragment or derivative thereof, may be described in reference to the amino acid positions in the amino acid sequence of SEQ ID NO: 69. In some embodiments, the IL-12 p40 subunit reference sequence can comprise a mature form an IL-12 p40 subunit, for example, the sequence of SEQ ID NO: 61. In some embodiments, the amino acid positions of the one or more amino acid substitutions and / or insertions and / or deletions at one or more locations in the amino acid sequence of the IL- 12 p40 subunit, or functional fragment or derivative thereof, may be described in reference to the amino acid positions in the amino acid sequence of SEQ ID NO: 61.

[0513] In some embodiments, the IL-12 p40 subunit of the present disclosure may include any amino acid sequence having an identity of at least about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more,99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more to its natural amino acid sequence or parental sequence, and having the activity of normally-occurring (i.e., natural) sequence or parental sequence.

[0514] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof comprises amino acid change(s) in one or more positions. Non-limiting examples of amino acid changes comprise amino acid substitutions and / or insertions and / or deletions. In some embodiments, the IL-12 p40 subunit, or the functional fragment or derivative thereof comprises amino acid substitution(s) in one or more positions. As a non-limiting example, the IL-12 p40 subunit, or the functional fragment or derivative thereof may comprise amino acid substitution(s) in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 or more positions.

[0515] In some embodiments, the IL- 12 p40 subunit described herein may comprise one or more amino acid substitutions and / or insertions and / or deletions. Amino acid substitution means that an amino acid residue is substituted for a replacement 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 immediately adjacent one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent another inserted amino acid residue. In various embodiments, one or more amino acids may be substituted and / or inserted and / or deleted from the sequence of SEQ ID NO: 27. In various embodiments, one or more amino acids may be substituted and / or inserted and / or deleted from the sequence of SEQ ID NO: 69. In various embodiments, one or more amino acids may be substituted and / or inserted and / or deleted from the sequence of SEQ ID NO: 12. In various embodiments, one or more amino acids may be substituted and / or inserted and / or deleted from the sequence of SEQ ID NO: 61.

[0516] A person with ordinary skill of the art will be able to identify the one or more amino acid changes (e.g., amino acid substitutions and / or insertions and / or deletions) in an IL-12 p40 subunit precursor form sequence described herein (e.g., SEQ ID NO: 12 or 27) within a shorter IL- 12 p40 subunit mature form sequence described herein (e.g., SEQ ID NO: 61 or 69) which lacks a signal peptide (e.g., SEQ ID NO: 31 or 65). Any of the amino acid change(s) described in reference to an IL- 12 p40 polypeptide comprising an IL- 12 p40 subunit mature form sequence described herein(e.g., SEQ ID NO: 69 or 61) may be applied to an IL-12 p40 polypeptide comprising a precursor form sequence of an IL-12 p40 subunit described herein (e.g., SEQ ID NO: 12 or 27), or vice versa.

[0517] A person with ordinary skill of the art will further be able to identify the one or more amino acid changes (e.g., amino acid substitutions and / or insertions and / or deletions) in an IL-12 p40 mature or precursor form sequence derived from a human described herein (e.g., SEQ ID NO: 61 or 12, respectively) within an IL- 12 p40 mature or precursor form sequence derived from a mouse described herein (e g., SEQ ID NO: 69 or 27, respectively). Any of the amino acid change(s) described in reference to an IL-12 p40 polypeptide comprising a mature or precursor form sequence of an IL-12 p40 subunit derived from a human (e.g., SEQ ID NO: 61 or 12, respectively), may also be applied to an IL-12 p40 polypeptide comprising a mature or precursor form sequence of an IL- 12 p40 subunit derived from a mouse (e.g., SEQ ID NO: 69 or 27, respectively), or vice versa.

[0518] In some embodiments, the IL-12 p40 subunit may comprise a substitution at any amino acid contained therein. In some embodiments, the IL-12 p40 subunit may comprise an insertion at any amino acid contained therein. In some embodiments, the IL- 12 p40 subunit may comprise a deletion at any amino acid contained therein.

[0519] In certain embodiments, amino acid changes (e.g., substitutions) to a protein or portion thereof are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, or (4) confer or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the normally-occurring (i.e., natural) sequence or parental sequence. In some embodiments, the amino acid sequence of the IL- 12 p40 subunit disclosed herein may be changed, for example, to increase activity and / or reduce toxicity of the IL-12 polypeptide disclosed herein. In some embodiments, the amino acid sequence of IL- 12 p40 subunit disclosed herein may be changed such that the activity of the IL- 12 polypeptide is attenuated, for example, for intravenous (IV) delivery.

[0520] By way of a non-limiting example, for the activity of the IL- 12 p40 subunit before the substitution, insertion, and / or deletion described herein, the activity of the IL-12 p40 subunit protein or amino acid sequence after the substitution, insertion, and / or deletion is increased by more than 2%, more than 5%, more than 10%, more than 20%, more than 40%, more than 60%,more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or 100% or more as compared to the activity before the substitution, deletion, insertion or addition. By way of another non-limiting example, for the toxicity of the IL- 12 p40 subunit before the substitution, insertion, and / or deletion described herein, the toxicity of the IL- 12 p40 subunit protein or amino acid sequence after the substitution, insertion, and / or deletion is reduced by more than 2%, more than 5%, more than 10%, more than 20%, more than 40%, more than 60%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or 100% or more as compared to the toxicity before the substitution, deletion, insertion and / or addition.

[0521] In some embodiments, the amino acid sequence of the IL- 12 p40 subunit may be modified to comprise one or more amino acid changes (e.g., substitutions), that may decrease susceptibility of the polypeptide to proteolysis and / or increase IL-12 biological half-life. In some embodiments, the amino acid sequence of the IL-12 p40 subunit may be modified to comprise one or more amino acid changes (e.g., substitutions), that may decrease susceptibility of the polypeptide to proteolysis and / or reduce IL-12 biological half-life.

[0522] In some embodiments, the IL-12 p40 subunit disclosed herein may also include conservative modifications and / or substitutions at other positions of IL-12 p40. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence. For example, amino acids belonging to one of the following groups represent conservative changes: Group I: Ala, Pro, Gly, Gin, Asn, Ser, Thr; Group II: Cys, Ser, Tyr, Thr; Group III: Vai, He, Leu, Met, Ala, Phe; Group IV: Lys, Arg, His; Group V: Phe, Tyr, Trp, His; and Group VI: Asp, Glu.

[0523] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof may comprise one or more amino acid substitutions at a position corresponding to an amino acid residue including, for example, without limitation positions 37, 39, 40, 41, 80, 81, 82, 106, 108, 115, 216, 217, 218, and 219 in the amino acid sequence of SEQ ID NO: 12. As a non-limiting example, the amino acid substitutions(s) may comprise an alanine (A) substitution, an arginine I substitution, an asparagine (N) substitution, an aspartic acid (D) substitution, a leucine (L) substitution, a lysine (K) substitution, a phenylalanine (F) substitution, a glutamine (Q) substitution, a glutamic acid I substitution, a serine (S) substitution, and a threonine (T) substitution, or any combination thereof.

[0524] In some embodiments, the one or more amino acid substitution is at a position corresponding to an amino acid residue selected from W37, P39, D40, A41, K80, E81, F82, K106, E108, DI 15, H216, K217, L218, and K219 in the amino acid sequence of SEQ ID NO: 12.

[0525] In some embodiments, the amino acid sequence of the IL-12 p40 subunit disclosed herein may further include one or more amino acid substitutions at a position corresponding to an amino acid residue selected from E81, F82, KI 06, K217, and K219 in the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amino acid sequence of the IL-12 p40 subunit disclosed herein may further include includes amino acid substitutions at positions corresponding to amino acid residues W37, P39, D40, E81, F82 in the amino acid sequence of SEQ ID NO: 12, or a combination thereof. In some embodiments, the amino acid sequence includes an amino acid substitution corresponding to amino acid residue E81, F82, K106, K217, and K219 in the amino acid sequence of SEQ ID NO: 12.

[0526] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof of the disclosure may include an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12, and further include an amino acid substitutions corresponding to the following amino acid substitutions: W37A, P39A, D40A, E81A, F82A, K106, D109A, K217A, E219A, E81A / F82A, W37A / E81A / F82A, E81A / F82A / K106A, E81A / F82A / K106A / K217A, E81F / F82A, E81K / F82A, E81L / F82A, E81H / F82A, E81S / F82A, E81A / F82A / K106N, E81A / F82A / K106Q,E81A / F82A / K106T, E81A / F82A / K106R, and / or P39A / D40A / E81A / F82A.

[0527] In some embodiments, the amino acid substitution(s) in the amino acid sequence of the IL-12 p40 subunit disclosed herein may comprise, for example, an alanine (A) substitution, an arginine I substitution, an asparagine (N) substitution, an aspartic acid (D) substitution, a leucine (L) substitution, a lysine (K) substitution, a phenylalanine (F) substitution, a glutamine (Q) substitution, a glutamic acid I substitution, a serine (S) substitution, and a threonine (T) substitution, or combinations of any thereof. Non-limiting examples of the amino acid substitutions in the recombinant IL-12 p40 polypeptides disclosed herein are provided in Table 2 below.Table 2. Example amino acid substitutions in the IL-12 p40 subunit

[0528] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof comprises amino acid change(s), e.g., substitution(s) in one or more positions selected from E81, F82, K106, and K217 of the amino acid sequence of SEQ ID NO: 27 or 12. In some embodiments, the amino acid substitution(s) in the amino acid sequence of the IL-12 p40 subunit disclosed herein comprises one or more alanine (A) substitutions. In some embodiments, the alanine (A) substitution comprises E81A, F82A, K106A, or K217A, or a combination thereof. In some embodiments, the alanine substitutions comprise E81 A / F82A (2xA). In some embodiments, the alanine substitutions comprise E81A / F82A / K106A (3xA). In some embodiments, the alanine substitutions comprise E81A / F82A / K106A / K217A (4xA).

[0529] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof may comprise one or more amino acid substitutions at a position corresponding to an amino acid residue including, for example, without limitation E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60, G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99, E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179, A180, E181, SI 83, Pl 85, El 87, N200, S204, F206, R208, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252, Q256, K258, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299 in the amino acid sequence of SEQ ID NO: 61. As a non-limiting example, the amino acid substitutions(s) may comprise an alanine (A) substitution, an arginine I substitution,an asparagine (N) substitution, an aspartic acid (D) substitution, a leucine (L) substitution, a lysine (K) substitution, a phenylalanine (F) substitution, a glutamine (Q) substitution, a glutamic acid I substitution, a serine (S) substitution, and a threonine (T) substitution, or any combination thereof.

[0530] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof of the disclosure may include an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 61, and further include an amino acid substitutions corresponding to the following amino acid substitutions: D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and / or E299Q with respect to the amino acid sequence of SEQ ID NO: 61.

[0531] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof of the disclosure may include an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 61, and further include an amino acid substitutions corresponding to the following amino acid substitutions: D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and / or E299Q with respect to the amino acid sequence of SEQ ID NO: 61.

[0532] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof of the disclosure may include an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 61, and further include an amino acid substitutions corresponding to the following amino acid substitutions: N103D / N113D / N200D / N281D, Q42E / E45Q, E45Q / Q56E, Q42E / E59Q, Q56E / E59Q,Q42E / E45Q / Q56E, E45Q / Q56E / E59Q, E32Q / E59Q, D34N / E59K, D34N / E59K / K99E, D34K / E59K / K99E, E32Q / D34N / E59K / K99E, E32K / D34N / E59K / K99E, D34N / E59Q,E59Q / E187Q, S43E / E59Q, S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E,E59Q / K260E, E59K / K99E, D18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144K, E59K / K99E / R159E,E59K / K99E / K264E, DI 8K / E59K / K99E / K264E, D18K / E59K / K99E / C252S,D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C252S, E59K / K99E / C252S / K264E,E59K / K99E / C252S, N103D / N113D, N103D / N200D, N103D / N281D, N113D / N200D, N113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D,N103D / N200D / N281D, N113D / N200D / N281D, N103Q / N113Q, N103Q / N200Q,N103Q / N281Q, N113Q / N200Q, N113Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N103Q / N113Q / N281Q, N103Q / N200Q / N281Q, N113Q / N200Q / N281Q,N 103 Q / N 113 Q / N200Q / N281 Q, E59K / K99E / N 103 Q / C252 S / K264E,E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / C252S / K264E,E59K / K99E / N281Q / C252S / K264E, E59K / K99E / N103Q / N113Q / C252S / K264E,E59K / K99E / N103Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N281Q / C252S / K264E,E59K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E,E59K / K99E / N200Q / N281Q / C252S / K264E, E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, and / or E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E with respect to the amino acid sequence of SEQ ID NO : 61.

