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WO2025231185A1 - Variants de glycoprotéines virales et leurs utilisations - Google Patents

Variants de glycoprotéines virales et leurs utilisations

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Publication number
WO2025231185A1
WO2025231185A1 PCT/US2025/027177 US2025027177W WO2025231185A1 WO 2025231185 A1 WO2025231185 A1 WO 2025231185A1 US 2025027177 W US2025027177 W US 2025027177W WO 2025231185 A1 WO2025231185 A1 WO 2025231185A1
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Prior art keywords
variant
cell
amino acid
recombinant virus
nucleic acid
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English (en)
Inventor
Guowei Fang
Baoming NIE
Xuning Wang
Debasish BORAL
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Legend Biotech Ireland Ltd
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Legend Biotech Ireland Ltd
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Publication of WO2025231185A1 publication Critical patent/WO2025231185A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4221—CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/29—Multispecific CARs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16045—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present disclosure relates to viral glycoprotein variants with reduced binding to low-density lipoprotein receptor (LDLR) family (e.g., LDL-R) compared to a reference viral glycoprotein.
  • LDLR low-density lipoprotein receptor
  • Viral vectors have become an important tool for gene therapy.
  • lentiviral vectors lentiviruses
  • LVs lentiviruses
  • LVs are attractive tools as they can stably integrate large gene fragments into target cells, and they have the ability to transduce both dividing and nondividing cells.
  • LVVs are typically produced using a packaging vector encoding viral structural proteins, an envelope vector encoding a viral glycoprotein, and a transfer vector encoding the transgene of interest.
  • the cells that LVVs target can be adapted by pseudotyping glycoproteins from different viruses which have varying cell tropism and target different receptors.
  • VSV-G vesicular stomatitis virus glycoprotein
  • VSV-G enhances gene transfer into multiple cell types including immune cells. Because VSV-G-pseudotyped LVV is stable and results in high titers, VSV-G- pseudotyped LVVs are used in current approved ex vivo CAR-T products. However, VSV-G- pseudotyped LVVs show limitations, such as inefficient transduction in quiescent blood cells, sensitivity to trypsin, and serum inactivation.
  • Cocal virus is in the Vesiculovirus genus, and is a causative agent of vesicular stomatitis in mammals.
  • the cocal virus glycoprotein (COV-G) shares around 74% identity at the amino acid level with VSV-G of the Indiana serotype.
  • Phylogenetic comparison of the envelope gene of vesiculoviruses shows that Cocal virus is serologically distinct from, but most closely related to, VSV-G Indiana among the vesiculoviruses.
  • Maraba virus MARAV
  • MARAV Maraba virus
  • MARAV-G shares around 80% identity at the amino acid level with VSV-G Indiana.
  • An associated risk of CAR-T cell production directly in vivo is the transduction of other cell types with the transgene induced by, e.g., the interaction between VSV-G and its receptor LDL-R.
  • the use of integrating vectors with broad cell tropism, e.g., LVV-pseudotyped with a VSV-G envelope protein, can represent a rare though serious safety concern.
  • Two main challenges for in vivo LVV gene therapy include: (1) lack of cell-specific targeting strategy; and (2) serum inactivation of the virus envelope glycoprotein mediated by the complement system.
  • the present application in one aspect provides a viral glycoprotein variant of a reference viral glycoprotein, wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations at amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331, and 354 in reference to a wildtype cocal virus glycoprotein (COV-G) sequence of SEQ ID NO: 1, and wherein the viral glycoprotein variant has reduced binding to low-density lipoprotein receptor (LDL-R) compared to the reference viral glycoprotein.
  • COV-G cocal virus glycoprotein
  • the viral glycoprotein variant has reduced binding to LDL-R and optionally retained (e.g., retained by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to the reference viral glycoprotein.
  • the reference viral glycoprotein is COV-G, vesicular stomatitis virus glycoprotein (VSV-G), or Maraba virus glycoprotein (MARAV-G).
  • the viral glycoprotein variant is a variant of COV-G, wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158, 182, 271-292, and 331, or one or more substitutions at any of amino acid positions of 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, 291, and 331, and wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1.
  • the COV-G variant comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, I144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, E154A, T155A, E156A, C158A, V182E, V182D, V182N, Y209A, I272E, I278E, I291E, 133 IE, 133 ID, 133 IK, and 1331R.
  • the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 4, 5, 8-10, 12, 13, 62-75, 82, 86, and 88-91 or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 4, 5, 8-10, 12, 13, and 62-75, 82, 86, and 88-91.
  • the viral glycoprotein variant is a variant of VSV-G, wherein the VSV-G variant comprises one or more deletions at amino acid positions 141-158 and 271-292, or one or more substitutions at any of amino acid positions of 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, and 291, and wherein the amino acid positions are in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2, 39, and 46.
  • the VSV-G variant comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, D156A or Y156A, C158A, I182E, Y209A, I272E, I278E, and I291E.
  • the VSV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 14-27, 54-59, and 76-81, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 14-27, 54-59, and 76-81.
  • the viral glycoprotein variant is a variant of MARAV-G, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and 271-292, or one or more substitutions at any of amino acid positions of 47,143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, 291, 331 and 354, and wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 40.
  • the MARAV-G variant comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of AG141-C158, AL271-R292, K47A, K47V, K47I, K47N, K47Q, K47D, K47E, K47H, W143A, I144A, D145A, S146A, Q147A, L148A, G150A, G151A, K152A, C153A, S154A, K155A, E156A, C158A, A182E, H209A, I272E, I278E, I291E, 133 IE, 133 ID, R354A, R354V, R354I R354N, R354Q, R354D, R354E and R354H.
  • the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 28-34, 47-52, 96-104, 111-112, and 118-125 or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 28-34, 47-52, 96-104, 111-112, and 118-125.
  • first nucleic acids encoding any of the viral glycoprotein variants described above.
  • vectors comprising any of the first nucleic acids described herein.
  • the present application in another aspect provides recombinant viruses comprising any of the viral glycoprotein variants described above.
  • the recombinant virus is a lentivirus.
  • the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on a target cell.
  • the envelope surface-bound targeting molecule is encoded by a second nucleic acid.
  • the envelope surface-bound targeting molecule further comprises a transmembrane domain (e.g., a transmembrane domain derived from CD8 (e.g., CD8a), CD4, CD28, 4- IBB, CD80, CD86, CD 152, and PD-1, such as CD8a) or membrane-anchoring domain.
  • the target cell is an immune cell, such as an immune cell selected from the group consisting of a monocyte, a dendritic cell, a macrophage, a B cell, a T cell, a natural killer (NK) cell, or a combination thereof.
  • the immune cell is a T cell.
  • the cell surface protein is selected from the group consisting of CD3, CD5, CD2, and CD7, such as CD3.
  • the envelope surface-bound targeting molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein on the target cell.
  • the antibody or antigen-binding fragment thereof is selected from the group consisting of a full- length antibody, a Fab, a Fab’, a (Fab’)2, an Fv, an scFv, and an sdAb, such as scFv.
  • the envelope surface-bound targeting molecule is an anti-CD3 scFv.
  • the envelope surface-bound targeting molecule is a T-cell activation molecule or a co-stimulation molecule.
  • the T-cell activation or co-stimulation molecule comprises CD86, CD80, CD137L, anti-CD28 antibody or antigen-binding fragment, or anti-4- IBB antibody or antigen-binding fragment.
  • the recombinant virus further comprises a third nucleic acid encoding a heterologous molecule (e.g., heterologous protein or RNA).
  • a heterologous molecule e.g., heterologous protein or RNA.
  • the heterologous molecule is an engineered receptor, such as a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), a chimeric TCR (cTCR), a T cell antigen coupler (TAC), or a TAC-like engineered receptor.
  • the engineered receptor is a CAR, wherein the CAR comprises an extracellular binding domain specifically recognizing a cancer-associated antigen, a cancerspecific antigen, or an autoimmune disease-associated antigen.
  • the extracellular binding domain specifically recognizes cancer-associated antigen or cancerspecific antigen selected from the group consisting of CD19, CD20, CD22, BCMA, DLL3, B7H3, PD-L1, PD-L2, CLL1, GPC3, GU2CYC, CD7, CD38, CD41, CD 123, Claudin 18.2, Claudin 6, NKG2D, GPRC5D, CD70, and any combination thereof.
  • the extracellular binding domain specifically recognizes CD 19 and/or CD20.
  • the first nucleic acid and the second nucleic acid are on the same vector.
  • the first nucleic acid and the second nucleic acid are under the control of a same promoter.
  • the first nucleic acid and the second nucleic acid are connected via a linking nucleic acid encoding a cleavable linker, such as a 2A peptide (e.g., P2A, T2A, E2A, etc.).
  • the first nucleic acid and the second nucleic acids are on two separate vectors.
  • the first nucleic acid, the second nucleic acid, and the third nucleic acid are on different vectors.
  • the present application in another aspect provides methods of specifically delivering a heterologous nucleic acid into a target cell, comprising contacting the target cell with a recombinant virus, wherein the recombinant virus comprises the heterologous nucleic acid to be delivered into the target cell and any of the viral glycoprotein variants described above.
  • the heterologous nucleic acid encodes a heterologous molecule, such as a CAR, an engineered TCR, a cTCR, a TAC, or a TAC-like engineered receptor.
  • the present application in another aspect provides methods of producing a recombinant virus comprising any of the viral glycoprotein variants described above, the method comprising introducing a first nucleic acid encoding the viral glycoprotein variant and a packaging vector comprising one or more nucleic acids encoding one or more packaging proteins into a producer cell, thereby producing the recombinant virus.
  • the method further comprises introducing a second nucleic acid encoding an envelope surface-bound targeting molecule into the producer cell, wherein the envelope surface-bound targeting molecule (e.g., scFv) specifically recognizes a cell surface protein on a target cell (e.g., CD3 on a T cell).
  • the method further comprises introducing a third nucleic acid encoding a heterologous molecule (e.g., CAR) into the producer cell.
  • a heterologous molecule e.g., CAR
  • compositions comprising any of the recombinant viruses described above, and a pharmaceutically acceptable excipient.
  • a disease or disorder e.g., cancer
  • an individual e.g., human
  • the method comprising administering to the individual a therapeutically effective amount of any of the pharmaceutical compositions described herein (e.g., pharmaceutical compositions comprising recombinant viruses expressing anti-cancer CAR).
  • FIG. 1 shows an amino acid sequence alignment between COV-G, VSV-G, and MARAV-G.
  • COVG indicates wildtype COV-G protein (SEQ ID NO: 1).
  • VSVG indicates reference VSV synthetic (syn)-G protein (SEQ ID NO: 46).
  • MARAV-G indicates wildtype MARAV-G protein (SEQ ID NO: 40). Amino acids boxed with solid lines indicate positions that were mutated, and amino acids boxed with dashed lines indicate deletion range.
  • FIG. 2 shows the transduction efficiency of LVs pseudotyped with various COV-G variants encoding anti-CD20+CD19 dual CAR on Jurkat T cells and Nalm6 B cells. 25 LVV copies per cell was used. Transduction was assessed by measuring the percentage of CAR + target cells by flow cytometry.
  • FIG. 3 shows the transduction efficiency of LVs pseudotyped with various COV-G variants and encoding anti-CD20+CD19 dual CAR on Jurkat T cells and Nalm6 B cells. 125 LVV copies per cell was used. Transduction was assessed by measuring the percentage of CAR + target cells by flow cytometry.
  • FIG. 4 shows the transduction of Jurkat cells with a titration of lentiviruses (LVs) pseudotyped with a WT or mutant COV-G, encoding a CD20+CD19 dual CAR transgene. Transduction was assessed by measuring the percentage of CAR + Jurkat cells by flow cytometry.
  • LVs lentiviruses
  • FIG. 5 shows infectious titer of lentiviruses (LVs) pseudotyped with a WT or mutant COV-G in Jurkat cells measured by transducing units (TU)/mL.
  • FIG. 6 shows the transduction of Jurkat cells with a titration of lentiviruses (LVs) pseudotyped with a WT or mutant MARAV-G, VSVG (WT) and COV-G (WT), encoding a CD20+CD19 dual CAR transgene. Transduction was assessed by measuring the percentage of CAR + Jurkat cells by flow cytometry.
  • LVs lentiviruses
  • FIG. 7 shows infectious titer of lentiviruses (LVs) pseudotyped with a WT or mutant MARAV-G, VSVG (WT) or COV-G (WT) in Jurkat cells measured by transducing units (TU)/mL.
  • FIG. 8 shows the transduction efficiency of LVVs pseudotyped with various COV-G variants encoding anti-CD20+CD19 dual CAR, with or without an envelope surface-bound anti-CD3 scFv.
  • Transduction of PBMCs was assessed by measuring the percentage of CAR + target cells by flow cytometry.
  • unT means untransduced control.
  • FIG. 9 shows the percentage of activated T cells transduced by LVVs pseudotyped with various COV-G variants encoding anti-CD20+CD19 dual CAR, with or without an envelope surface-bound anti-CD3 scFv. Activation was assessed by measuring the percentage of CD25 + target cells by flow cytometry.
  • FIG. 10 shows the percentage of B cells in a population exposed to T cells transduced with LVVs pseudotyped with various COV-G variants encoding anti-CD20+CD19 dual CAR. B cell depletion was analyzed by measuring the percentage of CD19 + B cells by flow cytometry.
  • FIG. 11 shows the rescue of Jurkat cell transduction by LVs pseudotyped with a WT or mutant COV-G along with an anti-CD3 scFv. Transduction was assessed by measuring the percentage of CAR + Jurkat cells by flow cytometry.
  • FIG. 12 shows rescued functional infectious titer of LVs pseudotyped with various COV-G variants with or without anti-CD3 scFv in Jurkat cells measured by transducing units (TU)/mL.
  • FIG. 13 shows the rescue of Jurkat cell transduction by LVs pseudotyped with a WT or mutant MARAV-G, VSVG (WT) or COV-G (WT) along with an anti-CD3 scFv. Transduction was assessed by measuring the percentage of CAR + Jurkat cells by flow cytometry.
  • FIG. 14 shows rescued functional infectious titer of LVs pseudotyped with a WT or mutant MARAV-G, VSVG (WT) or COV-G (WT) with or without anti-CD3 scFv in Jurkat cells measured by transducing units (TU)/mL.
  • FIG. 15 shows the rescue of PBMCs transduction by LVs pseudotyped with a WT or mutant MARAV-G, VSVG (WT) or COV-G (WT) along with an anti-CD3 scFv. Transduction was assessed by measuring the percentage of CAR + PBMCs by flow cytometry.
  • FIG. 16 shows rescued functional infectious titer of LVs pseudotyped with a WT or mutant MARAV-G, VSVG (WT) or COV-G (WT) with or without anti-CD3 scFv in Jurkat cells or PBMCs measured by transducing units (TU)/mL.
  • the present application in one aspect provides viral glycoprotein variants of a reference viral glycoprotein (e.g., COV-G, VSV-G, or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations at amino acid positions equivalent to 47,141-158, 182, 209, 270-300, 331 and 354 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein the viral glycoprotein variant has reduced binding to LDL-R compared to the reference viral glycoprotein.
  • the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on a target cell (e.g., CD3 on T cells), such as an envelope surface-bound antibody or antigen-binding fragment thereof (e.g., an anti-CD3 scFv).
  • the recombinant virus further comprises a nucleic acid encoding a heterologous molecule (e.g., heterologous protein or RNA), such as an engineered receptor (e.g., a CAR).
  • a heterologous molecule e.g., heterologous protein or RNA
  • an engineered receptor e.g., a CAR
  • LDL-R The broad tropism of LDL-R can lead to the non-specific transduction of target cells from recombinant viruses pseudotyped with glycoproteins such as VSV-G, COV-G, and MARAV-G.
  • glycoproteins such as VSV-G, COV-G, and MARAV-G.
  • inventors of the present application identified mutations in viral glycoproteins that can reduce binding to LDL-R. When LVVs were pseudotyped with these glycoprotein variants, non-specific transduction efficiency was greatly reduced, indicating these mutations were effective in reducing binding affinity to LDL-R.
  • the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to a viral glycoprotein receptor (e.g., LDL-R) compared to the reference viral glycoprotein.
  • a viral glycoprotein receptor e.g., LDL-R
  • the viral glycoprotein variant has retained (e.g., retained by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to the reference viral glycoprotein.
  • the recombinant virus Upon further engineering the recombinant virus to display an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) specific for the target cell of interest (e.g., T cells), it was observed that the recombinant viruses exhibited target cell-specific transduction, the efficiency of some of which was comparable or even higher than recombinant viruses pseudotyped with a reference viral glycoprotein but without the envelope surface-bound targeting molecule. Moreover, the envelope surface-bound targeting molecule not only promoted specific transduction, but also helped to activate the target cells simultaneously, where the activation efficiency was much higher than recombinant viruses pseudotyped with the reference viral glycoprotein or viral glycoprotein variant but without the envelope surfacebound targeting molecule.
  • an envelope surface-bound targeting molecule e.g., anti-CD3 scFv
  • nucleic acid of interest e.g., encoding a CAR
  • an envelope surface-bound targeting molecule e.g., anti-CD3 scFv
  • was functionally active in the transduced target cells e.g., inducing CAR-mediated cytotoxicity
  • the present application in one aspect provides a viral glycoprotein variant comprising one or more mutations as compared to a reference viral glycoprotein and having reduced binding to LDL-R compared to the reference viral glycoprotein.
  • a recombinant virus comprising any of the viral glycoprotein variants described herein, with or without expressing an envelope surface-bound targeting molecule described herein (e.g., anti-CD3 scFv).
  • viruses that further comprise a nucleic acid encoding a heterologous molecule described herein, such as a therapeutic protein (e.g., an engineered receptor, such as a CAR).
  • a therapeutic protein e.g., an engineered receptor, such as a CAR.
  • Percent (%) amino acid sequence identity and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • variant when used in reference to a polypeptide means a polypeptide that has one or more substitutions, deletions, or insertions relative to a parent or reference polypeptide.
  • the term may analogously be applied to polynucleotides that have one or more substitutions, deletions, or insertions relative to a parent or reference polynucleotide.
  • a “pseudotyped” recombinant virus is a recombinant viral particle having one or more envelope glycoproteins that are encoded by a virus that is distinct from the recombinant virus (e.g., lentiviral) genome.
  • the envelope glycoprotein may be modified, mutated, or engineered as described herein.
  • Recombinant viruses are those that are generated to incorporate modification(s) introduced into a specific gene/locus (or multiple loci) of the viral genome.
  • a recombinant virus may occur naturally or be produced by recombining pieces of DNA using recombinant DNA technology.
  • the term “specificity” refers to selective recognition of an antigen binding protein (such as a CAR or an antibody) for a particular epitope of an antigen. Natural antibodies, for example, are monospecific.
  • the term “multispecific” as used herein denotes that an antigen binding protein has two or more antigen-binding sites of which at least two bind different epitopes.
  • Bispecific as used herein denotes that an antigen binding protein has two different antigenbinding specificities.
  • the term “monospecific” as used herein denotes an antigen binding protein that has one or more binding sites each of which bind the same epitope.
  • antibody herein is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity.
  • an “antibody” may refer to an immunoglobulin molecule or a fragment thereof which is able to specifically bind to a specific epitope of an antigen.
  • Antibodies can be intact immunoglobulins derived from natural sources, or from recombinant sources and can be immunoreactive portions of intact immunoglobulins.
  • Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, antigen-binding fragments (such as Fv, Fab, Fab’, F(ab)2 and F(ab’)2), as well as single chain antibodies (scFv), heavy chain antibodies, such as camelid antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).
  • polyclonal antibodies monoclonal antibodies
  • antigen-binding fragments such as Fv, Fab, Fab’, F(ab)2 and F(ab’)2
  • single chain antibodies such as camelid antibodies
  • humanized antibodies Har
  • epitope refers to the specific group of atoms or amino acids on an antigen to which an antibody or antibody moiety binds.
  • a full-length antibody comprises two heavy chains and two light chains.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively.
  • the variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC- CDR3).
  • CDRs complementarity determining regions
  • CDR boundaries for antibodies and antigen-binding fragments may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991).
  • the three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops.
  • FRs framework regions
  • the constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions.
  • Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of a, 5, a, y, and p heavy chains, respectively.
  • IgGl yl heavy chain
  • lgG2 y2 heavy chain
  • lgG3 y3 heavy chain
  • lgG4 y4 heavy chain
  • IgAl al heavy chain
  • antigen-binding fragment refers to an antibody fragment including, for example, a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv’), a disulfide stabilized diabody (ds diabody), a single-chain Fv (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a single domain antibody (sdAb) (e.g., a camelized single domain antibody), a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure.
  • An antigen-binding fragment is capable of binding to the same
  • Fv is the minimum antibody fragment, which contains a complete antigenrecognition and -binding site. This fragment consists of a dimer of one heavy- and one lightchain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Single-chain Fv also abbreviated as “sFv” or “scFv,” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • Single domain antibody refers to a single monomeric variable antibody domain and which is capable of antigen binding.
  • Single domain antibodies include VHH domains as described herein. Examples of single domain antibodies include, but are not limited to, antibodies naturally devoid of light chains such as those from Camelidae species (e.g., llama), single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies.
  • Single domain antibodies e.g., VHH domains
  • Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, and bovine.
  • a single domain antibody can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; VHHs derived from such other species are within the scope of the disclosure.
  • the single domain antibody (e.g, VHH domain) provided herein has a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • Single domain antibodies may be genetically fused or chemically conjugated to another molecule (e.g, an agent).
  • Single domain antibodies may be part of a bigger binding molecule (e.g., a multispecific antibody or an engineered receptor).
  • variable region refers to the amino-terminal domains of the heavy or light chain of the antibody.
  • the variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.
  • Heavy-chain only antibodies from the Camelidae species have a single heavy chain variable region, which is referred to as “VHH”. VHH is thus a special type of VH.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, z.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or posttranslation modifications (e.g., isomerizations, amidations) that may be present in minor amounts.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • polyclonal antibody preparations which typically include different antibodies directed against different epitopes
  • each monoclonal antibody is directed against a single epitope on the antigen.
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • monoclonal antibodies may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nd ed.
  • CDR regions are well known to those skilled in the art and have been defined by well- known numbering systems.
  • Kabat CDRs are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra, Nick Deschacht et al., J Immunol 2010; 184:5696-5704).
  • Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)).
  • the end of the Chothia CDR- H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Diibel eds., 2d ed. 2010)).
  • IMGT ImMunoGeneTics
  • IG immunoglobulins
  • TCR T-cell receptors
  • MHC major histocompatibility complex
  • CDR complementary determining region
  • individual CDRs e.g., CDR-H1, CDR-H2
  • the scheme for identification of a particular CDR or CDRs is specified, such as the CDR as defined by the IMGT, Kabat, Chothia, or Contact method. In other cases, the particular amino acid sequence of a CDR is given.
  • CDR regions may also be defined by any combination of various numbering systems, e.g., a combination of Kabat and Chothia numbering systems, a combination of Kabat and AbM numbering systems, or a combination of Kabat and IMGT numbering systems. Therefore, the term such as “a CDR1 as set forth in a specific VH” includes any CDR1 as defined by the exemplary CDR numbering systems described above, but is not limited thereby.
  • a variable region e.g., a VH or VL
  • those skilled in the art would understand that CDRs within the region can be defined by different numbering systems or combinations thereof.
  • Binding affinity generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity that reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.
  • Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.
  • a variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure.
  • conservative amino acid substitution refers to substitutions in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Antibody variants having one or more conservative amino acid substitutions may be provided. Conservative substitutions are shown in Table B under the heading of “Preferred Substitutions”, while more substantial changes are provided in Table B under the heading “Exemplary Substitutions”. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity (e.g., retained/improved antigen binding).
  • Amino acids may be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Amino acids that influence chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.
  • Chimeric antigen receptor refers to genetically engineered receptors, which can be used to graft one or more antigen specificity onto immune effector cells, such as T cells. Some CARs are also known as “artificial T-cell receptors,” “chimeric T cell receptors,” or “chimeric immune receptors.” The CAR may comprise an extracellular antigen binding domain specific for one or more antigens (such as tumor antigens), a transmembrane domain, and an intracellular signaling domain of a T cell and/or other receptors. “CAR-T cell” refers to a T cell that expresses a CAR.
  • polypeptide and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.
  • polypeptides containing one or more analogs of an amino acid including but not limited to, unnatural amino acids, as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure may be based upon antibodies or other members of the immunoglobulin superfamily, a “polypeptide” can occur as a single chain or as two or more associated chains.
  • Polynucleotide or “nucleic acid,” as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.
  • Oligonucleotide refers to short, generally single- stranded, synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length.
  • oligonucleotide and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.
  • a cell that produces a binding molecule of the present disclosure may include a parent hybridoma cell, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies have been introduced.
  • the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction.
  • the direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5 ’ to the 5 ’ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3’ to the 3’ end of the RNA transcript are referred to as “downstream sequences.”
  • an “isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA, or a mixed nucleic acids, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence.
  • An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • an “isolated” nucleic acid molecule, such as a cDNA molecule can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the term embraces nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.
  • a substantially pure molecule may include isolated forms of the molecule.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • operatively linked when used in reference to nucleic acids or amino acids, refer to the operational linkage of nucleic acid sequences or amino acid sequence, respectively, placed in functional relationships with each other.
  • an operatively linked promoter, enhancer elements, open reading frame, 5’ and 3’ UTR, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA).
  • operatively linked nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (z.e., expression of the open reading frame).
  • an operatively linked peptide is one in which the functional domains are placed with appropriate distance from each other to impart the intended function of each domain.
  • vector refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) as described herein, in order to introduce a nucleic acid sequence into a host cell.
  • a vector for use can be expression vectors, plasmids, and viral vectors, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media.
  • Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art.
  • two or more nucleic acid molecules are to be co-expressed (e.g., both an antibody heavy and light chain or an antibody VH and VL)
  • both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • the introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art.
  • nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA
  • immunoblotting for expression of gene products or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product.
  • nucleic acid molecules are expressed in a sufficient amount to produce a desired product and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.
  • the term “host” as used herein can refer to any organism that can receive administration of a recombinant virus, such as an animal (e.g., a mammal, such as a human).
  • the term “host cell” as used herein refers to a particular subject cell that may be transduced with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transduced with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.
  • transducer cell refers to a particular subject cell that is capable of producing recombinant viral particles when the transfer vector, packaging vector(s), and envelope vector(s) are introduced into the cells.
  • Various methods of introducing the plasmids into the cells may be used, including transfection or electroporation.
  • autologous is meant to refer to any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • Allogeneic refers to a graft derived from a different individual of the same species.
  • transfected or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • pharmaceutically acceptable means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.
  • an effective amount or “therapeutically effective amount” as used herein refer to a sufficient amount of an agent to provide the desired biological result. That result can be reduction (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system.
  • a subject or an individual can be a mammal, such as a non-primate or a primate (e.g., human).
  • the individual is a human.
  • the individual is a mammal, e.g., a human, diagnosed with a disease or disorder, or at risk of developing a disease or disorder.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and/or prolonging survival.
  • delay means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.
  • Preventing includes providing prophylaxis with respect to the occurrence or recurrence of a disease in an individual that may be predisposed to the disease but has not yet been diagnosed with the disease.
  • activation refers to the state of a cell following sufficient cell surface moiety ligation to induce a noticeable biochemical or morphological change.
  • T cells such activation refers to the state of a T cell that has been sufficiently stimulated to induce cellular proliferation.
  • Activation of a T cell may also induce cytokine production and performance of regulatory or cytolytic effector functions. Within the context of other cells, this term infers either up or down regulation of a particular physicochemical process.
  • activated T cells indicates T cells that are currently undergoing cell division, cytokine production, performance of regulatory or cytolytic effector functions, and/or has recently undergone the process of “activation.”
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • novel viral glycoprotein variants have reduced (e.g., reducing at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) binding to a viral glycoprotein receptor (e.g., LDLR family receptor, such as LDL-R), compared to a reference viral glycoprotein (e.g., a corresponding wildtype viral glycoprotein).
  • a viral glycoprotein receptor e.g., LDLR family receptor, such as LDL-R
  • a reference viral glycoprotein e.g., a corresponding wildtype viral glycoprotein
  • the viral glycoprotein variants described herein in some embodiments comprise (and in some embodiments consist of or consisting essentially of) one or more mutations (e.g., insertion(s), deletion(s), and/or substitution(s)) at amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to a wildtype COV-G sequence of SEQ ID NO: 1.
  • the viral glycoprotein variant only contains a mutation within the region of amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to SEQ ID NO: 1.
  • the viral glycoprotein variant further contains one or more mutations outside the region of amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to SEQ ID NO: 1.
  • the viral glycoprotein variant has reduced (e.g., reducing at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more) binding to a viral glycoprotein receptor (e.g., LDLR family receptor), compared to the reference viral glycoprotein.
  • the viral glycoprotein variant has abolished binding (i.e., no binding, or no detectable binding) to a viral glycoprotein receptor (e.g., LDLR family receptor).
  • the viral glycoprotein receptor is LDL-R.
  • a viral glycoprotein variant of a reference viral glycoprotein wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and/or substitution(s)) at amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, and wherein the viral glycoprotein variant has reduced (e.g., reducing at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) binding to LDL-R compared to the reference viral glycoprotein.
  • the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and/or substitution(s)) at amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to
  • the viral glycoprotein variant is derived from a virus of the Rhabdoviridae family, such as the Vesiculovirus genus. Vesiculovirus is a genus of negativesense single-stranded (ss) RNA viruses.
  • the viral glycoprotein variant is derived from Cocal virus (COV), or a serotype thereof.
  • the viral glycoprotein variant is derived from vesicular stomatitis virus (VSV), or a serotype thereof.
  • the viral glycoprotein variant is derived from a strain of the VSV Indiana serotype, such as VSV Orsay strain (VSV Orsay) or VSV San Juan strain (VSV SJ).
  • the viral glycoprotein variant is derived from a synthetic vesicular stomatitis virus (VSV syn).