[0533] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof of the disclosure may include an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 61, and further include an amino acid substitutions corresponding to the following amino acid substitutions: N103D / N113D / N200D / N281D, Q42E / E45Q, E45Q / Q56E, Q42E / E59Q, Q56E / E59Q,Q42E / E45Q / Q56E, E45Q / Q56E / E59Q, E32Q / E59Q, D34N / E59K, D34N / E59K / K99E, D34K / E59K / K99E, E32Q / D34N / E59K / K99E, E32K / D34N / E59K / K99E, D34N / E59Q,E59Q / E187Q, S43E / E59Q, S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E,E59Q / K260E, E59K / K99E, D18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144K, E59K / K99E / R159E, E59K / K99E / K264E, D18K / E59K / K99E / K264E, D18K / E59K / K99E / C252S,D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C252S, E59K / K99E / C252S / K264E,E59K / K99E / C252S, N103D / N113D, N103D / N200D, N103D / N281D, N113D / N200D, N113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D,N103D / N200D / N281D, N113D / N200D / N281D, N103Q / N113Q, N103Q / N200Q,N103Q / N281Q, N113Q / N200Q, N113Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N103Q / N113Q / N281Q, N103Q / N200Q / N281Q, N113Q / N200Q / N281Q,N 103 Q / N 113 Q / N200Q / N281 Q, E59K / K99E / N 103 Q / C252 S / K264E,E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / C252S / K264E,E59K / K99E / N281Q / C252S / K264E, E59K / K99E / N103Q / N113Q / C252S / K264E,E59K / K99E / N103Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N281Q / C252S / K264E,E59K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E,E59K / K99E / N200Q / N281Q / C252S / K264E, E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, and / or E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E with respect to the amino acid sequence of SEQ ID NO : 61.

[0534] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof, of the present disclosure further comprises an IL-12 p35 subunit, or a functional fragment or derivative thereof. In some embodiments, the IL- 12 polypeptide, or the functional fragment or derivative thereof, of the present disclosure comprises an IL- 12 p35 subunit, or a functional fragment or derivative thereof.

[0535] In some embodiments, the IL-12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 71, 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 the amino acid sequence of SEQ ID NO: 71. In certain embodiments, the nucleotide sequence that encodes IL-12 p35 subunit protein comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 71, 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 about75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98% or at least about 99%, sequence identity with the amino acid sequence ofSEQ ID NO: 71. In certain embodiments, the nucleotide sequence that encodes the IL- 12 p35 subunit protein comprises the nucleotide sequence of SEQ ID NO: 72, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about70%, 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 the nucleotide sequence of SEQ ID NO: 72. In certain embodiments, the IL-12 p35 subunit protein comprises the amino acid sequence of SEQ ID NO: 71. In certain embodiments, the nucleotide sequence that encodes the IL-12 p35 subunit protein comprises the nucleotide sequence of SEQ ID NO: 72.

[0536] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 71. In some embodiments, the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 71. In some embodiments, the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 71.

[0537] In some embodiments, the IL-12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 63, 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 the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the nucleotide sequence that encodes IL-12 p35 subunit protein comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 63, 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 about75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98% or at least about 99%, sequence identity with the amino acid sequence ofSEQ ID NO: 63. In certain embodiments, the nucleotide sequence that encodes the IL- 12 p35 subunit protein comprises the nucleotide sequence of SEQ ID NO: 64, 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 the nucleotide sequence of SEQ ID NO: 64. In certain embodiments, the nucleotide sequence thatencodes the IL-12 p35 subunit protein comprises the nucleotide sequence of SEQ ID NO: 181 or 182, 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 the nucleotide sequence of SEQ ID NO: 181 or 182. In certain embodiments, the IL-12 p35 subunit protein comprises the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the nucleotide sequence that encodes the IL- 12 p35 subunit protein comprises the nucleotide sequence of SEQ ID NO: 64. In certain embodiments, the nucleotide sequence that encodes the IL-12 p35 subunit protein comprises the nucleotide sequence of SEQ ID NO: 181 or 182.

[0538] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the IL- 12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 63.

[0539] In some embodiments, the IL- 12 p35 subunit, or functional fragment or derivative thereof, may comprise one or more amino acid substitutions and / or insertions and / or deletions at one or more locations in the amino acid sequence, e.g., as compared to an amino acid sequence of a reference IL-12 p35 subunit. For example, the IL-12 p35 subunit may include a substitution(s) of one or more amino acids in the amino acid sequence of a parent IL- 12 p35 subunit with a similar or homologous amino acid(s) or a dissimilar amino acid(s).

[0540] In some embodiments, the amino acid positions of one or more amino acid substitutions and / or insertions and / or deletions at one or more locations in the amino acid sequence of an IL-12 p35 subunit, or a functional fragment or derivative thereof described herein, may be described in reference to the amino acid positions of an IL- 12 p35 subunit reference sequence. In some embodiments, the IL-12 p35 subunit reference sequence may comprise a precursor form of an IL- 12 p35 subunit described herein (i.e., an IL-12 p35 subunit precursor form). In some embodiments, the precursor form of the IL-12 p35 subunit may comprise a signal peptide described herein such as, but not limited to, the amino acid sequence MCQSRYLLFLATLALLNHLSLA (SEQ ID NO: 33), or a fragment or derivative thereof. In some embodiments, the precursor form of the IL- 12p35 subunit may comprise a signal peptide described herein such as, but not limited to, the amino acid sequence MCPARSLLLVATLVLLDHLSLA (SEQ ID NO: 67), or a fragment or derivative thereof. Non-limiting examples of precursor forms of the IL-12 p35 subunit include the sequences of SEQ ID NO: 29 and SEQ ID NO: 15.

[0541] In some embodiments, the IL-12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 29, 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 the amino acid sequence of SEQ ID NO: 29. In certain embodiments, the nucleotide sequence that encodes IL-12 p35 subunit protein comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 29, 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 about75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98% or at least about 99%, sequence identity with the amino acid sequence of SEQ ID NO: 29. In certain embodiments, the nucleotide sequence that encodes the IL- 12 p35 subunit protein comprises the nucleotide sequence of SEQ ID NO: 30, 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 the nucleotide sequence of SEQ ID NO: 30. In certain embodiments, the IL- 12 p35 subunit protein comprises the amino acid sequence of SEQ ID NO: 29. In certain embodiments, the nucleotide sequence that encodes the IL- 12 p35 subunit protein comprises the nucleotide sequence of SEQ ID NO: 30.

[0542] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 29.

[0543] In some embodiments, the IL-12 p35 subunit reference sequence comprises a precursor form of an IL-12 p35 subunit disclosed herein, for example, the sequence of SEQ ID NO: 15. In some embodiments, the amino acid positions of the one or more amino acid substitutions and / or insertions and / or deletions at one or more locations in the amino acid sequence of the IL-12 p35 subunit, or functional fragment or derivative thereof, may be described in reference to the amino acid positions in the amino acid sequence of SEQ ID NO: 15.

[0544] In some embodiments, the IL-12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 15, 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 the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the nucleotide sequence that encodes IL-12 p35 subunit protein comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 15, 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 about75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98% or at least about 99%, sequence identity with the amino acid sequence ofSEQ ID NO: 15. In certain embodiments, the nucleotide sequence that encodes the IL- 12 p35 subunit protein comprises the nucleotide sequence of SEQ ID NO: 16, 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 the nucleotide sequence of SEQ ID NO: 16. In certain embodiments, the IL- 12 p35 subunit protein comprises the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the nucleotide sequence that encodes the IL-12 p35 subunit protein comprises the nucleotide sequence of SEQ ID NO: 16.

[0545] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the IL-12 p35 subunit, or the functionalfragment or derivative thereof comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the IL- 12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 15.

[0546] In some embodiments, an IL-12 p35 subunit reference sequence may comprise a mature form sequence of an IL-12 p35 subunit described herein (i.e., an IL-12 p35 mature form). In some embodiments, the sequence of the mature form of the IL- 12 p35 subunit may be shorter in length than the sequence of the precursor form of the IL-12 p35 subunit described herein owing, at least in part, to the absence of a signal sequence from the mature form which may be present in the precursor form. In some embodiments, the IL-12 p35 reference sequence can comprise a mature form of an IL-12 p35 subunit, for example, the sequence of SEQ ID NO: 63. In some embodiments, the amino acid positions of the one or more amino acid substitutions and / or insertions and / or deletions at one or more locations in the amino acid sequence of the IL- 12 p35 subunit, or functional fragment or derivative, thereof may be described in reference to the amino acid positions in the amino acid sequence of SEQ ID NO: 63.

[0547] In some embodiments, the IL-12 p35 subunit of the present disclosure may include any amino acid sequence having an identity of at least about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more to its natural amino acid sequence or parental sequence, and having the activity of normally-occurring (i.e., natural) sequence or parental sequence.

[0548] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof comprises amino acid change(s) in one or more positions. Non-limiting examples of amino acid changes comprise amino acid substitutions and / or insertions and / or deletions. In some embodiments, the IL-12 p35 subunit, or the functional fragment or derivative thereof comprises amino acid substitution(s) in one or more positions. As a non-limiting example, the IL- 12 p35 subunit, or the functional fragment or derivative thereof may comprise amino acid substitution(s) in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 or more positions.

[0549] In some embodiments, the IL-12 p35 subunit described herein may comprise one or more amino acid substitutions and / or insertions and / or deletions. Amino acid substitution means that an amino acid residue is substituted for a replacement 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 immediately adjacent one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent another inserted amino acid residue. In various embodiments, one or more amino acids may be substituted and / or inserted and / or deleted from the sequence of SEQ ID NO: 29. In various embodiments, one of more amino acids may be substituted and / or inserted and / or deleted from the sequence of SEQ ID NO: 71. In various embodiments, one or more amino acids may be substituted and / or inserted and / or deleted from the sequence of SEQ ID NO: 15. In various embodiments, one of more amino acids may be substituted and / or inserted and / or deleted from the sequence of SEQ ID NO: 63.

[0550] A person with ordinary skill of the art will be able to identify the one or more amino acid changes (e.g., amino acid substitutions and / or insertions and / or deletions) in an IL-12 p35 precursor form sequence described herein (e.g., SEQ ID NO: 15 or 29) within a shorter IL- 12 p35 subunit mature form sequence described herein (e.g., SEQ ID NO: 63 or 71). Any of the amino acid change(s) described in reference to an IL-12 p35 polypeptide comprising a mature form sequence of an IL-12 p35 subunit described herein (e.g., SEQ ID NO: 63 or 71) may be applied to an IL-12 p35 polypeptide comprising a precursor form sequence of an IL-12 p35 subunit described herein (e.g., SEQ ID NO: 15 or 29), or vice versa.

[0551] A person with ordinary skill of the art will further be able to identify the one or more amino acid changes (e.g., amino acid substitutions and / or insertions and / or deletions) in an IL-12 p35 mature or precursor form sequence derived from a human described herein (e.g., SEQ ID NO: 63 or 15, respectively) within an IL- 12 p35 mature or precursor form sequence derived from a mouse described herein (SEQ ID NO: 71 or 29, respectively). Any of the amino acid change(s) described in reference to an IL-12 p35 polypeptide comprising a mature or precursor form sequence of an IL-12 p35 subunit derived from a human (e.g., SEQ ID NO: 63 or 15, respectively), may also be applied to an IL-12 p35 polypeptide comprising a mature or precursor form sequenceof an IL-12 p35 subunit derived from a mouse (e.g., SEQ ID NO: 71 or 29, respectively), or vice versa.

[0552] In some embodiments, the IL-12 p35 subunit may comprise a substitution at any amino acid contained therein. In some embodiments, the IL-12 p35 subunit may comprise an insertion at any amino acid contained therein. In some embodiments, the IL-12 p35 subunit may comprise a deletion at any amino acid contained therein.

[0553] In certain embodiments, amino acid changes (e.g., substitutions) to a protein or portion thereof are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, or (4) confer or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the normally-occurring (i.e., natural) sequence or parental sequence. In some embodiments, the amino acid sequence of the IL- 12 p35 subunit disclosed herein may be changed, for example, to increase activity and / or reduce toxicity of the IL- 12 polypeptide, or the functional fragment or derivative thereof disclosed herein. In some embodiments, the amino acid sequence of IL-12 p35 subunit disclosed herein may be changed such that the activity of the IL-12 polypeptide is attenuated, for example, for intravenous (IV) delivery.

[0554] By way of a non-limiting example, for the activity of the IL-12 p35 subunit before the substitution, insertion, and / or deletion described herein, the activity of the IL-12 p35 subunit protein or amino acid sequence after the substitution, insertion, and / or deletion is increased by more than 2%, more than 5%, more than 10%, more than 20%, more than 40%, more than 60%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or 100% or more as compared to the activity before the substitution, deletion, insertion or addition. By way of another non-limiting example, for the toxicity of the IL- 12 p35 subunit before the substitution, insertion, and / or deletion described herein, the toxicity of the IL- 12 p35 subunit protein or amino acid sequence after the substitution, insertion, and / or deletion is reduced by more than 2%, more than 5%, more than 10%, more than 20%, more than 40%, more than 60%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or 100% or more as compared to the toxicity before the substitution, deletion, insertion or addition.

[0555] In some embodiments, the amino acid sequence of the IL- 12 p35 subunit may be modified to comprise one or more amino acid changes (e.g., substitutions), that may decrease susceptibility of the polypeptide to proteolysis and / or increase IL-12 biological half-life. In some embodiments, the amino acid sequence of the IL-12 p35 subunit may be modified to comprise one or more amino acid changes (e g., substitutions), that may decrease susceptibility of the polypeptide to proteolysis and / or reduce IL-12 biological half-life.

[0556] In some embodiments, the IL-12 p35 subunit disclosed herein may also include conservative modifications and / or substitutions at other positions of IL-12 p35.

[0557] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof, may comprise one or more amino acid substitutions at a position corresponding to an amino acid residue including, for example, without limitation, of Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, 1171, R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196 in the amino acid sequence of SEQ ID NO: 63. As a non-limiting example, the amino acid substitutions(s) may comprise an alanine (A) substitution, an arginine I substitution, an asparagine (N) substitution, an aspartic acid (D) substitution, a leucine (L) substitution, a lysine (K) substitution, a phenylalanine (F) substitution, a glutamine (Q) substitution, a glutamic acid I substitution, a serine (S) substitution, and a threonine (T) substitution, or any combination thereof.