  • the viral glycoprotein variant is derived from Maraba virus (MARAV), or a serotype thereof.
  • a wildtype viral glycoprotein is used as a reference viral glycoprotein.
  • a synthetic viral glycoprotein is used as a reference viral glycoprotein.
  • the synthetic viral glycoprotein has the same sequence as a wildtype viral glycoprotein.
  • the synthetic viral glycoprotein has a different sequence than a wildtype viral glycoprotein.
  • the reference viral glycoprotein is wildtype COV-G comprising the amino acid sequence of SEQ ID NO: 1.
  • the reference viral glycoprotein is wildtype VSV Orsay-G comprising the amino acid sequence of SEQ ID NO: 2.
  • the reference viral glycoprotein is wildtype VSV SJ-G comprising the amino acid sequence of SEQ ID NO: 39.
  • the reference viral glycoprotein is VSV syn-G comprising the amino acid sequence of SEQ ID NO: 46.
  • the reference viral glycoprotein is wildtype MARAV-G comprising the amino acid sequence of SEQ ID NO: 40.
  • VSV-G The most common glycoprotein pseudotyped on viral vectors is VSV-G.
  • VSV-G refers to VSV-Gs in general, including all those VSV-Gs known in the art, either wildtype or synthetic.
  • Exemplary VSV-G include, but are not limited to, VSV Orsay-G, VSV SJ-G, and VSV syn-G (e.g., SEQ ID NO: 46).
  • VSV-G is a 511 amino acid protein made up of a fusion domain, pleckstrin homology domain, and trimerization domain.
  • VSV-G from the Indiana serotype is able to independently bind two distinct cysteine-rich (CR) domains (CR2 and CR3) of LDL- R, with VSV-G in complex with those domains reported in crystal structures (Nikolic J, Belot L, Raux H, Legrand P, Gaudin Y, A Albertini A. Nat Commun. 9(1): 1029 (2016)).
  • VSV-G forms trimers on the viral envelope, and upon binding to host receptors, mediates host cell entry through clathrin-mediated endocytosis (Rehman S, Bishnoi S, Roy R, Kumari A, Jayakumar H, Gupta S, Kar P, Pattnaik AK, Nayak D. ACS Omega. 7(37):32840-32848 (2022)).
  • COV-G and MARAV-G can also be used for pseudotyping viral vectors.
  • COV and MARAV are serologically distinct species from VSV, where COV-G and VSV-G share around 74% sequence identity, and MARAV-G and VSV-G share around 80% sequence identity at the amino acid level. While VSV-G, COV-G, and MARAV- G all exhibit a broad tropism and good stability, COV-G has been shown to have increased serum resistance (Trobridge GD, Wu RA, Hansen M, Ironside C, Watts KL, Olsen P, Beard BC, Kiem HP. Mol Ther. 18(4):725-33 (2010)).
  • VSV, COV, and MARAV are all negative- stranded RNA viruses in the Rhabdoviridae family and utilize LDL-R as the major entry receptor (Gutierrez-Guerrero A, Cosset FL, Verhoeyen E. Viruses. 2020 Sep 11 ; 12(9): 1016 (2020); Jayawardena N, Burga LN, Poirier IT, Bostina M. Oncolytic Virolher. 8:39-56 (2019)).
  • Receptors that bind viral glycoproteins include, but are not limited to, phosphatidylserine, nicotinic acetylcholine receptor, NPC1 (Niemann-Pick Cl), TfRl (transferrin receptor 1), ACE2 (angiotensin-converting enzyme 2), sialic acid, neuraminidase, and members of the LDLR family.
  • viral glycoprotein variants described herein have reduced (e.g., reducing at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) binding to one or more receptors that bind viral glycoproteins, such as LDL-R.
  • LDL-R is a type I transmembrane protein in the LDLR gene family involved in maintaining cholesterol homeostasis. This receptor binds cholesterol -rich lipoprotein ligands and transports them to the endosomal pathway (Go GW, Mani A. Yale J Biol Med. 85(1): 19- 28 (2012)).
  • the extracellular ligand binding domain consists of seven LDLR type A repeats, which are each comprised of 40 amino acid stretches that contain two internal disulfide bonds, mediated by six cysteines (Pedersen NB, Wang S, Narimatsu Y, Yang Z, Halim A, Schjoldager KT, Madsen TD, Seidah NG, Bennett EP, Levery SB, Clausen H.
  • Viral glycoproteins such as VSV-G binds LDL-R in the cysteine-rich regions of this extracellular ligand binding domain (Nikolic J, Belot L, Raux H, Legrand P, Gaudin Y, A Albertini A. Nat Commun. 9(1): 1029 (2016); Finkelshtein D, Werman A, Novick D, Barak S, Rubinstein M. Proc Natl Acad Sci U S A. 110(18):7306-l 1 (2013)).
  • LDL-R also contains epidermal growth factor (EGF)-like domains and a P-propeller domain, which are involved in allowing the release of bound lipoprotein ligands in the endosome in a pH-dependent manner (Zhao Z, Michaely P. J Biol Chem. 283(39):26528-37 (2008); Arias-Moreno X, Velazquez- Campoy A, Rodriguez JC, Pocovi M, Sancho J. J Biol Chem. 283(33):22670-9 (2008)).
  • LDL- R further contains an O-linked sugar domain next to the 22-amino acid single-pass transmembrane domain (Go GW, Mani A. Yale J Biol Med.
  • the cytoplasmic domain contains an NPxY motif important in signaling for endocytosis (Chen WJ, Goldstein JL, Brown MS. JBiolChem. 265(6):3116-23 (1990)).
  • LDL-R is the primary receptor (Finkelshtein D, Werman A, Novick D, Barak S, Rubinstein M. Proc Natl Acad Sci USA. 2013 Apr 30; 110(18):7306-l 1 (2013)). LDL-R has low levels of expression on un-activated T cells, B cells and hematopoietic stem cells (Amirache F, Levy C, Costa C, Mangeot PE, Torbett BE, Wang CX, Negre D, Cosset FL, Verhoeyen E. Blood. 2014 Feb 27; 123(9): 1422-4 (2014)).
  • LVVs pseudotyped with VSV-G (Gutierrez-Guerrero A, Cosset FL, Verhoeyen E. Viruses. 2020 Sep 11 ; 12(9): 1016 (2020)).
  • activation of T cells can result in increased expression of LDL-R on the cell surface, which can influence transduction efficiency of LVVs (Bonacina F, Moregola A, Svecla M, Coe D, Uboldi P, Fraire S, Beretta S, Beretta G, Pellegatta F, Catapano AL, Marelli-Berg FM, Norata GD. J Cell Biol. 221(1 l):e202202011 (2022)).
  • the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations at amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to a wildtype COV-G sequence of SEQ ID NO:
  • the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations at amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to a wildtype VSV Orsay-G sequence of SEQ ID NO:
  • the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations at amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to a wildtype VSV SJ-G sequence of SEQ ID NO: 39. In some embodiments, the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations at amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to a VSV syn-G sequence of SEQ ID NO: 46.
  • the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations at amino acid positions equivalent to 47, 141-158, 182, 209, 270- 300, 331 and 354 in reference to a wildtype MARAV-G sequence of SEQ ID NO: 40.
  • Amino acid positions “equivalent to” 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to any of SEQ ID NOs: 1, 2, 39, 40, and 46 in a viral glycoprotein X are amino acid positions in viral glycoprotein X that are homologous to the amino acid positions 47, 141-158, 182, 209, 270- 300, 331 and 354 in any of SEQ ID NOs: 1, 2, 39, 40, and 46, hereinafter also referred to as “equivalent amino acid positions”.
  • the equivalent amino acid positions in viral glycoprotein X can be at amino acid positions 47, 141-158, 182, 209, 270-300, 331 and 354 in viral glycoprotein X as well, or can be at a different location (e.g., at amino acid positions 272-302 of viral glycoprotein X); (ii) can be of the same length as amino acid positions 47, 141-158, 182, 209, 270-300, 331 and 354 in any of SEQ ID NOs: 1, 2, 39, 40, and 46, or can be longer or shorter; and/or (iii) can have the same, partially same, or different amino acid sequences compared to amino acid positions 47, 141-158, 182, 209, 270-300, 331 and 354 in any of SEQ ID NOs: 1, 2, 39, 40, and 46.
  • the equivalent amino acid positions in reference to amino acid positions 47, 141-158, 182, 209, 270-300, 331 and 354 of any of SEQ ID NOs: 1, 2, 39, 40, and 46 in a viral glycoprotein X have equivalent function(s) compared to amino acid positions 47, 141-158, 182, 209, 270-300, 331 and 354 of any of SEQ ID NOs: 1, 2, 39, 40, and 46, such as binding to LDL-R.
  • Methods of identifying such equivalent amino acid positions in a viral glycoprotein X are well-known in the art, such as by amino acid sequence alignment (e.g., see FIG. 1).
  • VSV-G 1182 in VSV-G is considered as an “equivalent amino acid position” in reference to VI 82 of the wildtype COV-G sequence of SEQ ID NO: 1.
  • H8 in VSV-G which is considered as an “equivalent amino acid position” in reference to Q8 of the wildtype COV-G sequence of SEQ ID NO: 1.
  • Alignment for purposes of determining the equivalent amino acid positions of a viral glycoprotein or a variant thereof can be achieved using any tool available in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGNTM (DNASTAR), or HMMER.
  • a viral glycoprotein variant comprising (or consisting essentially of, or consisting of) one or more mutations (e.g., insertion(s), deletion(s), and/or substitution(s)) at amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to a reference sequence of any of SEQ ID NOs: 1, 2, 39, 40, and 46, wherein the viral glycoprotein variant has reduced binding to LDL-R compared to a corresponding reference viral glycoprotein comprising the reference sequence of any of SEQ ID NOs: 1, 2, 39, 40, and 46.
  • one or more mutations e.g., insertion(s), deletion(s), and/or substitution(s)
  • the viral glycoprotein variant may or may not comprise further mutation(s) in addition to the amino acid positions equivalent to 47, 141-158, 182, 209, 270- 300, 331 and 354.
  • the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions equivalent to 47, 141-158, 271-292, 331 and 354, or one or more substitutions at amino acid positions equivalent to 47, 143-148, 150-156, 158, 182, 209, 272, 278, 291, 331 and 354 in reference to a reference sequence of any of SEQ ID NOs: 1, 2, 39, 40, and 46.
  • the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations at amino acid positions equivalent to 47A, 47V, 471, 47N, 47Q, 47D, 47E, 47H, A141-158, A271-292, 143A, 144A, 145A, 146A, 147A, 148A, 150A, 151A, 152A, 153A, 154A, 155A, 156A, 158A, 182E, 182D, 182N, 209A, 272E, 278E, 291E, 33 IE, 33 ID, 33 IK, 331R, 354A, 354V, 3541, 354N, 354Q, 354D, 354E and 354H in reference to a reference sequence of any of SEQ ID NOs: 1, 2, 39, 40, and 46.
  • the viral glycoprotein variant may or may not comprise further mutation(s) in addition to the one or more deletions at amino acid positions equivalent to 47, 141-158, 182, 271-292, 331 and/or 354, or one or more substitutions at amino acid positions equivalent to 47, 143-148, 150-156, 158, 182, 209, 272, 278, 291, 331 and 354.
  • the further mutation(s) can be within or outside of (or both) the regions of amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354.
  • the viral glycoprotein variant that comprises one or more mutations at amino acid positions equivalent to any of 47A, 47V, 471, 47N, 47Q, 47D, 47E, 47H, A141-158, A271-292, 143A, 144A, 145A, 146A, 147A, 148A, 150A, 151 A, 152A, 153 A, 154A, 155 A, 156A, 158A, 182E, 182D, 182N, 209A, 272E, 278E, 291E, 33 IE, 33 ID, 33 IK, 331R, 354A, 354V, 3541, 354N, 354Q, 354D, 354E and 354H further comprises one or more additional mutations within the region of amino acid positions equivalent to 47, 141-158, 182, 209, 270-300 (e.g., 271-292), 331 and 354 in reference to a reference sequence of any of SEQ ID NOs: 1, 2, 39, 40
  • the viral glycoprotein variant that comprises one or more mutations at amino acid positions equivalent to any of 47A, 47V, 471, 47N, 47Q, 47D, 47E, 47H, A141-158, A271- 292, 143 A, 144A, 145 A, 146A, 147A, 148A, 150A, 151 A, 152A, 153 A, 154A, 155 A, 156A, 158A, 182E, 182D, 182N, 209A, 272E, 278E, 291E, 33 IE, 33 ID, 33 IK, 331R, 354A, 354V, 3541, 354N, 354Q, 354D, R354E and R354H further comprises one or more additional mutations outside the region of amino acid positions equivalent to 47, 141-158, 182, 209, 270- 300, 331 and 354 in reference to a reference sequence of any of SEQ ID NOs: 1, 2, 39, 40, and 46.
  • the viral glycoprotein variant that comprises one or more mutations at amino acid positions equivalent to any of 47A, 47V, 471, 47N, 47Q, 47D, 47E, 47H, AMI- 158, A271-292, 143A, 144A, 145A, 146A, 147A, 148A, 150A, 151A, 152A, 153A, 154A, 155A, 156A, 158A, 182E, 182D, 182N, 209A, 272E, 278E, 291E, 33 IE, 33 ID, 33 IK, 331R, 354A, 354V, 3541, 354N, 354Q, 354D, R354E and R354H does not comprise any other mutations outside the region of amino acid positions equivalent to 47, 141-158, 182, 209, 270- 300, 331 and 354 in reference to a reference sequence of any of SEQ ID NOs: 1, 2, 39, 40, and 46.
  • the reference viral glycoprotein is COV-G.
  • the COV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and/or substitution(s)) at amino acid positions 141-158, 182, 209, 270-300, 331 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, and wherein the COV-G variant has reduced (e.g., reducing at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) binding to LDL-R compared to wildtype COV-G.
  • the COV-G variant may or may not comprise further mutation(s) in addition to the amino acid positions 141-158, 182, 209, 270-300, and 331.
  • the COV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158, 182, 271-292, and 331 or one or more substitutions at any of amino acid positions 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, and 291, and 331, wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1.
  • the COV-G variant may or may not comprise further mutation(s) in addition to the one or more deletions at amino acid positions 141-158, 182, 271-292, and 331, or one or more substitutions at any of amino acid positions of 143-148, 150-156, 158, 182, 209, 272, 278, 291, and 331.
  • the further mutation(s) can be within or outside of (or both) the regions of amino acid positions 141-158, 182, 209, 270-300, and 331 in reference to SEQ ID NO: 1.
  • the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions W143, 1144, D145, S146, Q147, F148, N150, G151, K152, C153, E154, T155, E156, C158, V182, Y209, 1272, 1278, 1291, and 1331.
  • the amino acid at any one of positions 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, 291, and 331 can be substituted for any other amino acid (preferably an amino acid with different side chain property), such as a hydrophilic amino acid (such as Cys, Ser, Thr, Asn, or Gin), an acidic amino acid (such as Asp or Glu), a basic amino acid (such as His, Lys, or Arg), an aromatic amino acid (such as Trp, Tyr, or Phe), an amino acid that influences chain orientation (such as Gly or Pro), or a hydrophobic amino acid (e.g., Norleucine, Met, Ala, Vai, He, Leu).
  • a hydrophilic amino acid such as Cys, Ser, Thr, Asn, or Gin
  • an acidic amino acid such as Asp or Glu
  • a basic amino acid such as His, Lys
  • the amino acid of any of positions 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, and 331 are substituted for a hydrophobic amino acid, such as an alanine (A).
  • the valine of position 182 is substituted for an acidic amino acid, such as a glutamic acid (E).
  • the isoleucine of any one of positions 272, 278 and 291 is substituted for an acidic amino acid, such as a glutamic acid (E).
  • COV-G variant wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, I144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, E154A, T155A, E156A, C158A, V182E, V182D, V182N, Y209A, I272E, I278E, I291E, 133 IE, 133 ID, 133 IK, and 1331R wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1.
  • COV-G variant comprising (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 4, 5, 8- 10, 12, 13, 62-75, 82, 86, and 88-91 or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 4, 5, 8-10, 12, 13, 62-75, 82, 86, and 88-91.
  • the COV-G variant that comprises one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, I144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, E154A, T155A, E156A, C158A, V182E, V182D, V182N, Y209A, I272E, I278E, I291E, 133 IE, 133 ID, 133 IK, and 1331R further comprises one or more additional mutations within the region of amino acid positions 141-158, 182, 209, 270-300 (e.g., 271-292), 331 in reference to the wildtype COV-G sequence of SEQ ID NO: 1.
  • the COV-G variant that comprises one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, I144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, E154A, T155A, E156A, C158A, V182E, V182D, V182N, Y209A, I272E, I278E, I291E, 133 IE, 133 ID, 133 IK, and 1331R further comprises one or more additional mutations outside the region of amino acid positions 141-158, 182, 209 270-300, and 331 in reference to the wildtype COV-G sequence of SEQ ID NO: 1.
  • the COV-G variant that comprises one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, I144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, E154A, T155A, E156A, C158A, V182E, V182D, V182N, Y209A, I272E, I278E, I291E, 133 IE, 133 ID, 133 IK, and 1331R does not comprise any other mutations outside the region of amino acid positions 141-158, 182, 209270-300, and 331 in reference to the wildtype COV-G sequence of SEQ ID NO: 1.
  • the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 4, 5, 8-10, 12, 13, 62-75, 82, 86, and 88
  • the reference viral glycoprotein is VSV-G, such as VSV Orsay- G, VSV SJ-G, or VSV syn-G.
  • VSV-G variant e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G
  • the VSV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and/or substitution(s)) at amino acid positions 141-158, 182, 209, and 270-300 in reference to a reference VSV-G sequence of any of SEQ ID NOs: 2, 39, and 46, and wherein the VSV-G variant has reduced (e.g., reducing at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) binding to LDL-R compared to a reference VSV- G (e.g., VSV Orsay- G, VSV SJ-G, or VSV syn
  • the VSV-G variant may or may not comprise further mutation(s) in addition to the amino acid positions 141-158, 182, 209, and 270-300.
  • the VSV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, and 291, wherein the amino acid positions are in reference to a reference VSV-G sequence of any of SEQ ID NOs: 2, 39, and 46.
  • the VSV-G variant may or may not comprise further mutation(s) in addition to the one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions of 143-148, 150-156, 158, 182, 209, 272, 278, and 291.
  • the further mutation(s) can be within or outside of (or both) the regions of amino acid positions 141-158, 182, 209, and 270-300 in reference to any of SEQ ID NOs: 2, 39, and 46.
  • the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions W143, V144, D145, S146, Q147, F148, N150, G151, K152, C153, S154, N155, D156 or Y156, C158, 1182, Y209, 1272, 1278, and 1291.
  • the amino acid at any one of positions 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, and 291 can be substituted for any other amino acid (preferably an amino acid with different side chain property), such as a hydrophilic amino acid (such as Cys, Ser, Thr, Asn, or Gin), an acidic amino acid (such as Asp or Glu), a basic amino acid (such as His, Lys, or Arg), an aromatic amino acid (such as Trp, Tyr, or Phe), an amino acid that influences chain orientation (such as Gly or Pro), or a hydrophobic amino acid (e.g., Norleucine, Met, Ala, Vai, He, Leu).
  • a hydrophilic amino acid such as Cys, Ser, Thr, Asn, or Gin
  • an acidic amino acid such as Asp or Glu
  • a basic amino acid such as His, Lys, or
  • the amino acid of positions 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, and 209 are substituted for a hydrophobic amino acid, such as an alanine (A).
  • the isoleucine of any one of positions 182, 272, 278, and 291 is substituted for an acidic amino acid, such as a glutamic acid (E).
  • the VSV-G variant comprises one or more mutations selected from the group consisting of A141-158, A271-292, 143A, 144A, 145A, 146A, 147A, 148A, 150A, 151A, 152A, 153A, 154A, 155A, 156A, 158A, 182E, 209A, 272E, 278E, and 291E in reference to a reference VSV-G sequence of any of SEQ ID NOs: 2, 39, and 46.
  • VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, D156A, C158A, I182E, Y209A, I272E, I278E, and I291E, wherein the amino acid positions are in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 2.
  • the VSV Orsay-G variant that comprises one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, D156A, C158A, I182E, Y209A, I272E, I278E, and 129 IE further comprises one or more additional mutations within the region of amino acid positions 141-158, 182, 209 and 270-300 (e.g., 271-292) in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 2.
  • the VSV Orsay-G variant that comprises one or more mutations selected from the group consisting of AG141- C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, D156A, C158A, I182E, Y209A, I272E, I278E, and I291E further comprises one or more additional mutations outside the regions of amino acid positions 141-158, 182, 209 and 270-300 in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 2.
  • the VSV Orsay-G variant that comprises one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, D156A, C158A, I182E, Y209A, I272E, I278E, and I291E does not comprise any other mutations outside the region of amino acid positions 141-158, 182, 209 and 270-300 in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 2.
  • VSV SJ-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, Y156A, C158A, I182E, Y209A, I272E, I278E, and I291E, wherein the amino acid positions are in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 39.
  • VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 21-27 and 54-59, or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 21-27 and 54-59.
  • the VSV SJ-G variant that comprises one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, Y156A, C158A, I182E, Y209A, I272E, I278E, and 129 IE further comprises one or more additional mutations within the region of amino acid positions 141-158, 182, 209, and 270-300 (e.g., 271-292) in reference to the wildtype VSV SJ- G sequence of SEQ ID NO: 39.
  • the VSV SJ-G variant that comprises one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, Y156A, C158A, I182E, Y209A, I272E, I278E, and I291E further comprises one or more additional mutations outside the regions of amino acid positions 141-158, 182, 209, and 270-300 in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 39.
  • the VSV SJ-G variant that comprises one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, Y156A, C158A, I182E, Y209A, I272E, I278E, and 129 IE does not comprise any other mutations outside the region of amino acid positions 141-158, 182, 209 and 270-300 in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 39.
  • the VSV SJ-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 21-27 and 54- 59.
  • a VSV syn-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, Y156A, C158A, I182E, Y209A, I272E, I278E, and I291E, wherein the amino acid positions are in reference to the reference VSV syn-G sequence of SEQ ID NO: 46.
  • VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 14-20 and 76-81, or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 14-20 and 76-81.
  • the VSV syn-G variant that comprises one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, Y156A, C158A, I182E, Y209A, I272E, I278E, and I291E further comprises one or more additional mutations within the region of amino acid positions 141-158, 182, 209, and 270-300 (e.g., 271-292) in reference to the reference VSV syn-G sequence of SEQ ID NO: 46.
  • the VSV syn-G variant that comprises one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, Y156A, C158A, I182E, Y209A, I272E, I278E, and 129 IE further comprises one or more additional mutations outside the regions of amino acid positions 141-158, 182, 209, and 270-300 in reference to the reference VSV syn-G sequence of SEQ ID NO: 46.
  • the VSV syn-G variant that comprises one or more mutations selected from the group consisting of AG141-C158, AL271- R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, Y156A, C158A, I182E, Y209A, I272E, I278E, and I291Edoes not comprise any other mutations outside the region of amino acid positions 141-158, 182, 209, and 270-300 in reference to the reference VSV syn-G sequence of SEQ ID NO: 46.
  • the VSV syn-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 14-20 and 76-81.
  • the reference viral glycoprotein is MARAV-G.
  • the MARAV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and/or substitution(s)) at amino acid positions 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to a wildtype MARAV-G sequence of SEQ ID NO: 40, and wherein the MARAV-G variant has reduced (e.g., reducing at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) binding to LDL-R compared to wildtype MARAV-G.
  • the MARAV-G variant may or may not comprise further mutation(s) in addition to the amino acid positions 47, 141-158, 182, 209, 270-300, 331 and 354.
  • the MARAV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 47, 141-158, 182, 271-292, 331 and/or 354 or one or more substitutions at any of amino acid positions 47, 143, 144, 145, 146, 147, 148, 150, 151,
  • the MARAV-G variant may or may not comprise further mutation(s) in addition to the one or more deletions at amino acid positions 47, 141-158, 182, 271-292, 331 and/or 354 or one or more substitutions at any of amino acid positions of 47, 143, 144, 145, 146, 147, 148, 150, 151, 152,
  • the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at amino acid positions K47, W143, 1144, D145, S146, Q147, L148, G150, G151, K152, C153, S154, K155, E156, C158, A182, H209, 1272, 1278, and 1291, 1331 and R354.
  • amino acid at any one of positions 47, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, 291, 331 and 354 can be substituted for any other amino acid (preferably an amino acid with different side chain property), such as a hydrophilic amino acid (such as Cys, Ser, Thr, Asn, or Gin), an acidic amino acid (such as Asp or Glu), a basic amino acid (such as His, Lys, or Arg), an aromatic amino acid (such as Trp, Tyr, or Phe), an amino acid that influences chain orientation (such as Gly or Pro), or a hydrophobic amino acid (e.g., Norleucine, Met, Ala, Vai, He, Leu).
  • a hydrophilic amino acid such as Cys, Ser, Thr, Asn, or Gin
  • an acidic amino acid such as Asp or Glu
  • a basic amino acid such as His
  • the amino acid of positions 47, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 331 and 354 are substituted for a hydrophobic amino acid, such as an alanine (A).
  • the alanine of position 182 is substituted for an acidic amino acid, such as a glutamic acid (E).
  • the isoleucine of any one of positions 272, 278, and 291 is substituted for an acidic amino acid, such as a glutamic acid (E).
  • MARAV-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of K47A, K47V, K47I, K47N, K47Q, K47D, K47E, K47H, AG141-C158, AL271-R292, W143A, I144A, D145A, S146A, Q147A, L148A, G150A, G151A, K152A, C153A, S154A, K155A, E156A, C158A, A182E, H209A, I272E, I278E, I291E, 133 IE, 133 ID, R354A, R354V, R354I, R354N, R354Q, R354D, R354E and R354H wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 40.
  • MARAV-G variant comprising (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 28-34, 47-52, 96-104, 111-112, and 118-125 or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 28-34, 47-52, 96-104, 111-112, and 118-125.
  • the MARAV-G variant that comprises one or more mutations selected from the group consisting of K47A, K47V, K47I, K47N, K47Q, K47D, K47E, K47H, AG141- C158, AL271-R292, W143A, I144A, D145A, S146A, Q147A, L148A, G150A, G151A, K152A, C153A, S154A, K155A, E156A, C158A, A182E, H209A, I272E, I278E, I291E, 133 IE, 133 ID, R354A, R354V, R354I, R354N, R354Q, R354D, R354E and R354H further comprises one or more additional mutations within the region of amino acid positions 47, 141- 158, 182, 209, 270-300 (e.g., 271-292), 331 and 354in reference to the wildtype MAR
  • the MARAV-G variant that comprises one or more mutations selected from the group consisting of K47A, K47V, K47I, K47N, K47Q, K47D, K47E, K47H, AG141-C158, AL271-R292, W143A, I144A, D145A, S146A, Q147A, L148A, G150A, G151A, K152A, C153A, S154A, K155A, E156A, C158A, A182E, H209A, I272E, I278E, I291E, 133 IE, 133 ID, R354A, R354V, R354I, R354N, R354Q, R354D, R354E and R354H further comprises one or more additional mutations outside the region of amino acid positions 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to the wildtype MARAV-G sequence of SEQ ID NO:
  • the MARAV-G variant that comprises one or more mutations selected from the group consisting of K47A, K47V, K47I, K47N, K47Q, K47D, K47E, K47H, AG141-C158, AL271-R292, W143A, I144A, D145A, S146A, Q147A, L148A, G150A, G151A, K152A, C153A, S154A, K155A, E156A, C158A, A182E, H209A, I272E, I278E, I291E, 133 IE, 133 ID, R354A, R354V, R354I, R354N, R354Q, R354D, R354E and R354H does not comprise any other mutations outside the region of amino acid positions47, 141-158, 182, 209, 270-300, 331 and 354 in reference to the wildtype MARAV-G sequence of SEQ ID NO: 40
  • the binding of the viral glycoprotein variant to a viral glycoprotein receptor can be measured by any known methods available in the art for detecting or measuring protein-protein binding, such as Western blot, dot blot, surface plasmon resonance (SPR) method, Bio-Layer Interferometry (BLI), FACS, and enzyme-linked immunosorbent assay (ELISA).
  • the dissociation constant (KD) between the viral glycoprotein variant and LDL-R is at least about 1.2-fold (e.g., such as at least about any of 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, or more) of the KD between the reference (e.g., wildtype) viral glycoprotein and LDL-R.
  • the binding between the viral glycoprotein variant and LDL-R is reduced by at least 20% (e.g., such as reduced by at least about any of 50%, 60%, 70%, 80%, 90%, 95%, or more) as compared to the binding between the reference (e.g., wildtype) viral glycoprotein and LDL-R.
  • Fusogenic activity can be evaluated, for example, by assessing efficiency of the viral glycoprotein variant to induce membrane fusion and CAR transduction.
  • the fusogenic activity of the viral glycoprotein variant is retained by at least 20% (e.g., such as retained by at least about any of 50%, 60%, 70%, 80%, 90%, 95%, or more) as compared to the fusogenic activity of the reference (e.g., wildtype) viral glycoprotein.
  • the infectivity of the virus pseudotyped with the viral glycoprotein variant can be restored (e.g., such as restored by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, or by at least about any of 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, or more) with an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on a target cell (e.g., an anti-CD3 scFv).
  • an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on a target cell
  • the infectivity of the virus pseudotyped with the viral glycoprotein variant cannot be restored (e.g., such as restored by no more than about 20%, 15%, 10%, 5%, 2%, or 0%) with an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on a target cell (e.g., an anti-CD3 scFv).
  • an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on a target cell.
  • isolated nucleic acids e.g., a first nucleic acid
  • isolated nucleic acids e.g., a first nucleic acid
  • vectors e.g., an envelope vector
  • the vector e.g., envelope vector
  • the vector further comprises a promoter (e.g., CMV promoter) upstream of the nucleic acid (e.g., first nucleic acid) encoding any of the viral glycoprotein variants described herein.