[0558] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof of the disclosure may include an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 63, and further include an amino acid substitutions corresponding to the following amino acid substitutions: N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L9A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, 1171 A, N195D, and / or N195Q with respect to the amino acid sequence of SEQ ID NO: 63.

[0559] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof of the disclosure may include an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 63, and further include an amino acid substitutions corresponding to the following amino acid substitutions:N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L9A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, Il 71 A, N195D, and / or N 195 Q with respect to the amino acid sequence of SEQ ID NO: 63.

[0560] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof of the disclosure may include an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 63, and further include an amino acid substitutions corresponding to the following amino acid substitutions: N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and / or N85D / N195D with respect to the amino acid sequence of SEQ ID NO: 63.

[0561] In some embodiments, the IL- 12 p35 subunit, or the functional fragment or derivative thereof of the disclosure may include an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 63, and further include an amino acid substitutions corresponding to the following amino acid substitutions: N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and / or N85D / N195D with respect to the amino acid sequence of SEQ ID NO: 63.

[0562] In some embodiments, the IL- 12 polypeptide, or the functional fragment or derivative thereof may further comprise a signal peptide. In some embodiments, the IL-12 polypeptide, or functional fragment of derivative thereof, may comprise one or more signal peptides. As a nonlimiting example, the IL-12 polypeptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or more, signal peptide(s). Non-limiting examples of signal peptides include the amino acid sequence of SEQ ID NOs: 31, 33, 65, or 67. In some embodiments, the IL- 12 polypeptide, or the functional fragment or derivative thereof may comprise a signal peptide which comprises the amino acid sequence of SEQ ID NOs: 31, 33, 65, or 67, 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 about96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with the amino acid sequence of SEQ ID NOs: 31, 33, 65, or 67. In certain embodiments, the nucleotide sequence that encodes the signal peptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NOs: 31, 33, 65, or 67, 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 the amino acid sequence of SEQ ID NOs: 31, 33, 65, or 67. In certain embodiments, the nucleotide sequence that encodes the signal peptide comprises the nucleotide sequence of SEQ ID NOs: 32, 34, 66, or 68, 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 the nucleotide sequence of SEQ ID NOs: 32, 34, 66, or 68. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NOs: 31, 33, 65, or 67. In certain embodiments, the nucleotide sequence that encodes the signal peptide comprises the nucleotide sequence of SEQ ID NOs: 32, 34, 66, or 68.

[0563] In some embodiments, the IL-12 p35 and the IL-12 p40 subunits may be oriented such that the IL- 12 p35 subunit, or the functional fragment or derivative thereof, may be operably linked to the signal peptide. In some embodiments, the IL- 12 p35 and the IL- 12 p40 subunits may be oriented such that the IL- 12 p40 subunit, or the functional fragment or derivative thereof, may be operably linked to the signal peptide.

[0564] In some embodiments, a signal peptide is operably linked to an IL-12 p40 subunit, or a functional fragment or derivative thereof, described herein, to form an IL- 12 p40 subunit precursor. In some embodiments, a signal peptide is operably linked to an IL-12 p35 subunit, or a functional fragment or derivative thereof, described herein, to form an IL- 12 p35 subunit precursor.

[0565] In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 31, 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 the amino acid sequence of SEQ ID NO: 31. In certain embodiments, the nucleotide sequence that encodes signal peptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 31, 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 about75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98% or at least about 99%, sequence identity with the amino acid sequence of SEQ ID NO: 31. In certain embodiments, the nucleotide sequence that encodes the signal peptide comprises the nucleotide sequence of SEQ ID NO: 32, 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 the nucleotide sequence of SEQ ID NO: 32. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 31. In certain embodiments, the nucleotide sequence that encodes the signal peptide comprises the nucleotide sequence of SEQ ID NO: 32.

[0566] In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence that has at least 80% sequence identity thereto. In some embodiments, the signal peptide consists of the amino acid sequence of SEQ ID NO: 31.

[0567] In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 33, 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 the amino acid sequence of SEQ ID NO: 33. In certain embodiments, the nucleotide sequence that encodes signal peptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 33, 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 about92%, 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 the amino acid sequence of SEQ ID NO: 33. In certain embodiments, the nucleotide sequence that encodes the signal peptide comprises the nucleotide sequence of SEQ ID NO: 34, 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 the nucleotide sequence of SEQ ID NO: 34. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 33. In certain embodiments, the nucleotide sequence that encodes the signal peptide comprises the nucleotide sequence of SEQ ID NO: 34.

[0568] In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that has at least 80% sequence identity thereto. In some embodiments, the signal peptide consists of the amino acid sequence of SEQ ID NO: 33.

[0569] In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 65, 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 the amino acid sequence of SEQ ID NO: 65. In certain embodiments, the nucleotide sequence that encodes signal peptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 65, 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 about75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98% or at least about 99%, sequence identity with the amino acid sequence ofSEQ ID NO: 65. In certain embodiments, the nucleotide sequence that encodes the signal peptide comprises the nucleotide sequence of SEQ ID NO: 66, 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 leastabout 97%, at least about 98% or at least about 99%, sequence identity with the nucleotide sequence of SEQ ID NO: 66. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 65. In certain embodiments, the nucleotide sequence that encodes the signal peptide comprises the nucleotide sequence of SEQ ID NO: 66.

[0570] In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence that has at least 80% sequence identity thereto. In some embodiments, the signal peptide consists of the amino acid sequence of SEQ ID NO: 65.

[0571] In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 67, 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 the amino acid sequence of SEQ ID NO: 67. In certain embodiments, the nucleotide sequence that encodes signal peptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 67, 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 about75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98% or at least about 99%, sequence identity with the amino acid sequence of SEQ ID NO: 67. In certain embodiments, the nucleotide sequence that encodes the signal peptide comprises the nucleotide sequence of SEQ ID NO: 68, 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 the nucleotide sequence of SEQ ID NO: 68. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 67. In certain embodiments, the nucleotide sequence that encodes the signal peptide comprises the nucleotide sequence of SEQ ID NO: 68.

[0572] In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence that has at least 80% sequence identity thereto. In some embodiments, the signal peptide consists of the amino acid sequence of SEQ ID NO: 67.

[0573] Given that IL-12 is a heterodimeric molecule composed of an alpha chain (the p35 subunit) and a beta chain (the p40 subunit), the simultaneous expression of the two subunits is necessary to produce the biologically active heterodimer. Recombinant IL-12 expression can be achieved with bicistronic vectors comprising the p40 and p35 subunits separated by an IRES (internal ribosome entry site) sequence to permit independent expression of both subunits from a single vector. As an alternative to bicistronic expression, functional single chain IL-12 fusion proteins have been generated by joining the p40 and p35 subunits with linkers, e.g., GlyeSer (SEQ ID NO: 35) or (Gly4Ser)3(SEQ ID NO: 18) linkers.

[0574] In some embodiments, the IL- 12 p40 subunit, or the functional fragment or derivative thereof and the IL- 12 p35 subunit, or the functional fragment or derivative thereof are connected by a first linker. In some embodiments, the first linker is from about 5 amino acids to about 30 amino acids in length. In some embodiments, the first peptide linker may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids. In some embodiments, the first linker may be absent. The first linker may comprise or consist of any of various linkers disclosed herein, or any combination thereof. For example, a first linker of the present disclosure may comprise or consist of any of the sequences of SEQ ID NO: 18-19, 36-49, 75-93, or any combination thereof.

[0575] In some embodiments, the first linker comprises the sequence (VPGVG)2 (SEQ ID NO: 17). In some embodiments, the first linker consists of the sequence (VPGVGfi (SEQ ID NO: 17).

[0576] In some embodiments, the first linker comprises the sequence (G4S)2 (SEQ ID NO: 40). In some embodiments, the first linker consists of the sequence (G4S)2 (SEQ ID NO: 40).

[0577] In some embodiments, the first linker comprises the sequence (G4S)3(SEQ ID NO: 18). In some embodiments, the first linker consists of the sequence (G4S)3(SEQ ID NO: 18).

[0578] In some embodiments, the first linker comprises the sequence (G4S)3GS (SEQ ID NO: 41). In some embodiments, the first linker consists of the sequence (G4S)3GS (SEQ ID NO: 41).

[0579] In some embodiments, the first linker comprises the sequence (G4S)4(SEQ ID NO: 42). In some embodiments, the first linker consists of the sequence (G4S)4(SEQ ID NO: 42).

[0580] In some embodiments, the first linker comprises the sequence (G4S)s (SEQ ID NO: 43). In some embodiments, the first linker consists of the sequence (G4S)s (SEQ ID NO: 43).

[0581] In some embodiments, the first linker comprises the sequence (G4S)e (SEQ ID NO: 44). In some embodiments, the first linker consists of the sequence (G4S)e (SEQ ID NO: 44).

[0582] In some embodiments, the first linker comprises the sequence (648)7 (SEQ ID NO: 45). In some embodiments, the first linker consists of the sequence (648)7 (SEQ ID NO: 45).

[0583] In some embodiments, the first linker comprises the sequence (648)8 (SEQ ID NO: 46). In some embodiments, the first linker consists of the sequence (648) (SEQ ID NO: 46).

[0584] In some embodiments, the first linker comprises the sequence (6666S)n, wherein n is an integer of 1-10, or more (SEQ ID NO: 47). In some embodiments, the first linker consists of the sequence (6666S)n, wherein n is an integer of 1-10, or more (SEQ ID NO: 47).

[0585] In some embodiments, the first linker comprises the sequence (638)2 (SEQ ID NO: 36). In some embodiments, the first linker consists of the sequence (638)2 (SEQ ID NO: 36).

[0586] In some embodiments, the first linker comprises the sequence (638)3 (SEQ ID NO: 37). In some embodiments, the first linker consists of the sequence (638)3 (SEQ ID NO: 37).

[0587] In some embodiments, the first linker comprises the sequence (638)4 (SEQ ID NO: 38). In some embodiments, the first linker consists of the sequence (638)4 (SEQ ID NO: 38).

[0588] In some embodiments, the first linker comprises the sequence (638)5 (SEQ ID NO: 39). In some embodiments, the first linker consists of the sequence (638)5 (SEQ ID NO: 39).

[0589] In some embodiments, the first linker comprises the sequence 666666S (SEQ ID NO: 35). In some embodiments, the first linker consists of the sequence 666666S (SEQ ID NO: 35).

[0590] In some embodiments, the first linker comprises the sequence 6S6SSR66S6S66S6666SK (SEQ ID NO: 48). In some embodiments, the first linker consists of the sequence 6S6SSR66S6S66S6666SK (SEQ ID NO: 48).

[0591] In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof is membrane bound. In some embodiments, the IL- 12 polypeptide, or the functional fragment or derivative thereof is not membrane bound. In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof is secreted. In some embodiments, the IL-12 polypeptide, or the functional fragment or derivative thereof is soluble.

[0592] As discussed below, the nucleic acid encoding the IL-12 polypeptide, or functional fragment or derivative thereof, may be positioned at any location within the VSV genome. Further, the nucleic acids encoding the IL-12 p35 and / or the IL-12 p40 subunits, or functional fragments or derivatives thereof, may be positioned at any location within the VSV genome.

[0593] In some embodiments, the nucleic acid encoding the IL-12 polypeptide, or functional fragment or derivative thereof, can be positioned upstream of a nucleic acid encoding a VSV N polypeptide, or functional fragment or derivative thereof. In some embodiments, the nucleic acid encoding the IL-12 p35 and / or the IL-12 p40 subunits, or functional fragments or derivatives thereof, can be positioned upstream of a nucleic acid encoding a VSV N polypeptide, or functional fragment or derivative thereof.

[0594] In some embodiments, the nucleic acid encoding the IL-12 polypeptide, or functional fragment or derivative thereof, can be positioned between the nucleic acid encoding the VSV N polypeptide, or functional fragment or derivative thereof, and the nucleic acid encoding a VSV P polypeptide, or functional fragment or derivative thereof. In some embodiments, the nucleic acid encoding the IL-12 p35 and / or the IL-12 p40 subunits, or functional fragments or derivatives thereof, can be positioned between the nucleic acid encoding the VSV N polypeptide, or functional fragment or derivative thereof, and the nucleic acid encoding a VSV P polypeptide, or functional fragment or derivative thereof.

[0595] In some embodiments, the nucleic acid encoding the IL-12 polypeptide, or functional fragment or derivative thereof, can be positioned between the nucleic acid encoding the VSV P polypeptide, or functional fragment or derivative thereof, and the nucleic acid encoding a VSV M polypeptide, or functional fragment or derivative thereof. In some embodiments, the nucleic acid encoding the IL-12 p35 and / or the IL-12 p40 subunits, or functional fragments or derivatives thereof, can be positioned between the nucleic acid encoding the VSV P polypeptide, or functional fragment or derivative thereof, and the nucleic acid encoding a VSV M polypeptide, or functional fragment or derivative thereof.