  • a promoter e.g., CMV promoter
  • retroviruses provide a convenient platform for gene delivery systems.
  • Heterologous nucleic acid can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to the target cell in vitro or ex vivo.
  • Vectors derived from retroviruses such as lentivirus are suitable tools to achieve long-term gene transfer, because they allow long-term, stable integration of a transgene and its propagation in progeny cells.
  • Lentiviral vectors also have low immunogenicity, and can transduce non-proliferating cells.
  • viruses that are enveloped (e.g., pseudotyped) with any of the viral glycoprotein variants described herein (e.g., any of the viral glycoprotein variants with reduced binding to LDL-R and comprising one or more deletions at amino acid positions equivalent to 141-158 and 271-292, or one or more substitutions at any of amino acid positions equivalent to 47, 143-148, 150-156, 158, 182, 209, 272, 278, 291, 331 and 354 in reference to any of SEQ ID NOs: 1, 2, 39, 40, and 46, such as any of SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, and 62-81).
  • enveloped e.g., pseudotyped
  • any of the viral glycoprotein variants described herein e.g., any of the viral glycoprotein variants with reduced binding to LDL-R and comprising one or more deletions at amino acid positions equivalent to 141-158 and 271-292, or one or more substitutions at any of amino acid positions equivalent to 47,
  • recombinant viruses include, but are not limited to, adenovirus, adeno-associated virus (AAV), baculovirus, lentivirus, retrovirus, herpes simplex virus, vaccinia virus, and derivatives thereof.
  • the recombinant virus is lentivirus.
  • the recombinant virus enveloped (e.g., pseudotyped) with any of the viral glycoprotein variants described herein is characterized by increased (e.g., increasing at least about any of 20%, 50%, 60%, 70%, 80%, 90%, 95%, 1.2-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, or more) stability, reduced (e.g., decreasing at least about any of 20%, 50%, 60%, 70%, 80%, 90%, 95%, 1.2-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 50- fold, 100-fold, 1000-fold, or more) serum activation, and/or reduced (e.g., decreasing at least about any of 20%, 50%, 60%, 70%, 80%, 90%, 95%, 1.2-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, or more) transduction efficiency.
  • increased e.g., increasing at least about any of 20%, 50%, 60%
  • Recombinant virus stability can be assessed by any method known in the art, such as thermostability, tolerability to varying pH or salt concentrations, resistance to serum, virus half-life, trypsin sensitivity, and freeze-thaw tolerance.
  • Serum sensitivity can be measured by any method known in the art, such as a serum neutralization assay.
  • Transduction efficiency can be assessed by using a recombinant virus carrying a selectable marker, such as an antibiotic resistance gene, or an easily detectable marker, such as a transgene encoding a protein that is readily detectable on cell surfaces (e.g., Flag-tagged engineered receptor, see Example 1 and Example 3).
  • a recombinant virus e.g., lentivirus
  • the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G)
  • the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and/or substitution(s)) at amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, and wherein the viral glycoprotein variant has reduced (e.g., reducing at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) binding to LDL-R compared to the
  • a reference viral glycoprotein e g
  • the viral glycoprotein variant (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), orMARAV- G variant) may or may not comprise any further mutations outside the region of amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to SEQ ID NO: 1.
  • the viral glycoprotein variant comprises one or more deletions at amino acid positions equivalent to 141-158 and 271-292, or one or more substitutions at any of amino acid positions equivalent to 47, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, 291, 331 and 354 in reference to the wildtype COV-G sequence of SEQ ID NO: 1.
  • the viral glycoprotein variant comprises one or more mutations at amino acid positions equivalent to 47V, 471, 47N, 47Q, 47D, 47E, 47H, A141-158, A271-292, 143A, 144A, 145A, 146A, 147A, 148A, 150A, 151A, 152A, 153A, 154A, 155A, 156A, 158A, 182E, 182D, 182N, 209A, 272E, 278E, 291E, 33 IE, 33 ID, 33 IK, 331R, 354A, 354V, 3541, 354N, 354Q, 354D, 354E and 354H in reference to the wildtype COV-G sequence of SEQ ID NO: 1.
  • the viral glycoprotein variant may or may not comprise further mutation(s) in addition to the one or more deletions at amino acid positions equivalent to 141-158 and/or 271-292, or one or more substitutions at amino acid positions equivalent to 47, 143-148, 150-156, 158, 182, 209, 272, 278, 291, 331, and 354 in reference to SEQ ID NO: 1.
  • the further mutation(s) can be within or outside of (or both) the regions of amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to SEQ ID NO: 1.
  • the viral glycoprotein is COV-G
  • the COV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and/or substitution(s)) at amino acid positions 141-158, 182, 209, 270-300, and 331 in reference to the wildtype COV-G sequence of SEQ ID NO: 1, and wherein the COV-G variant has reduced (e.g., reducing at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) binding to LDL-R compared to wildtype COV-G.
  • the COV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions of 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, 291, and 331 and wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1.
  • the COV- G variant comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, I144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, E154A, T155A, E156A, C158A, V182E, V182D, V182N, Y209A, I272E, I278E, I291E, 133 IE, 133 ID, 133 IK, and 1331R, in reference to SEQ ID NO: 1.
  • the COV-G variant consists of one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, I144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, E154A, T155A, E156A, C158A, V182E, V182D, V182N, Y209A, I272E, I278E, I291E, 133 IE, 133 ID, 133 IK, and I331Rin reference to SEQ ID NO: 1.
  • the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 4, 5, 8-10, 12, 13, 62-75, 82, 86, and 88-91 or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 4, 5, 8-10, 12, 13, 62-75, 82, 86, and 88-91.
  • the COV-G variant consists of the amino acid sequence of any of SEQ ID NOs: 4, 5, 8-10, 12, 13, 62-75, 82, 86, and 88-91.
  • the viral glycoprotein is VSV-G (e.g., VSV Orsay- G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and/or substitution(s)) at amino acid positions 141-158, 182, 209, and 270-300 in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2, 39, or 46, and wherein the VSV-G variant has reduced (e.g., reducing at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) binding to LDL-R compared to the corresponding reference VSV-G.
  • VSV-G e.g., VSV Orsay- G, VSV SJ-G, or VSV syn-G
  • the VSV-G variant comprises (or consists essentially of, or consists of) one or
  • the VSV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions of 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278 and 291, and wherein the amino acid positions are in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2, 39, and 46.
  • the VSV-G variant comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of A141-158, A271-292, 143A, 144A, 145A, 146A, 147A, 148A, 150A, 151 A, 152A, 153 A, 154A, 155 A, 156A, 158A, 182E, 209A, 272E, 278E, and 291E in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2, 39, and 46.
  • the VSV-G variant is a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, D156A, C158A, I182E, Y209A, I272E, I278E, and I291E in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 2.
  • the VSV Orsay-G variant consists of one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, D156A, C158A, I182E, Y209A, I272E, I278E, and I291E in reference to SEQ ID NO: 2.
  • the VSV-G variant is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, Y156A, C158A, I182E, Y209A, I272E, I278E, and I291E in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 39.
  • the VSV SJ-G variant that comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, Y156A, C158A, I182E, Y209A, I272E, I278E, and I291Eshares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the wildtype VSV SJ-G sequence of SEQ ID NO: 39.
  • the VSV SJ-G variant consists of one or more mutations selected from the group consisting ofAG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, Y156A, C158A, I182E, Y209A, I272E, I278E, and I291E in reference to SEQ ID NO: 39.
  • the VSV SJ-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 21-27 and 54-59, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 21-27 and 54-59. In some embodiments, the VSV SJ-G variant consists of the amino acid sequence of any of SEQ ID NOs: 21-27 and 54-59.
  • the VSV-G variant is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, Cl 53 A, S154A, N155A, Y156A, C158A, I182E, Y209A, I272E, I278E, and 129 IE in reference to the reference VSV syn-G sequence of SEQ ID NO: 46.
  • the VSV syn- G variant that comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of AG141-C158, AL271-R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, Y156A, C158A, I182E, Y209A, I272E, I278E, and I291Eshares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the reference VSV syn-G sequence of SEQ ID NO: 46.
  • the VSV syn-G variant consists of one or more mutations selected from the group consisting of AG141-C158, AL271- R292, W143A, V144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, S154A, N155A, Y156A, C158A, I182E, Y209A, I272E, I278E, and I291E in reference to SEQ ID NO: 46.
  • the VSV syn-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 14-20 and 76-81, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 14-20 and 76-81. In some embodiments, the VSV syn-G variant consists of the amino acid sequence of any of SEQ ID NOs: 14-20 and 76-81.
  • the viral glycoprotein is MARAV-G
  • the MARAV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and/or substitution(s)) at amino acid positions 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to the wildtype MARAV- G sequence of SEQ ID NO: 40, and wherein the MARAV-G variant has reduced (e.g., reducing at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) binding to LDL-R compared to wildtype MARAV-G.
  • the MARAV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and/or substitution(s)) at amino acid positions 47, 141-158, 182, 209, 270-300, 331 and 354 in reference
  • the MARAV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions of 47, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, 291, 331 and 354 and wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 40.
  • the MARAV-G variant comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of K47A K47V, K47I, K47N, K47Q, K47D, K47E, K47H, AG141-C158, AL271-R292, W143A, I144A, D145A, S146A, Q147A, L148A, G150A, G151A, K152A, C153A, S154A, K155A, E156A, C158A, A182E, H209A, I272E, I278E, I291E, 133 IE, 133 ID, R354A, R354V, R354I, R354N, R354Q, R354D, R354E and R354H in reference to SEQ ID NO: 40.
  • the MARAV-G variant that comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of K47A K47V, K47I, K47N, K47Q, K47D, K47E, K47H, AG141-C158, AL271-R292, W143A, I144A, D145A, S146A, Q147A, L148A, G150A, G151A, K152A, C153A, S154A, K155A, E156A, C158A, A182E, H209A, I272E, I278E, I291E, 133 IE, 133 ID, R354A, R354V, R354I, R354N, R354Q, R354D, R354E and R354H shares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
  • the MARAV-G variant consists of one or more mutations selected from the group consisting of K47A K47V, K47I, K47N, K47Q, K47D, K47E, K47H, AG141-C158, AL271-R292, W143A, I144A, D145A, S146A, Q147A, L148A, G150A, G151A, K152A, C153A, S154A, K155A, E156A, C158A, A182E, H209A, I272E, I278E, I291E, 133 IE, 133 ID, R354A, R354V, R354I, R354N, R354Q, R354D, R354E and R354H in reference to SEQ ID NO: 40.
  • the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 28-34, 47-52, 96-104, 111-112, and 118-125 or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 28-34 and 47-52, 96-104, 111-112, and 118-125.
  • the MARAV-G variant consists of the amino acid sequence of any of SEQ ID NOs: 28-34, 47-52, 96-104, 111-112, and 118-125.
  • the viral glycoprotein variant is encoded by a first nucleic acid.
  • the first nucleic acid is on an envelope vector.
  • the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on a target cell (e.g., anti-CD3 scFv).
  • the envelope surface-bound targeting molecule is encoded by a second nucleic acid, such as carried on an envelope vector (either same or different from the envelope vector carrying the first nucleic acid).
  • the recombinant virus further comprises a third nucleic acid encoding a heterologous molecule (e.g., heterologous protein or RNA), such as an engineered receptor (e.g., CAR).
  • the third nucleic acid is on a transfer vector.
  • a recombinant virus e.g., lentivirus
  • the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G)
  • the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and/or substitution(s)) at amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein the viral glycoprotein variant has reduced (e.g., reducing at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) binding to LDL-R compared to the reference
  • a reference viral glycoprotein e g
  • a recombinant virus comprising a COV-G variant
  • the COV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions of 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, 291, and 331 (e.g., W143A, I144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, E154A, T155A, E156A, C158A, V182E, V182D, V182N, Y209A, I272E, I278E, I291E, 133 IE, 133 ID,
  • the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs:4, 5, 8-10, 12, 13, 62-75, 82, 86, and 88-91.
  • a recombinant virus e.g., lentivirus
  • VSV-G variant e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G
  • the VSV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions of 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, and 291 (e.g., W143A, V144A, D145A, S146
  • VSV-G variant e.g., variant
  • the VSV-G variant is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 21-27 and 54- 59. In some embodiments, the VSV-G variant is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 14-20 and 76- 81.
  • a recombinant virus comprising a MARAV-G variant
  • the MARAV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions of 47, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, 291, 331 and 354 (e.g., K47A K47V, K47I, K47N, K47Q, K47D, K47E, K47H, W143A, I144A, D145A, S146A, Q147A, L148A, G150A, G151A, K152A, C153A, S154A, K155A, E156A, C158A, A182E, H209A
  • the MARAV-G variant comprises (or consists essentially of, or
  • the MARAV-G comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 28-34, 47-52, 96-104, 111-112, and 118-125.
  • the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain, e.g., a transmembrane domain derived from CD8.
  • the envelope surface-bound targeting molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein (e.g., CD3) on the target cell, such as Fab, scFv, or sdAb.
  • the envelope surface-bound targeting molecule comprises a T-cell activation molecule or a costimulation molecule, e.g., CD86, CD80, CD137L, anti-CD28 antibody or antigen-binding fragment, or anti-4- IBB antibody or antigen-binding fragment.
  • the viral glycoprotein variant is encoded by a first nucleic acid
  • the envelope surface-bound targeting molecule e.g., anti-CD3 scFv
  • the recombinant virus further comprises a third nucleic acid encoding a heterologous molecule, such as an engineered receptor (e.g., CAR).
  • the first and second nucleic acids can be on the same vector or different vectors (e.g., envelope vector). In some embodiments, the first and second nucleic acids are on the same vector (e.g., envelope vector), and the third nucleic acid is on a different vector (e.g., transfer vector). The first and second nucleic acids, when on the same vector, can be under the control of a same or different promoters.
  • the nucleic acids can be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A, E2A).
  • a linking nucleic acid sequence such as IRES
  • a linking nucleic acid encoding a cleavable linker e.g., 2A peptide, such as P2A, T2A, E2A.
  • a recombinant virus e.g., lentivirus
  • the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G)
  • the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and/or substitution(s)) at amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein the viral glycoprotein variant has reduced (e.g., reducing at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) binding to LDL-R compared to the reference
  • a reference viral glycoprotein e g
  • a recombinant virus comprising a COV-G variant
  • the COV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions of 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, 291, and 331 (e.g.,W143A, I144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, E154A, T155A, E156A, C158A, V182E, V182D, V182N, Y209A, I272E, I278E, I291E, 133 IE, 133 ID,
  • the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 4, 5, 8-10, 12, 13, 62-75, 82, 86, and 88-91.
  • a recombinant virus e.g., lentivirus
  • VSV-G variant e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G
  • the VSV- G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions of 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, and 291 (e.g., W143A, V144A, D145A, S146
  • VSV-G variant e.g., variant
  • the VSV-G variant is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 21-27 and 54-59. In some embodiments, the VSV-G variant is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 14-20 and 76-81.