[0596] In some embodiments, the nucleic acid encoding the IL-12 polypeptide, or functional fragment or derivative thereof, can be positioned downstream of a nucleic acid encoding a VSV M polypeptide, or functional fragment or derivative thereof. In some embodiments, the nucleic acid encoding the IL- 12 polypeptide, or functional fragment or derivative thereof, can be positioned downstream of a nucleic acid encoding a fusogenic polypeptide, e.g., a VSV G polypeptide, or functional fragment or derivative thereof. In some embodiments, the nucleic acid encoding the IL-12 p35 and / or the IL-12 p40 subunits, or functional fragments or derivatives thereof, can be positioned downstream of a nucleic acid encoding a VSV M polypeptide, or functional fragment or derivative thereof. In some embodiments, the nucleic acid encoding the IL-12 p35 and / or the IL-12 p40 subunits, or functional fragments or derivatives thereof, can be positioned downstream of a nucleic acid encoding a fusogenic polypeptide, e.g., a VSV G polypeptide, or functional fragment or derivative thereof.

[0597] In some embodiments, the nucleic acid encoding the IL-12 polypeptide, or functional fragment or derivative thereof, can be positioned between the nucleic acid encoding the VSV M polypeptide, or functional fragment or derivative thereof, and the nucleic acid encoding a VSV G polypeptide, or functional fragment or derivative thereof. In some embodiments, the nucleic acid encoding the IL-12 p35 and / or the IL-12 p40 subunits, or functional fragments or derivatives thereof, can be positioned between the nucleic acid encoding the VSV M polypeptide, or functional fragment or derivative thereof, and the nucleic acid encoding a VSV G polypeptide, or functional fragment or derivative thereof.

[0598] In some embodiments, the nucleic acid encoding the IL-12 polypeptide, or functional fragment or derivative thereof, can be positioned between the nucleic acid encoding the VSV M polypeptide, or functional fragment or derivative thereof, and the nucleic acid encoding a VSV L polypeptide, or functional fragment or derivative thereof. In some embodiments, the nucleic acid encoding the IL-12 polypeptide, or functional fragment or derivative thereof, can be positioned between the nucleic acid encoding the fusogenic polypeptide, e.g., a VSV G polypeptide, or functional fragment or derivative thereof, and the nucleic acid encoding the VSV L polypeptide, or functional fragment or derivative thereof. In some embodiments, the nucleic acid encoding the IL-12 p35 and / or the IL-12 p40 subunits, or functional fragments or derivatives thereof, can be positioned between the nucleic acid encoding the VSV M polypeptide, or functional fragment or derivative thereof, and the nucleic acid encoding a VSV L polypeptide, or functional fragment or derivative thereof. In some embodiments, the nucleic acid encoding the IL- 12 p35 and / or the IL- 12 p40 subunits, or functional fragments or derivatives thereof, can be positioned between the nucleic acid encoding the fusogenic polypeptide, e.g., a VSV G polypeptide, or functional fragment or derivative thereof, and the nucleic acid encoding the VSV L polypeptide, or functional fragment or derivative thereof.

[0599] In some embodiments, the nucleic acid encoding the IL-12 polypeptide, or functional fragment or derivative thereof, can be positioned downstream of the nucleic acid encoding the VSV L polypeptide, or functional fragment or derivative thereof. In some embodiments, the nucleic acid encoding the IL-12 p35 and / or the IL-12 p40 subunits, or functional fragments orderivatives thereof, can be positioned downstream of the nucleic acid encoding the VSV L polypeptide, or functional fragment or derivative thereof.

[0600] The IL-12 transgene position in the VSV genome can affect transcription levels of the IL-12 gene relative to other viral proteins. The positioning of the IL-12 transgene could impact a) replication kinetics of the virus; and b) production level of IL-12 by each infected cell. Replication kinetics, the amount of IL-12 produced per infected cell, and viral fitness can generally affect effectiveness, toxicity, therapeutic index, the type of tumors most effectively targeted, and other factors.

[0601] Without wishing to be bound by theory, a viral transcriptase can transcribe viral mRNA species using a virion RNA (e.g., a VSV genome) as a template. A single promoter site can be located at the 3 ' end of the viral genome, and polymerase can attach to the genomic RNA at this site. As the polymerase moves along the viral RNA it can encounter stop-start signals at the boundaries of each of the viral genes (e.g., genes encoding a nucleoprotein (N) polypeptide, a phosphoprotein (P) polypeptide, a matrix (M) polypeptide, a glycoprotein (G) polypeptide, and / or a large protein (L) polypeptide). Such mechanism can be referred to, for example, as stop-start (or stuttering) transcription, and can result in more mRNA being made from genes that are located at the 3’ end of the viral genome, and a gradient of progressively less mRNA is made from downstream genes (see, e.g., Figure 73A).

[0602] In some embodiments, insertion of the IL- 12 transgene within the VSV genome closer to the viral promoter can result in more mRNA being made from the IL- 12 transgene than from downstream genes.

[0603] In some embodiments, insertion of the IL- 12 transgene within the VSV genome closer to the viral promoter, e.g., downstream of the P gene, can impact the expression of the M gene and / or the G gene, thereby resulting in decreased levels of viral replication.

[0604] In various aspects, the present disclosure provides a recombinant rhabdovirus (e.g., a VSV) comprising a nucleotide encoding a recombinant protein comprising an IL-12 polypeptide, or a functional fragment or derivative thereof; the recombinant protein is not membrane bound; the IL- 12 polypeptide, or the functional fragment or derivative thereof, comprises an IL- 12 p40 subunit, or a functional fragment or derivative thereof, and an IL- 12 p35 subunit, or a functional fragment or derivative thereof, which are connected by a first linker; the rhabdovirus genome (e.g., VSV genome) comprises a gene encoding a fusogenic polypeptide (e.g., a G protein polypeptide),or a functional fragment or derivative thereof, and a gene encoding a large protein (L) polypeptide, or a functional fragment or derivative thereof; and, the nucleotide sequence encoding the recombinant protein is positioned between the gene encoding the fusogenic polypeptide, or the functional fragment or derivative thereof, and the gene encoding the L polypeptide, or the functional fragment or derivative thereof.

[0605] In some embodiments, upon infection of a subject in need thereof by a recombinant virus described herein, the recombinant virus may result in reduced, decreased, lowered, lessened, or abated serum interferon gamma (IFNy) release in the subject, as compared to a control, e.g., a control recombinant virus comprising a nucleotide sequence encoding an IL-12 polypeptide, or fragment or derivative thereof, that is not membrane bound. For example, serum interferon gamma (IFNy) release may be reduced, decreased, lowered, lessened, or abated in the subject by about 0.5%, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, or greater. In some embodiments, serum interferon gamma (IFNy) release may be reduced, decreased, lowered, lessened, or abated in the subject by from about 5% to about 10%, from about 10% to about 20%, from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%. In some embodiments, serum interferon gamma (IFNy) release may be reduced, decreased, lowered, lessened, or abated in the subject by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% or more. In some embodiments, the recombinant virus comprises a nucleotide sequence encoding an IL-12 polypeptide, or fragment or derivative thereof, which is membrane bound.

[0606] In some embodiments, upon infection of a subject in need thereof by a recombinant virus described herein, the recombinant virus does not result in body weight loss or result in reduced, decreased, lowered, lessened, or abated body weight loss in the subject, as compared to a control, e.g., a control recombinant virus comprising a nucleotide sequence encoding an IL-12 polypeptide that is not membrane bound. For example, body weight loss may be reduced, decreased, lowered, lessened, or abated in the subject by about 0.5%, about 1%, about 5%, about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, or greater. In some embodiments, body weight loss may be reduced, decreased, lowered, lessened, or abated in the subject by from about 5% to about 10%, from about 10% to about 20%, from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%. In some embodiments, body weight loss may be reduced, decreased, lowered, lessened, or abated in the subject by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% or more. In some embodiments, body weight loss in the subject is prevented. In some embodiments, the recombinant virus comprises a nucleotide sequence encoding an IL-12 polypeptide, or fragment or derivative thereof, which is membrane bound.

[0607] In some embodiments, an IL- 12 polypeptide, or functional fragment or derivative thereof can be connected to a transmembrane domain. In some embodiments, the IL- 12 p40 subunit of the IL- 12 polypeptide, or functional fragment or derivative thereof, is connected to the transmembrane domain via a second linker.

[0608] In some embodiments, the IL-12 p35 and the IL-12 p40 subunits may be oriented such that the IL-12 p35 subunit, or the functional fragment or derivative thereof, may be connected to the transmembrane domain via the second linker. In some embodiments, the IL-12 p35 and the IL- 12 p40 subunits may be oriented such that the IL- 12 p40 subunit, or the functional fragment or derivative thereof, may be connected to the transmembrane domain via the second linker.

[0609] In some embodiments of the above-described orientations of the IL- 12 p35 and the IL- 12 p40 subunits, cell surface clustering of the IL- 12 polypeptide described herein may be enhanced when IL-12 p35 versus the IL-12 p40 subunit is connected to the transmembrane via the second linker. In some embodiments of the above-described orientations of the IL-12 p35 and the IL-12 p40 subunits, cell surface clustering of the IL-12 polypeptide described herein may be diminished when IL-12 p35 versus the IL-12 p40 subunit is connected to the transmembrane via the second

[0610] In some embodiments, the second linker is from about 5 amino acids to about 50 amino acids in length. In some embodiments, the first linker may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 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. In some embodiments, the second linker may be absent. The second linker may comprise or consist of any of various linkers disclosed herein, or any combination thereof. For example, a second linker of the present disclosure may comprise or consist of any of the sequences of SEQ ID NO: 18-19, 36-49, 75-93, or any combination thereof.

[0611] In some embodiments, the second linker can be a flexible linker, a rigid linker, or a cleavable linker.

[0612] Flexible linkers can include glycine polymers, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between domains of polypeptides described herein (e.g., polypeptides comprising e.g., IL- 12 p35 unit, IL- 12 p40 subunit, transmembrane domain, and / or additional various polypeptides described herein). Glycine can access more phi-psi space than even alanine, and is much less restricted than residues with longer side chains. The ordinarily skilled artisan will recognize that design of polypeptide described herein can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for polypeptide structure.

[0613] Rigid linkers can exhibit relatively stiff structures by adopting a-helical structures or by containing multiple proline (pro) residues. The a-helix can be a rigid and stable structure, including intra-segment hydrogen bonds and a closely packed backbone. Various a-helical conformations can form rapidly such as during folding, which can allow for correct folding of connecting protein domains, e.g., without non-native interactions with the linker. Linkers in an a-helix structure can serve as rigid spacers to effectively separate protein domains as well as to reduce their unfavorable interactions. In the absence of an inherent rigid structure, non-helical linkers tend to be rich in pro, which can enhance the stiffness of a linker. Hence, non-helical linkers with pro-rich sequences can provide relatively rigid structures and can reduce inter-domain interference.

[0614] Cleavable linkers may be cleaved under specific conditions such as the presence of reducing reagents or proteases. This type of linker may improve bioactivity, reduce steric hindrance, or achieve independent actions / metabolism of individual domains of recombinant fusion proteins following linker cleavage. Cleavable linkers can be used to improve the bioactivityof chimeric proteins, or to specifically deliver prodrugs to target sites where the linkers are processed to activate bioactivity. In addition to reduction of disulfide bond, in vivo cleavage of cleavable linkers in recombinant fusion proteins may also be achieved by proteases that are expressed, e.g., under pathological conditions (e.g. in cancer), in specific cells (e.g., cancer / tumor cells) or tissues (e.g., tumors), or restricted within specific cellular compartments (e.g., the lysosome). Such cleavable linkers can be designed to be prone to a particular protease by incorporating specific protease-sensitive sequences. Unlike the reduction of a disulfide bond which can occur rapidly in the blood circulation, the specificity of many proteases can allow for slower cleavage of a cleavable linker, e.g., at specific sites in vivo. In vivo cleavable linkers sensitive to proteases that become active under certain physiological or pathological conditions can be used for drug targeting. Likewise, in vivo cleavable linkers which can be cleaved by proteases overexpressed in diseased cells / tissues can also be used for drug targeting. A non-limiting example of targeting intracellular compartments is the cleavable linker which can be specifically cleaved by furin, a cellular endoproteinase involved in the proteolytic activation of various precursor proteins as well as toxins. Cathepsin B, a protease present in the lysosome, has been applied for targeted intracellular activation of cytotoxic proteins.

[0615] Non-limiting examples of amino acid (aa) sequences of flexible, rigid, and cleavable linkers which may be used in the compositions of the present disclosure are provided in Table 3 (below).Table 3. Examples of Amino Acid Sequences of Flexible, Rigid, and Cleavable Linkers

[0616] In some embodiments, a cleavable linker can be a self-cleaving peptide such as a 2A selfcleaving peptide. Non-limiting examples of 2A self-cleaving peptides include a P2A peptide, an E2A peptide, an F2A peptide, and a T2A peptide. In some embodiments, the 2A self-cleaving peptide is a P2A peptide. As a non-limiting example, the P2A peptide may comprise or consist of the sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 109).

[0617] In some embodiments, a linker of the present disclosure may comprise or consist of a dipeptide linker such as, but not limited to, leucine-glutamic acid (LE) (SEQ ID NO: 91).

[0618] In some embodiments, the second linker comprises the sequence (648)3 (SEQ ID NO: 18). In some embodiments, the first linker consists of the sequence (648)3 (SEQ ID NO: 18).

[0619] In some embodiments, the second linker comprises the sequence A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 73). In some embodiments, the first linker consists of the sequence A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 73).

[0620] In some embodiments, the second linker comprises the sequence A(EAAAK)3A (SEQ ID NO: 74). In some embodiments, the first linker consists of the sequence A(EAAAK)3A (SEQ ID NO: 74).

[0621] Two basic types of transmembrane domains are alpha-helical and beta-barrels. Alphahelical transmembrane domains are located in the plasma membrane of eukaryotes. It is estimated that in humans, approximately 30% of all proteins may be alpha-helical membrane proteins. As an Example, one estimate of the human membrane proteome determined there are at least 2,925 unique integral alpha-helical transmembrane sequences encoded by the human genome. Betabarrel proteins are located in outer membranes of mitochondria, comprising a simple up-and-down topology that may reflect their common evolutionary origin and similar folding mechanism. Transmembrane domain classification by topology may refer to the position of the N- and C- terminal domains. Types I, II, and III can be single-pass molecules, whereas type IV can be multiple-pass molecules. Type I transmembrane domains may be anchored to the lipid membrane with a stop-transfer anchor sequence. The N-terminal domains of Type I transmembrane domainsare targeted to the ER lumen during synthesis, as well as to the extracellular space, in instances where mature forms may be located on cell surface. Type II and III transmembrane domains are anchored via a signal-anchor sequence. Type II transmembrane domains may be targeted to the endoplasmic reticulum (ER) lumen via the C-terminal domain. Type III transmembrane domains have their N-terminal domains targeted to the ER lumen. Type IV transmembrane domains are subclassified as either IV-A, with an N-terminal domain targeted to the cytosol, or IV-B, with an N-terminal domain targeted to the lumen. Non-limiting examples of transmembrane domains which may be used in the practice of the present disclosure include: single-pass transmembrane proteins (TMPs) (e.g., Type-I TMP including E3 Ubiquitin-Protein Ligase; Type-II TMP including 4F2 Cell-Surface Antigen Heavy Chain; Type-III TMP including Linker For Activation of T-cells Family Member 1; and, Type-IV TMP including Junctophilin-1); multi-pass TMPs, e.g., human calcitonin receptor; and, beta-barrel TMP.

[0622] In some embodiments, the transmembrane domain, or a functional fragment or derivative thereof, described herein may be derived from, e.g., tumor necrosis factor (TNF) alpha (TNFa), platelet-derived growth factor receptor (PDGFR), cluster of differentiation 4 (CD4) or fragments or derivatives thereof, or combinations thereof.

[0623] In some embodiments, a membrane-bound form of the IL- 12 polypeptide described herein may be expressed in a chimeric form using a transmembrane domain derived from, e.g., TNFa, PDGFR and / or CD4.

[0624] A suitable transmembrane-anchored sequence can be important in achieving stable and / or high expression of proteins, e.g., chimeric proteins on the plasma membrane. In particular, a high level of surface expression can be achieved using a transmembrane domain derived from a B7.1 antigen. In some embodiments, the transmembrane domain, or a functional fragment or derivative thereof, described herein may be derived from a B7.1 antigen. In some embodiments, the B7.1 antigen may be of mouse origin. In some embodiments, the B7. 1 antigen may be of human origin.

[0625] In various embodiments, the present disclosure provides a recombinant rhabdovirus comprising a nucleotide sequence encoding a recombinant protein, and the recombinant protein comprises: i. an IL-12 polypeptide comprising an IL-12 p35 subunit, or a functional fragment or derivative thereof, described herein;ii. a signal peptide operably described herein which can be linked to the IL-12 polypeptide; and iii. a transmembrane domain, e.g., a transmembrane domain derived from a B7.1 antigen, described herein.

[0626] In some embodiments, the transmembrane domain derived from a B7.1 antigen comprises the amino acid sequence of SEQ ID NO: 19, 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 about75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98% or at least about 99%, sequence identity with the amino acid sequence ofSEQ ID NO: 19. In certain embodiments, the nucleotide sequence that encodes transmembrane domain derived from a B7.1 antigen comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 19, 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 about80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about98% or at least about 99%, sequence identity with the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the transmembrane domain derived from a B7.1 antigen comprises the amino acid sequence of SEQ ID NO: 19.

[0627] In some embodiments, the B7.1 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 19, or a sequence which has at least 80% sequence identity thereto. In some embodiments, the B7.1 transmembrane domain consists of the amino acid sequence of SEQ ID NO: 19.

[0628] In some embodiments, the transmembrane domain derived from a B7.1 antigen comprises the amino acid sequence of SEQ ID NO: 20, 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 about75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98% or at least about 99%, sequence identity with the amino acid sequence ofSEQ ID NO: 20. In certain embodiments, the nucleotide sequence that encodes transmembrane domain derived from a B7.1 antigen comprises the nucleotide sequence that encodes the aminoacid sequence of SEQ ID NO: 20, 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 the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the transmembrane domain derived from a B7.1 antigen comprises the amino acid sequence of SEQ ID NO: 20.

[0629] In some embodiments, the B7.1 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 20 or a sequence which has at least 80% sequence identity thereto. In some embodiments, the B7.1 transmembrane domain consists of the amino acid sequence of SEQ ID NO: 20.

[0630] In some embodiments, the transmembrane domain derived from a B7.1 antigen comprises the amino acid sequence of SEQ ID NO: 23, 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 the amino acid sequence of SEQ ID NO: 23. In certain embodiments, the nucleotide sequence that encodes transmembrane domain derived from a B7.1 antigen comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 23, 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 the amino acid sequence of SEQ ID NO: 23. In certain embodiments, the transmembrane domain derived from a B7.1 antigen comprises the amino acid sequence of SEQ ID NO: 23.

[0631] In some embodiments, the B7.1 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 23, or a sequence which has at least 80% sequence identity thereto. In some embodiments, the B7.1 transmembrane domain consists of the amino acid sequence of SEQ ID NO: 23.

[0632] In some embodiments, the transmembrane domain derived from a B7.1 antigen comprises the amino acid sequence of SEQ ID NO: 24, 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 the amino acid sequence of SEQ ID NO: 24. In certain embodiments, the nucleotide sequence that encodes transmembrane domain derived from a B7.1 antigen comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 24, 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 the amino acid sequence of SEQ ID NO: 24. In certain embodiments, the transmembrane domain derived from a B7.1 antigen comprises the amino acid sequence of SEQ ID NO: 24.

[0633] In some embodiments, the B7.1 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 24, or a sequence which has at least 80% sequence identity thereto. In some embodiments, the B7.1 transmembrane domain consists of the amino acid sequence of SEQ ID NO: 24.

[0634] In some embodiments, the transmembrane domain derived from a B7.1 antigen comprises the amino acid sequence of SEQ ID NO: 26, 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 the amino acid sequence of SEQ ID NO: 26. In certain embodiments, the nucleotide sequence that encodes transmembrane domain derived from a B7.1 antigen comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about98% or at least about 99%, sequence identity with the amino acid sequence of SEQ ID NO: 26. In certain embodiments, the transmembrane domain derived from a B7.1 antigen comprises the amino acid sequence of SEQ ID NO: 26.

[0635] In some embodiments, the B7.1 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 26, or a sequence which has at least 80% sequence identity thereto. In some embodiments, the B7.1 transmembrane domain consists of the amino acid sequence of SEQ ID NO: 26.

[0636] In certain embodiments, a topological domain may be operably linked to a transmembrane domain disclosed herein (e.g., a B7.1 transmembrane domain). The topological domain may be derived, for example, without limitation, from a B7.1 antigen, tumor necrosis factor (TNF) alpha (TNFa), platelet-derived growth factor receptor (PDGFR), or cluster of differentiation 4 (CD4).

[0637] In some embodiments, the topological domain comprises the amino acid sequence of SEQ ID NO: 21 or 22, 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 the amino acid sequence of SEQ ID NO: 21 or 22. In certain embodiments, the nucleotide sequence that encodes topological domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 21 or 22, 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 the amino acid sequence of SEQ ID NO: 21 or 22. In certain embodiments, the topological domain comprises the amino acid sequence of SEQ ID NO: 21 or 22.

[0638] In some embodiments, the topological domain comprises the amino acid sequence of SEQ ID NO: 21 or 22, or a sequence which has at least 80% sequence identity thereto. In some embodiments, the topological domain consists of the amino acid sequence of SEQ ID NO: 21 or 22.

[0639] In some embodiments, the topological domain comprises the amino acid sequence of SEQ ID NO: 25, 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 the amino acid sequence of SEQ ID NO: 25. In certain embodiments, the nucleotide sequence that encodes topological domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 25, 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 the amino acid sequence of SEQ ID NO: 25. In certain embodiments, the topological domain comprises the amino acid sequence of SEQ ID NO: 25.

[0640] In some embodiments, the topological domain comprises the amino acid sequence of SEQ ID NO: 25, or a sequence which has at least 80% sequence identity thereto. ...

Claims

CLAIMSWhat is claimed is:

1. A recombinant rhabdovirus comprising a nucleotide sequence encoding a recombinant protein, wherein the recombinant protein comprises an IL-12 polypeptide, or a functional fragment or derivative thereof.

2. The recombinant rhabdovirus of claim 1, wherein the recombinant rhabdovirus comprises a rhabdovirus genome, said rhabdovirus genome comprising a gene encoding a fusogenic polypeptide, or a functional fragment or derivative thereof, and a gene encoding a large protein (L) polypeptide, or a functional fragment or derivative thereof, and wherein the nucleotide sequence encoding the recombinant protein is positioned between the gene encoding the fusogenic polypeptide, or a functional fragment or derivative thereof, and the gene encoding the L polypeptide, or a functional fragment or derivative thereof.

3. The recombinant rhabdovirus of claim 1 or 2, wherein the nucleotide sequence encoding the recombinant protein is a template for a positive sense transcript encoding said recombinant protein.

4. The recombinant rhabdovirus of any one of claims 1-3, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises an IL-12 p40 subunit, or a functional fragment or derivative thereof.

5. The recombinant rhabdovirus of claim 4, wherein the IL- 12 polypeptide, or the functional fragment or derivative thereof, further comprises an IL-12 p35 subunit, or a functional fragment or derivative thereof.

6. The recombinant rhabdovirus of any one of claims 1-4, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises an IL-12 p35 subunit, or a functional fragment or derivative thereof.

7. The recombinant rhabdovirus of any one of claims 4-6, wherein the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence which has at least 80% sequence identity thereto.

8. The recombinant rhabdovirus of claim 7, wherein the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 69.

9. The recombinant rhabdovirus of claim 8, wherein the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 69.

10. The recombinant rhabdovirus of any one of claims 4-6, wherein the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence which has at least 80% sequence identity thereto.

11. The recombinant rhabdovirus of claim 10, wherein the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61.

12. The recombinant rhabdovirus of claim 11, wherein the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 61.

13. The recombinant rhabdovirus of any one of claims 5-12, wherein the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence which has at least 80% sequence identity thereto.

14. The recombinant rhabdovirus of claim 13, wherein the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 71.

15. The recombinant rhabdovirus of claim 14, wherein the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 71.

16. The recombinant rhabdovirus of claims 5-12, wherein the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence which has at least 80% sequence identity thereto.

17. The recombinant rhabdovirus of claim 16, wherein the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63.

18. The recombinant rhabdovirus of claim 17, wherein the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 63.

19. The recombinant rhabdovirus of any one of claims 5-18, wherein the IL-12 p40 subunit, or the functional fragment or derivative thereof, and the IL- 12 p35 subunit, or the functional fragment or derivative thereof, are connected by a linker.

20. The recombinant rhabdovirus of claim 19, wherein the linker is from about 5 amino acids to about 30 amino acids in length.

21. The recombinant rhabdovirus of claim 20, wherein the linker comprises the amino acid sequence (VPGVG)2(SEQ ID NO: 17).

22. The recombinant rhabdovirus of claim 21, wherein the linker consists of the amino acid sequence (VPGVG)2(SEQ ID NO: 17).

23. The recombinant rhabdovirus of any one of claims 5-22, wherein the recombinant protein further comprises a signal peptide operably linked to the IL-12 polypeptide.

24. The recombinant rhabdovirus of claim 23, wherein the signal peptide is operably linked to the IL-12 p40 subunit, or the functional fragment or derivative thereof, and / or the IL-12 p35 subunit, or the functional fragment or derivative thereof.

25. The recombinant rhabdovirus of claim 24, wherein the signal peptide is operably linked tothe IL-12 p40 subunit, or the functional fragment or derivative thereof, to form an IL-12 p40 subunit precursor, or a functional fragment or derivative thereof.

26. The recombinant rhabdovirus of claim 25, wherein the signal peptide operably linked to the IL-12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence that has at least 80% sequence identity thereto.

27. The recombinant rhabdovirus of claim 26, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that has at least 80% sequence identity thereto.

28. The recombinant rhabdovirus of claim 27, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 27.

29. The recombinant rhabdovirus of claim 28, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 27.

30. The recombinant rhabdovirus of claims 26-29, wherein the IL-12 p40 subunit precursor, or a functional fragment or derivative thereof, comprises one or more amino acid changes at positions E81, F82, K106, or K217 of the amino acid sequence of SEQ ID NO: 27.

31. The recombinant rhabdovirus of claim 30, wherein the one or more amino acid changes comprises one or more alanine (A) substitutions.

32. The recombinant rhabdovirus of claim 25, wherein the signal peptide operably linked to the IL-12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence that has at least 80% sequence identity thereto.

33. The recombinant rhabdovirus of claim 32, wherein the IL-12 p40 subunit precursor, or thefunctional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that has at least 80% sequence identity thereto.