  • a recombinant virus comprising a MARAV- G variant
  • the MARAV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions of 47, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, 291, 331 and 354 (e.g., W143A, I144A, D145A, S146A, Q147A, L148A, G150A, G151A, K152A, C153A, S154A, K155A, E156A, C158A, A182E, H209A, I272E, I278E, I291E, 133 IE, 133 ID), wherein the amino acid positions
  • the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 28-34, 47-52, 96-104, 111-112, and 118- 125.
  • the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain, e.g., a transmembrane domain derived from CD8.
  • the envelope surface-bound targeting molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein (e.g., CD3) on the target cell, such as Fab, scFv, or sdAb.
  • the envelope surface-bound targeting molecule comprises a T-cell activation molecule or a costimulation molecule, e.g., CD86, CD80, CD137L, anti-CD28 antibody or antigen-binding fragment, or anti -4- IBB antibody or antigen-binding fragment.
  • the recombinant virus further comprises a third nucleic acid encoding a heterologous molecule, such as an engineered receptor (e.g., CAR), wherein the third nucleic acid is on a different vector (e.g., transfer vector).
  • the first and second nucleic acids can be under the control of a same or different promoters.
  • the first and second nucleic acids are under the control of a single/ same promoter, and the first and second nucleic acids are connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A).
  • a linking nucleic acid sequence such as IRES
  • a linking nucleic acid encoding a cleavable linker e.g., 2A peptide, such as P2A, T2A.
  • the first and second nucleic acids are under the control of a single/same promoter and connected via a linking nucleic acid encoding a P2A cleavable linker.
  • a recombinant virus e.g., lentivirus
  • the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G)
  • the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and/or substitution(s)) at amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein the viral glycoprotein variant has reduced (e.g., reducing at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) binding to LDL-R compared to the reference
  • a reference viral glycoprotein e g
  • a recombinant virus comprising a COV-G variant
  • the COV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions of 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, 291, and 331 (e.g., W143A, I144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, E154A, T155A, E156A, C158A, V182E, V182D, V182N, Y209A, I272E, I278E, I291E, 133 IE, 133 ID,
  • the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 4, 5, 8-10, 12, 13, 62-75, 82, 86, and 88-91.
  • a recombinant virus e.g., lentivirus
  • VSV- G variant e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G
  • the VSV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions of 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, and 291 (e.g., W143A, V144A, D145A, S146
  • the VSV-G is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 21-27 and 54-59. In some embodiments, the VSV-G is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 14-20 and 76-81.
  • a recombinant virus comprising a MARAV- G variant
  • the MARAV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions of 47, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, 291, 331 and 354 (e.g., K47A K47V, K47I, K47N, K47Q, K47D, K47E, K47H, W143A, I144A, D145A, S146A, Q147A, L148A, G150A, G151A, K152A, C153A, S154A, K155A, E156A, C158A, A182E, H209A
  • the MARAV-G variant comprises (or consists essentially of, or
  • the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 28-34 , 47-52, 96-104, 111-112, and 118-125.
  • the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain, e.g., a transmembrane domain derived from CD8 (e.g., CD8a).
  • the envelope surface-bound targeting molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein (e.g., CD3) on the target cell, such as Fab, scFv, or sdAb.
  • the envelope surface-bound targeting molecule comprises a T-cell activation molecule or a co-stimulation molecule, e.g., CD86, CD80, CD137L, anti-CD28 antibody or antigen-binding fragment, or anti-4-lBB antibody or antigen-binding fragment.
  • the CAR comprises from N-terminus to C-terminus: (a) an extracellular antigen binding domain (e.g., an anti -CD 19 antigen-binding fragment and/or an anti-CD20 antigen-binding fragment); (b) an optional hinge domain; (c) a transmembrane domain; and (d) an intracellular signaling domain.
  • the CAR further comprises an intracellular co-stimulatory signaling domain, either between the transmembrane domain and the intracellular signaling domain, or at the C-terminus of the intracellular signaling domain.
  • the extracellular antigen binding domain comprises two or more antibody moieties connected in tandem.
  • the first and second nucleic acids can be on the same vector or different vectors (e.g., envelope vector). In some embodiments, the first and second nucleic acids are on the same vector (e.g., envelope vector), and the third nucleic acid is on a different vector (e.g., transfer vector). The first and second nucleic acids, when on the same vector, can be under the control of a same or different promoters.
  • the nucleic acids can be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A, E2A).
  • the recombinant virus comprises two or more units of third nucleic acids encoding two or more CARs (e.g., one anti-CD19 CAR, one anti-CD20 CAR).
  • the two or more units of third nucleic acids can be on the same vector or different vectors (e.g., transfer vector).
  • the two or more units of third nucleic acids when on the same vector, can be under the control of a same or different promoters.
  • the nucleic acids can be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A, E2A).
  • a recombinant virus e.g., lentivirus
  • the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G)
  • the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and/or substitution(s)) at amino acid positions equivalent to 47, 141-158, 182, 209, 270-300, 331 and 354 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein the viral glycoprotein variant has reduced (e.g., reducing at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) binding to LDL-R compared to the reference
  • a reference viral glycoprotein e g
  • a recombinant virus comprising a COV-G variant
  • the COV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions of 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, 291, and 331 (e.g., W143A, I144A, D145A, S146A, Q147A, F148A, N150A, G151A, K152A, C153A, E154A, T155A, E156A, C158A, V182E, V182D, V182N, Y209A, I272E, I278E, I291E, 133 IE, 133 ID,
  • the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 4, 5, 8-10, 12, 13, 62-75, 82, 86, and 88-91.
  • a recombinant virus e.g., lentivirus
  • VSV-G variant e.g., variant of VSV Orsay - G, VSV SJ-G, or VSV syn-G
  • the VSV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions of 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, and 291 (e.g., W143A, V144A, D145A, SEQ ID NOs: 4, 5, 8-10, 12, 13, 62
  • the VSV-G variant is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 21-27 and 54-59. In some embodiments, the VSV-G variant is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 14-20 and 76-81.
  • a recombinant virus comprising a MARAV-G variant
  • the MARAV-G variant comprises (or consists essentially of, or consists of) one or more deletions at amino acid positions 141-158 and/or 271-292, or one or more substitutions at any of amino acid positions of 47, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 209, 272, 278, 291, 331 and 354 (e.g., K47A K47V, K47I, K47N, K47Q, K47D, K47E, K47H, W143A, I144A, D145A, S146A, Q147A, L148A, G150A, G151A, K152A, C153A, S154A, K155A, E156A, C158A, A182E, H209A
  • the MARAV-G variant comprises (or consists essentially of, or
  • the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 28-34 , 47-52, 96-104, 111-112, and 118-125.
  • the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain, e.g., a transmembrane domain derived from CD8.
  • the envelope surface-bound targeting molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein (e.g., CD3) on the target cell, such as Fab, scFv, or sdAb.
  • the envelope surface-bound targeting molecule comprises a T-cell activation molecule or a co-stimulation molecule, e.g., CD86, CD80, CD137L, anti-CD28 antibody or antigen-binding fragment, or anti-4-lBB antibody or antigen-binding fragment.
  • the CAR comprises from N-terminus to C-terminus: (a) an extracellular antigen binding domain (e.g., an anti -CD 19 antigen-binding fragment and/or an anti-CD20 antigen-binding fragment); (b) an optional hinge domain; (c) a transmembrane domain; and (d) an intracellular signaling domain.
  • the CAR further comprises an intracellular co-stimulatory signaling domain, either between the transmembrane domain and the intracellular signaling domain, or at the C-terminus of the intracellular signaling domain.
  • the extracellular antigen binding domain comprises two or more antibody moieties connected in tandem.
  • the first and second nucleic acids can be under the control of a same or different promoters. In some embodiments, the first and second nucleic acids are under the control of a single/same promoter and connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A, E2A).
  • the first and second nucleic acids are under the control of a single/same promoter and connected via a linking nucleic acid encoding a P2A cleavable linker.
  • the recombinant virus comprises two or more units of third nucleic acids encoding two or more CARs (e.g., one anti-CD19 CAR, one anti-CD20 CAR).
  • the two or more units of third nucleic acids can be on the same vector or different vectors (e.g., transfer vector).
  • the two or more units of third nucleic acids, when on the same vector can be under the control of a same or different promoters.
  • the nucleic acids can be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A, E2A).
  • a linking nucleic acid sequence such as IRES
  • a linking nucleic acid encoding a cleavable linker e.g., 2A peptide, such as P2A, T2A, E2A.
  • Recombinant viruses enveloped e.g., pseudotyped
  • any of the viral glycoprotein variants described herein e.g., any of the viral glycoprotein variants with reduced binding to LDL-R and comprising one or more deletions at amino acid positions equivalent to 141-158 and 271-292, or one or more substitutions at any of amino acid positions equivalent to 47, 143- 148, 150-156, 158, 182, 209, 272, 278, 291, 331 and 354 in reference to any of SEQ ID NOs: 1, 2, 39, 40, and 46, such as any of SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, 62-82, 86, 88-91, 96-104, 111-112, and 118-125) can have reduced binding affinity between the viral glycoprotein variant and LDL-R.
  • recombinant viruses can be engineered to further comprise an envelope surface-bound targeting molecule.
  • the envelope surface-bound targeting molecule specifically recognizes a cell surface protein on a target cell, such as anti-CD3 scFv that can target T cells.
  • the envelope surface-bound targeting molecule further comprises a transmembrane domain (e.g., CD8-derived transmembrane domain) or membrane-anchoring domain.
  • the envelope surface-bound targeting molecule is encoded by a second nucleic acid.
  • the envelope surface-bound targeting molecule may comprise an antibody or antigenbinding fragment thereof which can enhance recombinant virus targeting to any target cell of interest.
  • the target cell can be dividing or non-dividing, such as hematopoietic cells, lymphoid cells, myeloid cells, epithelial cells, fibroblasts, muscle cells, and neuronal cells.
  • the target cell is a disease cell (e.g., cancer cell) to be treated.
  • the target cell is an immune cell, such as an immune effector cell.
  • the immune cell is selected from the group consisting of a monocyte, a dendritic cell, a macrophage, a B cell, a T cell, a natural killer (NK) cell, or a combination thereof.
  • the immune cell is a T cell, such as a killer T cell (Tc), cytotoxic T lymphocyte (CTL), a helper T cell (Th), a regulatory T cell (Treg), an aP T cell, a y5 T cell, and a natural killer T (NKT) cell.
  • Cell surface protein on a target cell can be any antigen displayed on the surface of the target cell.
  • a cell surface protein specific for the target cell can be chosen to be targeted by the envelope surface-bound targeting molecule.
  • the cell surface protein of a T cell can be any antigen displayed on the surface of a T cell.
  • a T-cell specific antigen can be used.
  • the T cell surface protein is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD31, CD38, CD45, CD56, CD58, CD69, CD95, CD103, CD122, CD127, CD161, CCR4, CCR5, CCR6, CCR7, CCR10, CTLA4, CXCR3, CD 11 a, and CD40L.
  • the envelope surface-bound antibody or antigenbinding fragment thereof specifically recognizes CD3.
  • Exemplary anti-CD3 antibodies or antigen-binding fragments include but are not limited to, clones OKT3 (e.g., Invitrogen), UCHT1 (e.g., Invitrogen), SP7 (e.g., Invitrogen), SK7 (e.g., Invitrogen), F7.2.38 (e.g., Invitrogen), HIT3A (e.g., Invitrogen), 7D6 (e.g., Invitrogen), S4.1 (e.g., Invitrogen), RIV9 (e.g., Invitrogen), MEM-57 (e.g., Invitrogen), 3F3A1 (e.g., Proteintech), UMAB54 (e.g., OriGene), GT0013 (e.g., Invitrogen), APA1/1 (e.g., Invitrogen), 4E2 (e.g., Invitrogen), 4D10A6 (e.g., Invitrogen
  • the antibody or antigen-binding fragment thereof comprised by the envelope surfacebound targeting molecule can be of any format.
  • the envelope surfacebound antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, a Fab, a Fab’, a (Fab’k, an Fv, an scFv, and an sdAb.
  • the antibody or antigen-binding fragment thereof is an scFv.
  • recombinant viruses expressing the envelope surface-bound targeting molecule can transduce and activate immune cells (e.g., T cells) at the same time.
  • the target cell is a T cell
  • the activation of the T cell comprises upregulation (e.g., upregulating at least about 20%, 50%, 90%, 1-fold, 2-fold, 10-fold, or more) of a marker selected from one or more of CD25, CD38 CD69, CD71, GITR (glucocorticoid- induced TNFR-related receptor), CTLA-4, NTB-A, and HLA-DR.
  • upregulation e.g., upregulating at least about 20%, 50%, 90%, 1-fold, 2-fold, 10-fold, or more
  • a marker selected from one or more of CD25, CD38 CD69, CD71, GITR (glucocorticoid- induced TNFR-related receptor), CTLA-4, NTB-A, and HLA-DR.
  • the target cell is an NK cell
  • the activation of the NK cell comprises upregulation (e.g., upregulating at least about 20%, 50%, 90%, 1-fold, 2-fold, 10-fold, or more) of a marker selected from one or more of CD16, CD27, CD56, CD69, Sca-1, NKG2D, and KLRG1 (killer cell lectin-like receptor Gl).
  • the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain.
  • the membrane-anchoring domain is a glycosylphosphatidylinositol (GPI) anchor.
  • the transmembrane domain is derived from a transmembrane protein selected from the group consisting of CD8, CD4, CD28, 4-1BB, CD80, CD86, CD152, and PD-1.
  • the transmembrane domain is derived from CD8, such as CD8a.
  • the envelope surface-bound targeting molecule further comprises a domain C- terminal to the transmembrane domain.
  • the domain C-terminal to the transmembrane domain of the envelope surface-bound targeting molecule can derive from an intracellular portion of any transmembrane protein, which can be a primary intracellular signaling domain, intracellular co-stimulatory signaling domain, or a cytoplasmic portion without signaling function.
  • the domain C-terminal to the transmembrane domain of the envelope surface-bound targeting molecule can derive from any of CD3( ⁇ , FcRy, FcRP, CD3y, CD35, CD3s, TCRa, TCRp, TCRy, TCR5, CD5, CD22, CD79a, CD79b, CD66d, and 4-1BB.
  • the envelope surface-bound targeting molecule thereof comprises an intracellular domain derived from 4- IBB C-terminal to the transmembrane domain.
  • the envelope surface-bound targeting molecule comprises from N’ to C’ : an antibody or antigen-binding fragment thereof that specifically recognizes a cell surface protein on a target cell (e.g., anti-CD3 scFv), a transmembrane domain or membrane-anchoring domain (e.g., CD8-derived transmembrane domain), and optionally a domain C-terminal to the transmembrane domain or membrane-anchoring domain (e.g., intracellular domain of 4- IBB or fragment thereof).
  • a target cell e.g., anti-CD3 scFv
  • a transmembrane domain or membrane-anchoring domain e.g., CD8-derived transmembrane domain
  • a domain C-terminal to the transmembrane domain or membrane-anchoring domain e.g., intracellular domain of 4- IBB or fragment thereof.
  • the envelope surface-bound targeting molecule binds to the cell surface protein on target cell with a KD of ⁇ lOpM, ⁇ IpM, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g., about 10' 5 M or less, such as any of from about 10' 5 M to about 10' 13 M, from about 10' 8 M to about 10' 13 M, from about 10' 9 M to about 10' 13 M, from about 10' 10 M to about 10' 13 M, or from about 10' 11 M to about 10' 13 M).
  • a KD ⁇ lOpM, ⁇ IpM, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g., about 10' 5 M or less, such as any of from
  • a variety of methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure, including by RIA, for example, performed with the an antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81); by biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays by Octet®, using, for example, an Octet®Red96 system, or by Biacore®, using, for example, a Biacore®TM-2000 or a Biacore®TM-3000.
  • RIA for example, performed with the an antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81)
  • BLI biolayer interferometry
  • SPR surface plasmon resonance
  • an “on-rate” or “rate of association” or “association rate” or “k on ” may also be determined with the same BLI or SPR techniques described above using, for example, the Octet®Red96, the Biacore®TM-2000, or the Biacore®TM-3000 system.
  • the envelope surface-bound targeting molecule comprises an immune cell (e.g., T-cell) activation molecule or co-stimulation molecule.
  • the immune cell activation molecule or co-stimulation molecule activates and/or co-stimulates T cells.
  • T cell activation or co-stimulation molecules include, but are not limited to, CD3, CD8a, CD27, CD28, CD80, CD86, FCRy, 0X40, ICOS, CD40 ligand (CD40L), 4-1BB, and CD137 ligand (CD137L).
  • the T cell activation or co-stimulation molecule is selected from the group consisting of an anti-CD28 antibody or antigen-binding fragment thereof, anti-CD137 antibody or antigen-binding fragment thereof, an anti-4-lBB antibody or antigen-binding fragment thereof, CD86, CD80, and CD137L.
  • the T cell activation or co-stimulation molecule comprises a transmembrane domain (e.g., derived from CD8) or membrane-anchoring domain.
  • the envelope surface-bound targeting molecule comprises from N’ to C’ : an immune cell activation molecule or co-stimulation molecule (e.g., anti-CD28 or anti-4-lBB antigen-binding fragment), a transmembrane domain or membrane-anchoring domain (e.g., CD8-derived transmembrane domain), and optionally a domain C-terminal to the transmembrane domain or membrane-anchoring domain (e.g., intracellular domain of 4- IBB or fragment thereof).
  • the envelope surface-bound targeting molecule is a full length CD86, CD80, or CD137L.
  • the envelope surface-bound targeting molecule improves transduction efficiency of the recombinant virus comprising any of the viral glycoprotein variants disclosed herein (e.g., any of the viral glycoprotein variants with reduced binding to LDL-R and comprising one or more deletions at amino acid positions equivalent to 141-158 and 271-292, or one or more substitutions at any of amino acid positions equivalent to 47, 143-148, 150-156, 158, 182, 209, 272, 278, 291, 331 and 354 in reference to any of SEQ ID NOs: 1, 2, 39, 40, and 46, such as any of SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, 62- 82, 86, 88-91, 96-104, 111-112, and 118-125) by specifically targeting the recombinant virus to the target cell of interest (e.g., T cell), such as improving at least about 5%
  • the target cell of interest e.g., T cell
  • a recombinant virus comprising any of the viral glycoprotein variants disclosed herein (e.g., any of the viral glycoprotein variants with reduced binding to LDL-R and comprising one or more deletions at amino acid positions equivalent to 141-158 and 271-292, or one or more substitutions at any of amino acid positions equivalent to 47, 143-148, 150-156, 158, 182, 209, 272, 278, 291, 331 and 354 in reference to any of SEQ ID NOs: 1, 2, 39, 40, and 46, such as any of SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, 62-82, 86, 88-91, 96-104, 111-112, and 118-125) and an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) has reduced transduction of non-target cells (e.g., non-T cell), such as reducing at least about 5% (e.g., at least about any of 10%,
  • the transduction efficiency is reflected by the expression of a heterologous nucleic acid (e.g., encoding a CAR) comprised in the recombinant virus by the target cell.
  • the transduction efficiency is reflected by the expression and/or function of a heterologous nucleic acid (e.g., encoding a CAR or siRNA) comprised in the recombinant virus in the target cell, such as CAR-T mediated cytotoxicity, or gene knock-down in the target cell.
  • the recombinant virus comprises a heterologous nucleic acid encoding a CAR (e.g., anti-CD20+CD19 CAR), and any of the viral glycoprotein variants disclosed herein (e.g., any of the viral glycoprotein variants with reduced binding to LDL-R and comprising one or more deletions at amino acid positions equivalent to 141-158 and 271- 292, or one or more substitutions at any of amino acid positions equivalent to 47, 143-148, 150-156, 158, 182, 209, 272, 278, 291, 331 and 354 in reference to any of SEQ ID NOs: 1, 2, 39, 40, and 46, such as any of SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, 62-82, 86, 88-91, 96-104, 111-112, and 118-125) and an envelope surface-bound targeting molecule (e.g., anti- CD3 scFv), wherein the target cell (e.g., anti- CD
  • the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) of the recombinant virus can activate the transduced target cell (e.g., T cell) simultaneously with transduction.
  • the transduced target cell e.g., T cell
  • at least about 20% (such as at least about any of 50%, 80%, 90%, 95%, 99%, or 100%) of transduced target cells are activated (e.g., upregulating CD25 expression) simultaneously with transduction.
  • a recombinant virus comprising any of the viral glycoprotein variants disclosed herein (e.g., any of the viral glycoprotein variants with reduced binding to LDL-R and comprising one or more deletions at amino acid positions equivalent to 141-158 and 271-292, or one or more substitutions at any of amino acid positions equivalent to 47, 143-148, 150-156, 158, 182, 209, 272, 278, 291, 331 and 354 in reference to any of SEQ ID NOs: 1, 2, 39, 40, and 46, such as any of SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, 62-82, 86, 88-91, 96-104, 111-112, and 118-125) and an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) increases activation of target cells (e.g., T cell), such as increasing at least about 10% (e.g., at least about any of 20%, 50%, 90%, 1-fold,
  • target cells
  • the recombinant virus further comprises a nucleic acid (e.g., a third nucleic acid) that encodes a heterologous molecule (e.g., heterologous protein or RNA), such as a therapeutic protein or RNA (e.g., siRNA).
  • a heterologous molecule e.g., heterologous protein or RNA
  • a therapeutic protein or RNA e.g., siRNA
  • heterologous nucleic acids or molecules encoded thereof refer to a nucleic acid or a molecule to be introduced to the target cell by the recombinant viruses described herein.
  • the target cell may or may not have an endogenous version of this to-be-introduced nucleic acid or molecule.
  • the recombinant virus further comprises a nucleic acid (e.g., a third nucleic acid) that encodes a functional or wildtype version of an RNA or protein, which can compensate for the expression and/or function of a corresponding RNA or protein (e.g., mutant protein) in the target cell to be transduced.
  • the heterologous molecule is a therapeutic protein.
  • the therapeutic protein is an immunomodulator, such as immunostimulatory or immunosuppressant protein. Immunomodulators include but are not limited to, immune checkpoint inhibitors, immune checkpoint stimulators, cytokines, and immunocytokines.
  • the therapeutic protein is an antagonist (e.g., antagonist antibody) of an inhibitory checkpoint molecule, such as an inhibitory checkpoint molecule selected from the group consisting of PD-1, PD-L1, PD- L2, CTLA-4, LAG-3, TIM- 3, HHLA2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT, Siglec7, Siglec9, and VISTA.
  • an inhibitory checkpoint molecule selected from the group consisting of PD-1, PD-L1, PD- L2, CTLA-4, LAG-3, TIM- 3, HHLA2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT, Siglec7, Siglec9, and VISTA.
  • the therapeutic protein is an agonist (e.g., agonist antibody) of a stimulatory immune checkpoint molecule, such as an stimulatory immune checkpoint molecule selected from the group consisting of CD27, CD28, 0X40, ICOS, GITR, 4-1BB, CD27, CD40, CD3, and HVEM.
  • a stimulatory immune checkpoint molecule selected from the group consisting of CD27, CD28, 0X40, ICOS, GITR, 4-1BB, CD27, CD40, CD3, and HVEM.
  • the therapeutic protein is an immunostimulatory cytokine, such as an immunostimulatory cytokine selected from the group consisting of IL-1, IL-2, IL-3, IL- 4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-a, IFN-P, IFN-y, TNF-a, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF, and GM-CSF.
  • an immunostimulatory cytokine selected from the group consisting of IL-1, IL-2, IL-3, IL- 4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-a
  • the therapeutic protein is an immunosuppressive cytokine, such as an immunosuppressive cytokine selected from the group consisting of IL-IRa, IL-4, IL-5, IL-6, IL-10, IL-11, IL-13, IL-27, IL-33, IL-35, IL-37, IL-39, IFN-a, LIF, and TGF-p.
  • an immunosuppressive cytokine selected from the group consisting of IL-IRa, IL-4, IL-5, IL-6, IL-10, IL-11, IL-13, IL-27, IL-33, IL-35, IL-37, IL-39, IFN-a, LIF, and TGF-p.
  • the therapeutic protein is an antibody construct (e.g., a monoclonal antibody, a multispecific antibody, a bispecific antibody, a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a dsFv, a (dsFv)2, a bispecific dsFv, an scFv, an sdAb, a nanobody, or a bi-specific T-cell engager (BiTE)) or an engineered receptor.
  • the heterologous molecule is an engineered receptor.
  • the engineered receptor comprises i) an extracellular antigen binding domain specifically recognizing a target antigen, and ii) a transmembrane domain.
  • the engineered receptor is a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), a chimeric TCR (cTCR), a T cell antigen coupler (TAC), or a TAC-like engineered receptor.
  • the engineered receptor is a CAR, such as any CAR known in the art or described herein.
  • the CAR comprises i) an extracellular antigen binding domain specifically recognizing a target antigen, ii) a transmembrane domain, and iii) an intracellular signaling domain (e.g., derived from CD3Q.
  • the CAR comprises from N-terminus to C-terminus: (a) an extracellular antigen binding domain; (b) an optional hinge domain; (c) a transmembrane domain; (d) an optional intracellular costimulatory signaling domain; and (e) an intracellular signaling domain.
  • the CAR comprises from N-terminus to C-terminus: (a) an extracellular antigen binding domain; (b) an optional hinge domain; (c) a transmembrane domain; (d) an intracellular signaling domain; and (e) an optional intracellular co- stimulatory signaling domain.
  • the extracellular antigen binding domain of the CAR may be selected from the group consisting of a Fab, a Fab’, a (Fab’)2, an Fv, an scFv, an sdAb, and a peptide ligand specifically binding to the target antigen, or a combination thereof.
  • the extracellular antigen binding domain can be monospecific or multispecific.
  • the recombinant virus comprises a third nucleic acid (e.g., on transfer vector) encoding a CAR.
  • a third nucleic acid e.g., on transfer vector
  • Many CARs targeting different tumor antigens known in the field can be used herein, such as CAR targeting CD 19 and CD20.
  • the third nucleic acid encodes for two CARs (e.g., CD20+CD19 dual CAR), either expressed on the same vector or on different vectors (e.g., a first unit of a third nucleic acid encoding a first CAR is on one vector, a second unit of a third nucleic acid encoding a second CAR is on another vector).
  • a first unit of a third nucleic acid encoding a first CAR and a second unit of a third nucleic acid encoding a second CAR are carried on a single vector, either under the control of a single promoter or different promoters.
  • the first unit of the third nucleic acid encoding the first CAR and the second unit of the third nucleic acid encoding the second CAR are carried on a single vector and under the control of a single promoter, wherein the first unit of the third nucleic acid and the second unit of the third nucleic acid are connected via a linking sequence (e.g., IRES, or encoding a cleavable linker such as 2A peptide).
  • a linking sequence e.g., IRES, or encoding a cleavable linker such as 2A peptide
  • the third nucleic acid encodes for a single CAR (e.g., CD20+CD 19 tandem CAR).
  • CARs that can be used herein include, but are not limited to, those described in WO2013123061, W02016100232, W02019028051, and WO2019126724, the contents of each of which are incorporated herein by reference in their entirety.
  • the extracellular antigen binding domain comprises an antibody moiety specifically recognizing a target epitope or antigen.
  • the extracellular antigen binding domain comprises two or more antibody moieties specifically recognizing one or more target epitopes or antigens, such as connected in tandem.
  • the two or more antibody moieties can be the same or different.
  • the two or more antibody moieties can recognize the same epitope or different epitopes.
  • the extracellular antigen binding domain recognizes a pathogen-specific antigen, such as a viral antigen, a bacterial antigen, or a parasitic antigen.
  • the extracellular antigen binding domain recognizes an antigen associated with an autoimmune or inflammatory disease.
  • the extracellular antigen binding domain recognizes a tumor associated antigen (e.g., cancer-associated antigen, or cancer-specific antigen), including but not limited to GPC2, GPRC5D, CD7, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD70, CD123, CD228, CD 138, BCMA, AFP, GPC3, CEA, Claudin 18.2, CLL1, CD33, folate receptor (FRa), mesothelin (MSLN), DLL3, CD276, gplOO, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, NKG2D ligand, ULBP, and HER-2.
  • a tumor associated antigen e.g., cancer-associated antigen, or cancer-specific antigen
  • Cancer-associated antigen can include any molecule (e.g., peptide) that can be found in some healthy cells (e.g., at low levels or during a specific developmental stage or stage of cellular differentiation) and that is perturbed in cancer cells, for example the overexpression of a specific peptide.
  • Cancer-specific antigen can be a molecule (e.g., peptide) that is only found in tumor cells and is not present in normal cells.
  • the extracellular antigen binding domain specifically recognizes CD 19 and/or CD20.
  • the extracellular antigen binding domain comprises an anti-CD19 antibody moiety (e.g., sdAb or scFv) and an anti-CD20 antibody moiety (e.g., sdAb or scFv) connected in tandem.
  • an anti-CD19 antibody moiety e.g., sdAb or scFv
  • an anti-CD20 antibody moiety e.g., sdAb or scFv
  • Anti-CD19 antibody moi eties can be derived from any anti-CD19 antibody clone, including not limited to, HIB19 (e.g., Invitrogen), 6OMP31 (e.g., Invitrogen), SJ25C1 (e.g., Invitrogen), LC1 (e.g., Invitrogen), 4G7 (e.g., Invitrogen), LE-CD19 (e.g., Invitrogen), SP110 (e.g., Invitrogen), 1C10A1 (e.g., Proteintech), OTI2F6 (e.g., OriGene), 2E2B6B10 (e.g., Invitrogen), LT19 (e.g., Invitrogen), 2E2 (e.g., Invitrogen), HD37 (e.g., Invitrogen), UMAB103 (e.g., OriGene), OTI2B11 (e.g., OriGene), OTI
  • Anti-CD20 antibody moieties can be derived from any anti-CD20 antibody clone, including not limited to, 2H7 (e.g, Invitrogen), L26 (e.g, Invitrogen), SP32 (e.g, Invitrogen), B9E9 (e.g, Invitrogen), HI47 (e.g, Invitrogen), LT20 (e.g, Invitrogen), 4A7G3 (e.g.