34. The recombinant rhabdovirus of claim 33, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 12.

35. The recombinant rhabdovirus of claim 34, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 12.

36. The recombinant rhabdovirus of any one of claims 32-35, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises one or more amino acid changes at positions E81, F82, K106, or K217 of the amino acid sequence of SEQ ID NO: 12.

37. The recombinant rhabdovirus of claim 36, wherein the one or more amino acid changes comprises one or more alanine (A) substitutions.

38. The recombinant rhabdovirus of any one of claims 24-27, wherein the signal peptide is operably linked to the IL- 12 p35 subunit, or the functional fragment or derivative thereof, to form an IL- 12 p35 subunit precursor, or a functional fragment or derivative thereof.

39. The recombinant rhabdovirus of claim 38, wherein the signal peptide operably linked to the IL-12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that has at least 80% sequence identity thereto.

40. The recombinant rhabdovirus of claim 38, wherein the signal peptide operably linked to the IL-12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence that has at least 80% sequence identity thereto.41 . The recombinant rhabdovirus of any one of claims 1 -40, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence that has at least 80% sequence identity thereto.

42. The recombinant rhabdovirus of claim 41, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 159.

43. The recombinant rhabdovirus of claim 42, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 159.

44. The recombinant rhabdovirus of any one of claims 1-40, wherein the IL-12 polypeptide, or a functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 154, or an amino acid sequence that has at least 80% sequence identity thereto.

45. The recombinant rhabdovirus of claim 44, wherein the IL- 12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 154.

46. The recombinant rhabdovirus of claim 45, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 154.

47. A recombinant rhabdovirus comprising a nucleotide encoding a recombinant protein comprising an IL-12 polypeptide, wherein the IL-12 polypeptide comprises an IL-12 p40 subunit, or a functional fragment or derivative thereof, and an IL- 12 p35 subunit, or a functional fragment or derivative thereof, wherein the IL- 12 p40 subunit, or a functional fragment or derivative thereof, and the IL- 12 p35 subunit, or a functional fragment or derivative thereof, are connected by a linker, wherein the recombinant rhabdovirus comprises a rhabdovirus genome, said rhabdovirus genome comprising a gene encoding a fusogenic polypeptide, or a functional fragment or derivative thereof, and a gene encoding a large protein (L) polypeptide, or a functional fragment or derivative thereof, and wherein the nucleotide sequence encoding the recombinant protein is positioned between the gene encoding the fusogenic polypeptide, or a functional fragment or derivative thereof, and thegene encoding the L polypeptide, or a functional fragment or derivative thereof.

48. The recombinant rhabdovirus of claim 47, wherein the nucleotide sequence encoding the recombinant protein is a template for a positive sense transcript encoding said recombinant protein.

49. The recombinant rhabdovirus of claim 47 or 48, wherein the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence which has at least 80% sequence identity thereto.

50. The recombinant rhabdovirus of claim 49, wherein the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 69.

51. The recombinant rhabdovirus of claim 50, wherein the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 69.

52. The recombinant rhabdovirus of claim 47 or 48, wherein the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence which has at least 80% sequence identity thereto.

53. The recombinant rhabdovirus of claim 52, wherein the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61.

54. The recombinant rhabdovirus of claim 53, wherein the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 61.

55. The recombinant rhabdovirus of any one of claims 47-54, wherein the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence which has at least 80% sequence identity thereto.

56. The recombinant rhabdovirus of claim 55, wherein the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 71.

57. The recombinant rhabdovirus of claim 56, wherein the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 71.

58. The recombinant rhabdovirus of any one of claims 47-54, wherein the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence which has at least 80% sequence identity thereto.

59. The recombinant rhabdovirus of claim 58, wherein the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63.

60. The recombinant rhabdovirus of claim 59, wherein the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 63.

61. The recombinant rhabdovirus of any one of claims 47-60, wherein the linker is from about 5 amino acids to about 30 amino acids in length.

62. The recombinant rhabdovirus of claim 61, wherein the linker comprises the amino acid sequence (VPGVG)2(SEQ ID NO: 17).

63. The recombinant rhabdovirus of claim 62, wherein the linker consists of the amino acid sequence (VPGVG)2(SEQ ID NO: 17).

64. The recombinant rhabdovirus of any one of claims 47-63, wherein the recombinant protein further comprises a signal peptide operably linked to the IL-12 p40 subunit, or the functional fragment or derivative thereof, to form an IL- 12 p40 subunit precursor, or a functional fragment or derivative thereof.

65. The recombinant rhabdovirus of claim 64, wherein the signal peptide operably linked tothe IL-12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence that has at least 80% sequence identity thereto.

66. The recombinant rhabdovirus of claim 65, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that has at least 80% sequence identity thereto.

67. The recombinant rhabdovirus of claim 66, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 27.

68. The recombinant rhabdovirus of claim 67, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 27.

69. The recombinant rhabdovirus of any one of claims 65-68, wherein the IL-12 p40 subunit precursor, or a functional fragment or derivative thereof, comprises one or more amino acid changes at positions E81, F82, K106, or K217 of the amino acid sequence of SEQ ID NO: 27.

70. The recombinant rhabdovirus of claim 69, wherein the one or more amino acid changes comprises one or more alanine (A) substitutions.

71. The recombinant rhabdovirus of claim 64, wherein the signal peptide operably linked to the IL-12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence that has at least 80% sequence identity thereto.

72. The recombinant rhabdovirus of claim 71, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that has at least 80% sequence identity thereto.

73. The recombinant rhabdovirus of claim 72, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 12.

74. The recombinant rhabdovirus of claim 73, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 12.

75. The recombinant rhabdovirus of any one of claims 71-74, wherein the IL- 12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises one or more amino acid changes at positions E81, F82, K106, or K217 of the amino acid sequence of SEQ ID NO: 12.

76. The recombinant rhabdovirus of claim 75, wherein the one or more amino acid changes comprises one or more alanine (A) substitutions.

77. The recombinant rhabdovirus of any one of claims 47-76, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence that has at least 80% sequence identity thereto.

78. The recombinant rhabdovirus of claim 77, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 159.

79. The recombinant rhabdovirus of claim 78, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 159.

80. The recombinant rhabdovirus of any one of claims 47-76, wherein the IL-12 polypeptide, or a functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 154, or an amino acid sequence that has at least 80% sequence identity thereto.

81. The recombinant rhabdovirus of claim 80, wherein the IL- 12 polypeptide, or the functionalfragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 154.

82. The recombinant rhabdovirus of claim 81, wherein the IL- 12 polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 154.

83. The recombinant rhabdovirus of any one of claims 1-82, wherein the recombinant rhabdovirus is a vesiculovirus.

84. The recombinant rhabdovirus of claim 83, wherein the vesiculovirus is a vesicular stomatitis virus (VSV).

85. The recombinant rhabdovirus of claim 84, wherein the rhabdovirus genome further comprises a gene encoding a VSV nucleoprotein (N) polypeptide, or a functional fragment or derivative thereof.

86. The recombinant rhabdovirus of any one of claims 83-85, wherein the rhabdovirus genome further comprises a gene encoding a VSV phosphoprotein (P) polypeptide, or a functional fragment or derivative thereof.

87. The recombinant rhabdovirus of any one of claims 83-86, wherein the rhabdovirus genome further comprises a gene encoding a VSV matrix (M) polypeptide, or a functional fragment or derivative thereof.

88. The recombinant rhabdovirus of any one of claims 83-87, wherein the L polypeptide is a VSV large protein (L) polypeptide, or a functional fragment or derivative thereof.

89. The recombinant rhabdovirus of claim 88, wherein the rhabdovirus genome comprises a gene encoding a VSV nucleoprotein (N) polypeptide, or a functional fragment or derivative thereof, a gene encoding a VSV phosphoprotein (P) polypeptide, or a functional fragment or derivative thereof, a gene encoding a VSV matrix (M) polypeptide, or a functional fragment or derivative thereof, a gene encoding a fusogenic polypeptide, or a functionalfragment or derivative thereof, and a gene encoding a VSV large protein (L) polypeptide, or a functional fragment or derivative thereof.

90. The recombinant rhabdovirus of claim 89, wherein the recombinant rhabdovirus comprises an RNA molecule, wherein the RNA molecule comprises a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, or a functional fragment or derivative thereof, and a nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein.

91. The recombinant rhabdovirus of claim 90, wherein the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein is positioned between the nucleotide sequence that is a template for a positive sense transcript encoding the fusogenic polypeptide, or the functional fragment or derivative thereof, and the nucleotide sequence that is a template for a positive sense transcript encoding the VSV L polypeptide, or the functional fragment or derivative thereof.

92. The recombinant rhabdovirus of claim 91, wherein the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein comprises, from the 3’ end to the 5’ end of said nucleotide sequence, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSVP polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template fora positive sense transcript encoding a fusogenic polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein, and a nucleotide sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, or a functional fragment or derivative thereof.

93. The recombinant rhabdovirus of any one of claims 87-92, wherein the VSV M polypeptide is a wild-type VSV M polypeptide, or a functional fragment or derivative thereof.

94. The recombinant rhabdovirus of claim 93, wherein the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that has at least 80% sequence identity thereto.

95. The recombinant rhabdovirus of claim 94, wherein the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 3.

96. The recombinant rhabdovirus of claim 95, wherein the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 3.

97. The recombinant rhabdovirus of any one of claims 87-92, wherein the VSV M polypeptide is a mutant VSV M polypeptide, or a functional fragment or derivative thereof.

98. The recombinant rhabdovirus of claim 97, wherein the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises an amino acid change at position M51 of the amino acid sequence of SEQ ID NO: 3.

99. The recombinant rhabdovirus of claim 98, wherein the amino acid change comprises a methionine (M) substitution.

100. The recombinant rhabdovirus of claim 99, wherein the methionine (M) substitution comprises a methionine (M) to an arginine (R) substitution (M51R).

101. The recombinant rhabdovirus of any one of claims 97-100, wherein the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that has at least 80% sequence identity thereto.

102. The recombinant rhabdovirus of claim 101, wherein the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 5.

103. The recombinant rhabdovirus of claim 102, wherein the mutant VSV M polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 5.

104. The recombinant rhabdovirus of any one of claims 89-103, wherein the fusogenic polypeptide is a VSV glycoprotein (G) polypeptide, or a functional fragment or derivative thereof.

105. The recombinant rhabdovirus of any one of claims 47-104, wherein the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence that has at least 80% sequence identity thereto.

106. The recombinant rhabdovirus of claim 105, wherein the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 49.

107. The recombinant rhabdovirus of claim 106, wherein the recombinant rhabdovirus consists of the amino acid sequence of SEQ ID NO: 49.

108. The recombinant rhabdovirus of any one of claims 47-104, wherein the recombinantrhabdovirus comprises the amino acid sequence of SEQ ID NO: 113, or an amino acid sequence that has at least 80% sequence identity thereto.

109. The recombinant rhabdovirus of claim 108, wherein the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 113.

110. The recombinant rhabdovirus of claim 109, wherein the recombinant rhabdovirus consists of the amino acid sequence of SEQ ID NO: 113.

111. The recombinant rhabdovirus of any one of claims 1-110, wherein upon infection of a cell with the recombinant rhabdovirus, the cell expresses the recombinant protein comprising the IL- 12 polypeptide, or the functional fragment or derivative thereof.

112. The recombinant rhabdovirus of claim 111, wherein the cell is a human cell.

113. The recombinant rhabdovirus of claim 111 or 112, wherein the cell is a cancer cell.

114. The recombinant rhabdovirus of any one of claims 1-113, wherein the recombinant rhabdovirus is a replication-competent virus.

115. The recombinant rhabdovirus of any one of claims 1-113, wherein the recombinant rhabdovirus is a non-replicative.

116. The recombinant rhabdovirus of any one of claims 1-115, wherein the recombinant rhabdovirus is an oncolytic virus.

117. A recombinant polynucleotide comprising: i. a nucleotide sequence encoding a vesicular stomatitis virus (VSV) nucleoprotein (N) polypeptide, or a functional fragment or derivative thereof; ii. a nucleotide sequence encoding a VSV phosphoprotein (P) polypeptide, or a functional fragment or derivative thereof;iii. a nucleotide sequence encoding a VSV matrix (M) polypeptide, or a functional fragment or derivative thereof; iv. a nucleotide sequence encoding a fusogenic polypeptide, or a functional fragment or derivative thereof; v. a nucleotide sequence encoding a VSV large protein (L) polypeptide, or a functional fragment or derivative thereof; and vi. a nucleotide sequence encoding a recombinant protein comprising an IL-12 polypeptide, or a functional fragment or derivative thereof.

118. The recombinant polynucleotide of claim 117, wherein the nucleotide sequence encoding the recombinant protein is positioned between the nucleotide sequence encoding the fusogenic polypeptide, or a functional fragment or derivative thereof, and the nucleotide sequence encoding the L polypeptide, or a functional fragment or derivative thereof.

119. The recombinant polynucleotide of claim 117 or 118, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises an IL-12 p40 subunit, or a functional fragment or derivative thereof.

120. The recombinant polynucleotide of claim 119, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, further comprises an IL- 12 p35 subunit, or a functional fragment or derivative thereof.

121. The recombinant polynucleotide of any one of claims 117-119, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises an IL-12 p35 subunit, or a functional fragment or derivative thereof.

122. The recombinant polynucleotide of any one of claims 119-121, wherein the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence which has at least 80% sequence identity thereto.

123. The recombinant polynucleotide of claim 122, wherein the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 69.

124. The recombinant polynucleotide of claim 123, wherein the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 69.

125. The recombinant polynucleotide of any one of claims 119-121, wherein the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence which has at least 80% sequence identity thereto.

126. The recombinant polynucleotide of claim 125, wherein the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61.

127. The recombinant polynucleotide of claim 126, wherein the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 61.

128. The recombinant polynucleotide of any one of claims 120-127, wherein the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence which has at least 80% sequence identity thereto.

129. The recombinant polynucleotide of claim 128, wherein the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 71.

130. The recombinant polynucleotide of claim 129, wherein the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 71.

131. The recombinant polynucleotide of any one of claims 120-127, wherein the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence which has at least 80% sequence identity thereto.

132. The recombinant polynucleotide of claim 131, wherein the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63.

133. The recombinant polynucleotide of claim 132, wherein the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 63.

134. The recombinant polynucleotide of any one of claims 120-133, wherein the IL-12 p40 subunit, or the functional fragment or derivative thereof, and the IL-12 p35 subunit, or the functional fragment or derivative thereof, are connected by a linker.

135. The recombinant polynucleotide of claim 134, wherein the linker is from about 5 amino acids to about 30 amino acids in length.

136. The recombinant polynucleotide of claim 135, wherein the linker comprises the amino acid sequence (VPGVG)2(SEQ ID NO: 17).

137. The recombinant polynucleotide of claim 136, wherein the linker consists of the amino acid sequence (VPGVG)2(SEQ ID NO: 17).

138. The recombinant polynucleotide of any one of claims 120-137, wherein the recombinant protein further comprises a signal peptide operably linked to the IL- 12 polypeptide.

139. The recombinant polynucleotide of claim 138, wherein the signal peptide is operably linked to the IL-12 p40 subunit, or the functional fragment or derivative thereof, and / or the IL-12 p35 subunit, or the functional fragment or derivative thereof.

140. The recombinant polynucleotide of claim 139, wherein the signal peptide is operably linked to the IL- 12 p40 subunit, or the functional fragment or derivative thereof, to form an IL- 12 p40 subunit precursor, or a functional fragment or derivative thereof.

141. The recombinant polynucleotide of claim 140, wherein the signal peptide operably linked to the IL-12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence that has at least 80% sequence identity thereto.

142. The recombinant polynucleotide of claim 141, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that has at least 80% sequence identity thereto.

143. The recombinant polynucleotide of claim 142, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 27.

144. The recombinant polynucleotide of claim 143, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 27.

145. The recombinant polynucleotide of any one of claims 141-144, wherein the IL- 12 p40 subunit precursor, or a functional fragment or derivative thereof, comprises one or more amino acid changes at positions E81, F82, K106, or K217 of the amino acid sequence of SEQ ID NO: 27.

146. The recombinant polynucleotide of claim 145, wherein the one or more amino acid changes comprises one or more alanine (A) substitutions.

147. The recombinant polynucleotide of claim 140, wherein the signal peptide operably linked to the IL-12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 65, or anamino acid sequence that has at least 80% sequence identity thereto.

148. The recombinant polynucleotide of claim 147, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that has at least 80% sequence identity thereto.

149. The recombinant polynucleotide of claim 148, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 12.

150. The recombinant polynucleotide of claim 149, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 12.

151. The recombinant polynucleotide of any one of claims 147-150, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises one or more amino acid changes at positions E81, F82, KI 06, or K217 of the amino acid sequence of SEQ ID NO: 12.

152. The recombinant polynucleotide of claim 151, wherein the one or more amino acid changes comprises one or more alanine (A) substitutions.

153. The recombinant polynucleotide of any one of claims 139-152, wherein the signal peptide is operably linked to the IL- 12 p35 subunit, or the functional fragment or derivative thereof, to form an IL- 12 p35 subunit precursor, or a functional fragment or derivative thereof.

154. The recombinant polynucleotide of claim 153, wherein the signal peptide operably linked to the IL-12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that has at least 80% sequence identity thereto.

155. The recombinant polynucleotide of claim 153, wherein the signal peptide operably linkedto the IL-12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence that has at least 80% sequence identity thereto.

156. The recombinant polynucleotide of any one of claims 117-155, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence that has at least 80% sequence identity thereto.

157. The recombinant polynucleotide of claim 156, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 159.

158. The recombinant polynucleotide of claim 157, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 159.

159. The recombinant polynucleotide of any one of claims 117-155, wherein the IL-12 polypeptide, or a functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 154, or an amino acid sequence that has at least 80% sequence identity thereto.

160. The recombinant polynucleotide of claim 159, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 154.

161. The recombinant polynucleotide of claim 160, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 154.

162. The recombinant polynucleotide of any one of claims 117-161, comprising a nucleotide sequence encoding, from the 5’ end to the 3’ end of said nucleotide sequence, a VSV Npolypeptide, or a functional fragment or derivative thereof, a VSV P polypeptide, or a functional fragment or derivative thereof, a VSV M polypeptide, or a functional fragment or derivative thereof, a fusogenic polypeptide, or a functional fragment or derivative thereof, the recombinant protein, and a VSV L polypeptide, or a functional fragment or derivative thereof.

163. The recombinant polynucleotide of any one of claims 117-162, wherein the VSV M polypeptide is a wild-type VSV M polypeptide, or a functional fragment or derivative thereof.

164. The recombinant polynucleotide of claim 163, wherein the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that has at least 80% sequence identity thereto.

165. The recombinant polynucleotide of claim 164, wherein the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 3.

166. The recombinant polynucleotide of claim 165, wherein the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 3.

167. The recombinant polynucleotide of any one of claims 117-162, wherein the VSV M polypeptide is a mutant VSV M polypeptide, or a functional fragment or derivative thereof.

168. The recombinant polynucleotide of claim 167, wherein the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises an amino acid change at position M51 of the amino acid sequence of SEQ ID NO: 3.

169. The recombinant polynucleotide of claim 168, wherein the amino acid change comprises a methionine (M) substitution.

170. The recombinant polynucleotide of claim 169, wherein the methionine (M) substitution comprises a methionine (M) to an arginine (R) substitution (M51R).

171. The recombinant polynucleotide of any one of claims 167-170, wherein the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that has at least 80% sequence identity thereto.

172. The recombinant polynucleotide of claim 171, wherein the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 5.

173. The recombinant polynucleotide of claim 172, wherein the mutant VSV M polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 5.

174. The recombinant polynucleotide of any one of claims 117-173, wherein the fusogenic polypeptide is a VSV glycoprotein (G) polypeptide, or a functional fragment or derivative thereof.

175. The recombinant polynucleotide of any one of claims 117-174, wherein the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence that has at least 80% sequence identity thereto.

176. The recombinant polynucleotide of claim 175, wherein the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 49.

177. The recombinant polynucleotide of claim 176, wherein the recombinant rhabdovirus consists of the amino acid sequence of SEQ ID NO: 49.

178. The recombinant polynucleotide of any one of claims 1 17-174, wherein the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 113, or an amino acid sequence that has at least 80% sequence identity thereto.

179. The recombinant polynucleotide of claim 178, wherein the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 113.

180. The recombinant polynucleotide of claim 179, wherein the recombinant rhabdovirus consists of the amino acid sequence of SEQ ID NO: 113.

181. The recombinant polynucleotide of any one of claims 117-180, wherein the polynucleotide is DNA.

182. The recombinant polynucleotide of claim 181, wherein the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 50, or a nucleotide sequence which has at least 65% sequence identity thereto.

183. The recombinant polynucleotide of claim 182, wherein the recombinant polynucleotide comprises the amino acid sequence of SEQ ID NO: 50.

184. The recombinant polynucleotide of claim 183, wherein the recombinant polynucleotide consists of the amino acid sequence of SEQ ID NO: 50.

185. The recombinant polynucleotide of claim 181, wherein the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 112, or a nucleotide sequence which has at least 65% sequence identity thereto.

186. The recombinant polynucleotide of claim 185, wherein the recombinant polynucleotide comprises the amino acid sequence of SEQ ID NO: 112.

187. The recombinant polynucleotide of claim 186, wherein the recombinant polynucleotideconsists of the amino acid sequence of SEQ ID NO: 112.

188. The recombinant polynucleotide of any one of claims 117-180, wherein the polynucleotide is RNA.

189. A recombinant polynucleotide, comprising a nucleotide sequence encoding, from the 5’ end to the 3’ end of said nucleotide sequence, a VSV N polypeptide, or a functional fragment or derivative thereof, a VSV P polypeptide, or a functional fragment or derivative thereof, a VSV M polypeptide, or a functional fragment or derivative thereof, a fusogenic polypeptide, or a functional fragment or derivative thereof, a recombinant protein, and a VSV L polypeptide, or a functional fragment or derivative thereof, wherein: i. the VSV M polypeptide, or the functional fragment or derivative thereof, is a mutant VSV M polypeptide, or a functional fragment or derivative thereof, said mutant VSV M polypeptide comprising the amino acid sequence of SEQ ID NO: 5; ii. the fusogenic polypeptide, or the functional fragment thereof, is a VSV glycoprotein (G) polypeptide, or a functional fragment or derivative thereof; and iii. the recombinant protein comprises an IL-12 polypeptide, or a functional fragment or derivative thereof, said IL- 12 polypeptide comprising the amino acid sequence of SEQ ID NO: 159 or SEQ ID NO: 154.

190. The recombinant polynucleotide of claim 189, wherein the IL-12 polypeptide comprises the amino acid sequence of SEQ ID NO: 159, and the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 49.

191. The recombinant polynucleotide of claim 189, wherein the IL-12 polypeptide comprises the amino acid sequence of SEQ ID NO: 154, and the recombinant protein comprises the amino acid sequence of SEQ ID NO: 113.

192. A recombinant polynucleotide, wherein the recombinant polynucleotide is an RNAmolecule comprising: i. a nucleotide sequence that is a template for a positive sense transcript encoding a VSV nucleoprotein (N) polypeptide, or a functional fragment or derivative thereof; ii. a nucleotide sequence that is a template for a positive sense transcript encoding a VSV phosphoprotein (P) polypeptide, or a functional fragment or derivative thereof; iii. a nucleotide sequence that is a template for a positive sense transcript encoding a VSV matrix (M) polypeptide, or a functional fragment or derivative thereof; iv. a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide, or a functional fragment or derivative thereof; v. a nucleotide sequence that is a template for a positive sense transcript encoding a VSV large protein (L) polypeptide, or a functional fragment or derivative thereof; and vi. a nucleotide sequence that is a template for a positive sense transcript encoding a recombinant protein comprising an IL-12 polypeptide, or a functional fragment or derivative thereof.

193. The recombinant polynucleotide of claim 192, wherein the nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein is positioned between the nucleotide sequence that is a template for a positive sense transcript encoding the fusogenic polypeptide, or the functional fragment or derivative thereof, and the nucleotide sequence that is a template for a positive sense transcript encoding the VSV L polypeptide, or a functional fragment or derivative thereof.

194. The recombinant polynucleotide of claim 192 or 193, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises an IL-12 p40 subunit, or a functional fragment or derivative thereof.

195. The recombinant polynucleotide of claim 194, wherein the IL- 12 polypeptide, or the functional fragment or derivative thereof, further comprises an IL-12 p35 subunit, or a functional fragment or derivative thereof.

196. The recombinant polynucleotide of any one of claims 192-194, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises an IL-12 p35 subunit, or a functional fragment or derivative thereof.

197. The recombinant polynucleotide of any one of claims 194- 196, wherein the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence which has at least 80% sequence identity thereto.

198. The recombinant polynucleotide of claim 197, wherein the IL- 12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 69.

199. The recombinant polynucleotide of claim 198, wherein the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 69.

200. The recombinant polynucleotide of any one of claims 194-196, wherein the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence which has at least 80% sequence identity thereto.

201. The recombinant polynucleotide of claim 200, wherein the IL-12 p40 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 61.

202. The recombinant polynucleotide of claim 201, wherein the IL-12 p40 subunit consists of the amino acid sequence of SEQ ID NO: 61.

203. The recombinant polynucleotide of any one of claims 195-202, wherein the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acidsequence of SEQ ID NO: 71, or an amino acid sequence which has at least 80% sequence identity thereto.

204. The recombinant polynucleotide of claim 203, wherein the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 71.

205. The recombinant polynucleotide of claim 204, wherein the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 71.

206. The recombinant polynucleotide of any one of claims 195-202, wherein the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence which has at least 80% sequence identity thereto.

207. The recombinant polynucleotide of claim 206, wherein the IL-12 p35 subunit, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 63.

208. The recombinant polynucleotide of claim 207, wherein the IL-12 p35 subunit consists of the amino acid sequence of SEQ ID NO: 63.

209. The recombinant polynucleotide of any one of claims 195-208, wherein the IL-12 p40 subunit, or the functional fragment or derivative thereof, and the IL- 12 p35 subunit, or the functional fragment or derivative thereof, are connected by a linker.

210. The recombinant polynucleotide of claim 209, wherein the linker is from about 5 amino acids to about 30 amino acids in length.

211. The recombinant polynucleotide of claim 210, wherein the linker comprises the amino acid sequence (VPGVG)2(SEQ ID NO: 17).

212. The recombinant polynucleotide of claim 211, wherein the linker consists of the amino acid sequence (VPGVG)2(SEQ ID NO: 17).

213. The recombinant polynucleotide of any one of claims 195-212, wherein the recombinant protein further comprises a signal peptide operably linked to the IL-12 polypeptide.

214. The recombinant polynucleotide of claim 213, wherein the signal peptide is operably linked to the IL-12 p40 subunit, or the functional fragment or derivative thereof, and / or the IL-12 p35 subunit, or the functional fragment or derivative thereof.

215. The recombinant polynucleotide of claim 214, wherein the signal peptide is operably linked to the IL-12 p40 subunit, or the functional fragment or derivative thereof, to form an IL-12 p40 subunit precursor, or a functional fragment or derivative thereof.

216. The recombinant polynucleotide of claim 215, wherein the signal peptide operably linked to the IL-12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence that has at least 80% sequence identity thereto.

217. The recombinant polynucleotide of claim 216, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that has at least 80% sequence identity thereto.

218. The recombinant polynucleotide of claim 217, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 27.

219. The recombinant polynucleotide of claim 218, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 27.

220. The recombinant polynucleotide of any one of claims 216-219, wherein the IL-12 p40 subunit precursor, or a functional fragment or derivative thereof, comprises one or more amino acid changes at positions E81, F82, K106, or K217 of the amino acid sequence of SEQ ID NO: 27.

221. The recombinant polynucleotide of claim 220, wherein the one or more amino acid changes comprises one or more alanine (A) substitutions.

222. The recombinant polynucleotide of claim 215, wherein the signal peptide operably linked to the IL-12 p40 subunit comprises the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence that has at least 80% sequence identity thereto.

223. The recombinant polynucleotide of claim 222, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that has at least 80% sequence identity thereto.

224. The recombinant polynucleotide of claim 223, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 12.

225. The recombinant polynucleotide of claim 224, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 12.

226. The recombinant polynucleotide of any one of claims 222-225, wherein the IL-12 p40 subunit precursor, or the functional fragment or derivative thereof, comprises one or more amino acid changes at positions E81, F82, K106, or K217 of the amino acid sequence of SEQ ID NO: 12.

227. The recombinant polynucleotide of claim 226, wherein the one or more amino acid changes comprises one or more alanine (A) substitutions.

228. The recombinant polynucleotide of any one of claims 214-227, wherein the signal peptide is operably linked to the IL-12 p35 subunit, or the functional fragment or derivative thereof, to form an IL- 12 p35 subunit precursor, or a functional fragment or derivative thereof.

229. The recombinant polynucleotide of claim 228, wherein the signal peptide operably linked to the IL-12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that has at least 80% sequence identity thereto.

230. The recombinant polynucleotide of claim 228, wherein the signal peptide operably linked to the IL-12 p35 subunit comprises the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence that has at least 80% sequence identity thereto.

231. The recombinant polynucleotide of any one of claims 192-230, wherein the IL- 12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence that has at least 80% sequence identity thereto.

232. The recombinant polynucleotide of claim 231, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 159.

233. The recombinant polynucleotide of claim 232, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 159.

234. The recombinant polynucleotide of any one of claims 192-230, wherein the IL-12 polypeptide, or a functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 154, or an amino acid sequence that has at least 80% sequence identity thereto.

235. The recombinant polynucleotide of claim 234, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 154.

236. The recombinant polynucleotide of claim 235, wherein the IL-12 polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 154.

237. The recombinant polynucleotide of any one of claims 192-236, wherein the recombinant polynucleotide comprises, from the 3’ end to the 5’ end of said recombinant polynucleotide, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding the recombinant protein, and a nucleotide sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, or a functional fragment or derivative thereof.

238. The recombinant polynucleotide of any one of claims 192-237, wherein the VSV M polypeptide is a wild-type VSV M polypeptide, or a functional fragment or derivative thereof.

239. The recombinant polynucleotide of claim 238, wherein the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that has at least 80% sequence identity thereto.

240. The recombinant polynucleotide of claim 239, wherein the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQID NO: 3.

241. The recombinant polynucleotide of claim 240, wherein the wild-type VSV M polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 3.

242. The recombinant polynucleotide of any one of claims 192-237, wherein the VSV M polypeptide is a mutant VSV M polypeptide, or a functional fragment or derivative thereof.

243. The recombinant polynucleotide of claim 242, wherein the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises an amino acid change at position M51 of the amino acid sequence of SEQ ID NO: 3.

244. The recombinant polynucleotide of claim 243, wherein the amino acid change comprises a methionine (M) substitution.

245. The recombinant polynucleotide of claim 244, wherein the methionine (M) substitution comprises a methionine (M) to an arginine (R) substitution (M51R).

246. The recombinant polynucleotide of any one of claims 242-245, wherein the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that has at least 80% sequence identity thereto.

247. The recombinant polynucleotide of claim 246, wherein the mutant VSV M polypeptide, or the functional fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 5.

248. The recombinant polynucleotide of claim 247, wherein the mutant VSV M polypeptide, or the functional fragment or derivative thereof, consists of the amino acid sequence of SEQ ID NO: 5.

249. The recombinant polynucleotide of any one of claims 192-248, wherein the fusogenic polypeptide is a VSV glycoprotein (G) polypeptide, or a functional fragment or derivative thereof.

250. The recombinant polynucleotide of any one of claims 192-249, wherein the recombinant protein comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence that has at least 80% sequence identity thereto.

251. The recombinant polynucleotide of claim 250, wherein the recombinant protein comprises the amino acid sequence of SEQ ID NO: 49.

252. The recombinant polynucleotide of claim 251, wherein the recombinant protein consists of the amino acid sequence of SEQ ID NO: 49.

253. The recombinant polynucleotide of any one of claims 192-249, wherein the recombinant protein comprises the amino acid sequence of SEQ ID NO: 113, or an amino acid sequence that has at least 80% sequence identity thereto.

254. The recombinant polynucleotide of claim 253, wherein the recombinant protein comprises the amino acid sequence of SEQ ID NO: 113.

255. The recombinant polynucleotide of claim 254, wherein the recombinant protein consists of the amino acid sequence of SEQ ID NO: 113.

256. A recombinant polynucleotide, wherein the recombinant polynucleotide is an RNA molecule comprising, from the 3’ end to the 5’ end of said recombinant polynucleotide, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sensetranscript encoding a VSV M polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a fusogenic polypeptide, or a functional fragment or derivative thereof, a nucleotide sequence that is a template for a positive sense transcript encoding a recombinant protein, and a nucleotide sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, or a functional fragment or derivative thereof, wherein: i. the VSV M polypeptide, or the functional fragment or derivative thereof, is a mutant VSV M polypeptide, or a functional fragment or derivative thereof, said mutant VSV M polypeptide comprising the amino acid sequence of SEQ ID NO: 5; ii. the fusogenic polypeptide, or the functional fragment thereof, is a VSV glycoprotein (G) polypeptide, or a functional fragment or derivative thereof; and iii. the recombinant protein comprises an IL-12 polypeptide, or a functional fragment or derivative thereof, said IL- 12 polypeptide comprising the amino acid sequence of SEQ ID NO: 159 or SEQ ID NO: 154.

257. The recombinant polynucleotide of claim 256, wherein the IL-12 polypeptide comprises the amino acid sequence of SEQ ID NO: 159, and the recombinant rhabdovirus comprises the amino acid sequence of SEQ ID NO: 49.

258. The recombinant polynucleotide of claim 256, wherein the IL-12 polypeptide comprises the amino acid sequence of SEQ ID NO: 154, and the recombinant protein comprises the amino acid sequence of SEQ ID NO: 113.

259. A vector comprising the recombinant polynucleotide of any one of claims 117-258.

260. A recombinant vesicular stomatitis virus (VSV) comprising the recombinant polynucleotide of any one of claims 117-258.

261. A composition comprising the recombinant rhabdovirus of any one of claims 1-116, and acarrier and / or excipient.

262. A composition comprising the recombinant polynucleotide of any one of claims 117-258, and a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

263. A product of manufacture comprising the recombinant rhabdovirus of any one of claims 1 - 116 or the composition of claim 261 or 262 in a sterile vial, ampoule or syringe.

264. A host cell comprising the recombinant rhabdovirus of any one of claims 1-116.

265. A host cell comprising the recombinant polynucleotide of any one of claims 117-258.

266. A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the recombinant rhabdovirus of any one of claims 1-116 or the composition of claim 262.

267. The method of claim 266, wherein the administration of the recombinant rhabdovirus or composition to the subject is under conditions wherein the recombinant rhabdovirus infects cancer cells of the subject to form infected cancer cells, wherein the infected cancer cells express the recombinant protein comprising the IL-12 polypeptide, or the functional fragment or derivative thereof, and wherein the number of cancer cells within the subject is reduced following the administration.

268. The method of claim 266, wherein the administration of the recombinant rhabdovirus or composition to the subject is under conditions wherein the recombinant rhabdovirus infects cancer cells of the subject to form infected cancer cells, wherein the infected cancer cells express the recombinant protein comprising the IL- 12 polypeptide, or the functional fragment or derivative thereof, and wherein the number of cancer cells within the subject does not increase following the administration.

269. A method for inducing a cancer regression in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effective amount of the recombinant rhabdovirus of any one of claims 1-116 or the composition of claim 262.

270. The method of claim 269, wherein the cancer regression produces a reduced tumor size.

271. The method of any one of claims 266-270, wherein the recombinant rhabdovirus or composition is administered via local delivery.

272. The method of claim 271, wherein the local delivery is intratumoral delivery.

273. The method of claim 271 or 272, wherein the recombinant rhabdovirus or composition is administered at a dose of from about lxlO6 / mL TCID50 / mL to about11TCID50 / m1Lx.10274. The method of claim 273, wherein the recombinant rhabdovirus or composition is administered at a dose of from about7TCID1x501 / 0mL to about10TCID50 / m1Lx.10275. The method of claim 273 or 274, wherein the recombinant rhabdovirus or composition is administered at a dose of about7TC1xID1050 / mL, about 3xl07TCID50 / mL, about IxlO8TCIDjo / mL, about 3xl08TCID50 / mL, about9TC1xID1050 / mL, about 3xl09TCID50 / mL, or about 1x1100TCID50 / mL.

276. The method of claim 275, wherein the recombinant rhabdovirus or composition is administered at a dose of about lxlOloTCID5o / mL.

277. The method of any one of claims 266-276, wherein the recombinant rhabdovirus comprises a nucleotide sequence encoding a mutant VSV matrix (M) polypeptide, or a functional fragment or derivative thereof, said mutant VSV M polypeptide comprising a methionine (M) to an arginine (R) substitution at position M51 of the amino acid sequence of SEQ ID NO: 3, and wherein the administration of the recombinant rhabdovirus to the subject produces a reduced serum interferon gamma (IFN-y) release in the subject as compared to a control recombinant rhabdovirus comprising a nucleotide sequence encoding a wild-typeVSV M polypeptide, or a functional fragment or derivative thereof.

278. The method of any one of claims 266-277, wherein the recombinant rhabdovims comprises a nucleotide sequence encoding a mutant VSV matrix (M) polypeptide, or a functional fragment or derivative thereof, said mutant VSV M polypeptide comprising a methionine (M) to an arginine (R) substitution at position M51 of the amino acid sequence of SEQ ID NO: 3, and wherein the administration of the recombinant rhabdovirus to the subject produces a maintained or increased body weight in the subject as compared to a control recombinant rhabdovirus comprising a nucleotide sequence encoding a wild-type VSV M polypeptide, or a functional fragment or derivative thereof.

279. The method of any one of claims 266-278, wherein the administration of the recombinant rhabdovirus to the subject produces an enhanced tumor-specific CD8 T cell response in the subject as compared to a control recombinant rhabdovirus which does not comprise a nucleotide sequence encoding a recombinant protein comprising an IL-12 polypeptide, or a functional fragment or derivative thereof.

280. The method of claim 266, wherein the enhanced tumor-specific CD8 T cell response comprises an increased serum interferon gamma (ZFN-y) release.

281. The method of any one of claims 266-280, wherein the cancer is selected from head and neck cancer, colon cancer, lung cancer, prostate cancer, ovarian cancer, hepatocellular carcinoma, pancreatic cancer, kidney cancer, melanoma, brain cancer, lymphoma, myeloma, lymphocytic leukemia, myelogenous leukemia, and breast cancer.

282. The method of any one of claims 266-281, wherein the method further comprises administering to the subject a therapeutically effective amount of one or more immune checkpoint inhibitors.

283. The method of claim 282, wherein the immune checkpoint inhibitors comprise one of more of an anti-PD-1 antibody, an anti-PDLl antibody, or an anti-CTLA-4 antibody.

284. The method of claim 283, wherein the immune checkpoint inhibitor comprises an anti-PD- 1 antibody.

285. The method of any one of claims 282-284, wherein the immune checkpoint inhibitor is administered via systemic delivery.

286. The method of claim 285, wherein the immune checkpoint inhibitor is administered via parenteral delivery.

287. The method of claim 286, wherein the parenteral delivery is subcutaneous, intraperitoneal, intradermal, intramuscular, or intravenous delivery.

288. The method of claim 287, wherein the parenteral delivery is intravenous delivery.

289. The method of any one of claims 285-288, wherein the immune checkpoint inhibitor is administered at a dose from about 200 mg to about 400 mg.

290. The method of claim 289, wherein the immune checkpoint inhibitor is administered at a dose of about 350 mg.

291. The method of any one of claims 266-290, wherein the subject is human.

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