  • OTI4B4 e.g, OriGene
  • UMAB37 e.g, OriGene
  • UMAB38 e.g, OriGene
  • UMAB39 e.g, OriGene
  • 3E9D3C1G3 e.g, Invitrogen
  • MEM-97 e.g, Invitrogen
  • FMC7 e.g, Invitrogen
  • MS4A1/3409 e.g, NeoBiotechnologies
  • MS4A1/3410 e.g. NeoBiotechnologies
  • MS4A1/3411 e.g., NeoBiotechnologies
  • MS4A1/4655 e.g.,
  • NeoBiotechnologies e.g., NeoBiotechnologies
  • IGEL/773 e.g., NeoBiotechnologies
  • SPM618 e.g.,
  • NeoBiotechnologies OTI11F7 (e.g., OriGene), 0TI1H4 (e.g., OriGene), OTI3C4 (e.g., OriGene), OTI2D3 (e.g., OriGene), OTI10A5 (e.g., OriGene), OTI2C11 (e.g., OriGene), OTI1C12 (e.g., OriGene), 743X69 (e.g., OriGene), 743X78 (e.g., OriGene), 743AB30 (e.g., OriGene), 743Y4 (e.g., OriGene), 743X56 (e.g., OriGene), 743X65 (e.g., OriGene), 743X45 (e.g., OriGene), 743AB71 (e.g., OriGene), 13.6E12 (e.g., AbboMax), ZY
  • the transmembrane domain that can be directly or indirectly fused to the extracellular antigen binding domain.
  • the transmembrane domain may be derived either from a natural or from a synthetic source.
  • the transmembrane domain of the CAR is derived from a molecule selected from the group consisting of: an a, P or C, chain of a TCR, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, 4- 1BB (CD 137), CD 154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD 11 a, CD 18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 160, Claudin-6, IL-2RP, IL-2Ry, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD l id, ITGAE, CD 103, ITGAL
  • the CAR of the present disclosure may comprise a hinge domain that is located between the extracellular antigen binding domain and the transmembrane domain.
  • a hinge domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen binding domain relative to the transmembrane domain of the effector molecule can be used.
  • the hinge domain can be a peptide linker, or a hinge domain of antibodies, such as an IgG, IgA, IgM, IgE, or IgD.
  • the hinge domain can be a hinge domain of a naturally occurring protein (e.g., derived from CD8a), or a non-naturally occurring peptide.
  • the hinge domain between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain is a peptide linker, such as a (GGGGS)n linker (e.g., SEQ ID NO: 43), wherein n can be an integer of at least 1, e.g., 1, 2, 3, 4, or more; or a (GxS)n linker (SEQ ID NO: 126), wherein x and n, independently can be an integer of at least 1, such as between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (e.g., SEQ ID NO: 44).
  • the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 43-45.
  • the CAR comprises an intracellular signaling domain consisting essentially of (or consisting of) a primary intracellular signaling domain of an immune effector cell.
  • Primary intracellular signaling domain refers to intracellular signaling sequence that acts in a stimulatory manner to induce immune effector functions.
  • the primary intracellular signaling domain contains a signaling motif known as immunoreceptor tyrosine-based activation motif, or ITAM.
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITAMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule.
  • ITAMs may also function as docking sites for other proteins involved in signaling pathways.
  • Exemplary ITAM-containing primary intracellular signaling sequences include those derived from CD3( ⁇ , FcsRip, FcsRIy, CD3y, CD35, CD3s, DAP12, CNAIP/NFAM1, STAM-1, STAM-2, Moesin, CD5, CD22, CD79a, CD79b, and CD66d (CEACAM3).
  • the intracellular signaling domain of the CAR comprises (or consists essentially of, or consists of) an CD3( ⁇ intracellular signaling domain.
  • the CAR comprises at least one intracellular co-stimulatory signaling domain.
  • intracellular co-stimulatory signaling domain refers to at least a portion of a protein that mediates a secondary or co-stimulatory signal transduction within a cell to induce an immune response such as an effector function.
  • the intracellular co-stimulatory signaling domain can act in an antigen-independent manner to provide a secondary or co-stimulatory signal to immune cells.
  • the intracellular co-stimulatory signaling domain of the CAR can be an intracellular signaling domain from a co-stimulatory protein, which transduces a secondary or co-stimulatory signal and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils.
  • co-stimulatory molecule refers to a cognate binding partner on an immune cell (such as T cell) that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the immune cell, such as, but not limited to, proliferation and survival.
  • Activation of an intracellular co-stimulatory signaling domain in a host cell e.g.
  • an immune cell may induce the cell to increase or decrease the production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival, and/or cytotoxicity.
  • the intracellular co-stimulatory signaling domain of any co- stimulatory molecule may be compatible for use in the CAR described herein.
  • the type(s) of intracellular co-stimulatory signaling domain is selected based on factors such as the type of the immune effector cells in which the CAR would be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effect).
  • the co-stimulatory molecule is selected from the group consisting of CD27, CD28, CD137, 0X40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP 10, DAP 12, CD83, and ligands of CD83.
  • the CAR comprises an intracellular co-stimulatory signaling domain derived from 4- IBB.
  • the CAR of the present disclosure may further comprise a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide.
  • signal peptides are peptide sequences that target a polypeptide to the desired site in a cell.
  • the signal peptide targets the engineered receptor to the secretory pathway of the cell and will allow for integration and anchoring of the CAR into the lipid bilayer. During or following translocation of the polypeptide to the cell membrane, the signal peptide may be cleaved.
  • the signal peptide is derived from a molecule selected from the group consisting of CD8a, GM-CSF receptor a, and IgGl heavy chain.
  • the signal peptide is derived from a CD8a propeptide, e.g., human CD8a.
  • the CAR may comprise one or more peptide linkers, such as between different components of the engineered receptor (e.g., between two or more intracellular co-stimulatory signaling domains, between intracellular co-stimulatory signaling domain and primary intracellular signaling domain, or between the extracellular antigen binding domain and the transmembrane domain), and/or within one engineered receptor component (e.g., extracellular antigen binding domain, such as within an scFv, or for connecting two or more antibody moieties in tandem).
  • the engineered receptor e.g., between two or more intracellular co-stimulatory signaling domains, between intracellular co-stimulatory signaling domain and primary intracellular signaling domain, or between the extracellular antigen binding domain and the transmembrane domain
  • extracellular antigen binding domain such as within an scFv, or for connecting two or more antibody moieties in tandem
  • Each peptide linker in a CAR may have the same or different length and/or sequence depending on the structural and/or functional features of the antibody moieties and/or the various domains. Each peptide linker may be selected and optimized independently. The length, the degree of flexibility and/or other properties of the peptide linker(s) used in the engineered receptors may have some influence on properties, including but not limited to the affinity, specificity or avidity for one or more particular antigens or epitopes.
  • the peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO 1996/34103.
  • the peptide linker is a flexible linker.
  • exemplary flexible linkers include but not limited to glycine polymers (G)n, glycineserine polymers, glycine-alanine polymers, alanine-serine polymers, threonine-serine, and other flexible linkers known in the art.
  • Other linkers known in the art for example, as described in WO2016014789, WO2015158671, WO2016102965, US20150299317, WO2018067992, US7741465, Colcher et al., J. Nat. Cancer Inst. 82: 1191-1197 (1990), and Bird et al., Science 242:423-426 (1988) may also be included in the chimeric receptors provided herein, the disclosure of each of which is incorporated herein by reference in their entirety.
  • the peptide linker is (GGGGS)n (SEQ ID NO: 43), wherein n is an integer of at least 1 (e.g., 1, 2, 3, 4, or more).
  • the peptide linker is (GxS)n (SEQ ID NO: 126), wherein x and n independently can be an integer of at least 1, such as between 3 and 12 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).
  • the (GxS)n linker comprises the amino acid sequence of SEQ ID NO: 44.
  • the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 43-45.
  • the engineered receptor is an engineered TCR.
  • the engineered TCR may comprise i) an extracellular antigen binding domain that comprises a Va and a VP (or a V5 and a Vy) derived from a wildtype TCR together specifically recognizing a target antigen, wherein the Va, the VP, or both (or the V5, the Vy, or both), comprise one or more mutations (e.g., insertions, deletions, or substitutions, such as conservative substitutions) in one or more CDRs relative to the wild type TCR; and ii) a transmembrane domain derived from a TCR molecule (e.g., TCRa/TCRp, or TCRS/TCRy).
  • the engineered TCR can be a single chain TCR (scTCR) or a dimeric TCR (dTCR).
  • the engineered TCR may bind to the same cognate peptide-MHC bound by the wildtype TCR.
  • the engineered TCR may bind to the same cognate peptide-MHC with higher affinity compared to that bound by the wildtype TCR.
  • the engineered TCR may bind to the same cognate peptide-MHC with lower affinity compared to that bound by the wildtype TCR.
  • the engineered TCR may bind to a non-cognate peptide- MHC not bound by the wildtype TCR.
  • the engineered TCR may not comprise an intracellular signaling domain (e.g., the cytoplasmic domain of TCRa, TCRP, TCR5, or TCRy).
  • the engineered TCR may further comprise a hinge domain (or a connecting domain) between the TCR Ig-like constant domain and the TCR transmembrane domain, such as a hinge domain derived from TCRa/TCRp or TCRS/TCRy.
  • the engineered receptor is a chimeric T cell receptor (cTCR).
  • the cTCR may comprise: i) an extracellular antigen binding domain comprising an antibody or antigen-binding fragment thereof (e.g., scFv, Fab, or sdAb) that specifically recognizes a target antigen, and ii) a full-length TCR subunit, wherein the TCR subunit is selected from the group consisting of TCRa, TCRP, TCRy, TCR6, CD3y, CD3s, and CD36; wherein the extracellular antigen binding domain is fused (directly or indirectly) to the N-terminus of the full-length TCR subunit.
  • an extracellular antigen binding domain comprising an antibody or antigen-binding fragment thereof (e.g., scFv, Fab, or sdAb) that specifically recognizes a target antigen
  • a full-length TCR subunit wherein the TCR subunit is selected from the group consisting of TCRa,
  • the cTCR may comprise an optional extracellular domain (ECD) or portion thereof derived from a TCR subunit.
  • ECD extracellular domain
  • the cTCR can be incorporated into a functional TCR complex along with other endogenous TCR subunits and confer antigen specificity to the TCR complex.
  • the extracellular antigen binding domain of the cTCR may be selected from the group consisting of a Fab, a Fab’, a (Fab’)2, an Fv, an scFv, and an sdAb.
  • the cTCR extracellular antigen binding domain may be fused to the N-terminus of the full-length or a portion thereof of CD3s, CD3y, or CD35.
  • the cTCR extracellular antigen binding domain may be fused to the N-terminus of a TCRa molecule (with or without the Va domain), and/or the N-terminus of a TCRP molecule (with or without the VP domain).
  • the cTCR extracellular antigen binding domain may be fused to the N-terminus of a TCRy molecule (with or without the Vy domain), and/or the N-terminus of a TCR5 molecule (with or without the V5 domain).
  • the cTCR may or may not comprise an intracellular signaling domain.
  • the cTCR may comprise an intracellular signaling domain, such as the intracellular signaling domain of CD3y, CD3s, or CD35.
  • the cTCR intracellular signaling domain and the cTCR transmembrane domain can be derived from the same TCR subunit, e.g., both from CD3s, both from CD3s, or both from CD35.
  • the cTCR extracellular antigen binding domain and the TCR subunit can be fused via a linker (such as a GS linker).
  • the extracellular antigen binding domain is fused to the N-terminus of the transmembrane domain via an optional linker or hinge domain.
  • the cTCR may further comprise a hinge domain between the ECD portion derived from a first TCR subunit and a transmembrane domain derived from a second TCR subunit.
  • the cTCR comprises from N-terminus to C-terminus: (a) an extracellular antigen binding domain comprising an antibody or antigen-binding fragment thereof (e.g., scFv, Fab, or sdAb) that specifically recognizes a target antigen; (b) an optional linker, (c) an optional extracellular domain of a first TCR subunit or a portion thereof, (d) an optional hinge domain, (e) a transmembrane domain derived from a second TCR subunit, and (f) an optional cytoplasmic domain (e.g., optional intracellular signaling domain); wherein the first and second TCR subunits are independently selected from the group consisting of TCRa, TCRP, TCRy, TCR5, CD3s, CD3y, and CD35.
  • the first and second TCR subunits can be the same or different.
  • TAC T cell antisen coupler
  • the engineered receptor is a T cell antigen coupler (TAC) comprising: (a) an extracellular antigen binding domain comprising an antigen-binding fragment (e.g., scFv, sdAb) that specifically recognizes a target antigen; (b) an optional first linker; (c) an extracellular TCR binding domain that specifically recognizes the extracellular domain of a TCR subunit (e.g., CD3s); (d) an optional second linker; (e) an optional extracellular domain derived from a first TCR co-receptor (such as CD4, CD28, or CD8); (f) a transmembrane comprising a transmembrane derived from a second TCR co-receptor (such as CD4, CD28, or CD8); and (g) an optional intracellular signaling domain comprising an intracellular signaling domain derived from a third TCR co-receptor (such as CD4, CD28, or CD8).
  • TAC T cell antigen coupler
  • the first, second, and third TCR co-receptors are the same (e.g., all CD4). In some embodiments, the first, second, and third TCR co-receptors are different.
  • the TAC comprises: (a) an extracellular antigen binding domain comprising an antigen-binding fragment (e.g., scFv, sdAb) that specifically recognizes one or more epitopes of a tumor antigen; (b) an optional first linker; (c) an extracellular TCR binding domain that specifically recognizes the extracellular domain of a TCR subunit (e.g., CD3s); (d) an optional second linker; and (e) a full length TCR co-receptor (e.g., CD4, CD8, or CD28).
  • an extracellular antigen binding domain comprising an antigen-binding fragment (e.g., scFv, sdAb) that specifically recognizes one or more epitopes of a tumor antigen
  • an optional first linker
  • the engineered receptor is a TAC-like engineered receptor comprising: (a) an extracellular antigen binding domain comprising an antigen-binding fragment (e.g., scFv, sdAb) that specifically recognizes one or more epitopes of a target antigen; (b) an optional first linker; (c) an extracellular TCR binding domain that specifically recognizes the extracellular domain of a first TCR subunit; (d) an optional second linker; (e) an optional extracellular domain derived from a second TCR subunit or a portion thereof; (f) a transmembrane domain comprising a transmembrane domain derived from a third TCR subunit; and (g) an optional intracellular signaling domain comprising an intracellular signaling domain derived from a fourth TCR subunit; wherein the first, second, third, and fourth TCR subunits are all selected from the group consisting of TCRa, TCRP, TCRy, TCR5, CD3s,
  • the first, second, third, and fourth TCR subunits are the same. In some embodiments, the second, third, and fourth TCR subunits are the same. In some embodiments, the first, second, third, and fourth TCR subunits are different. In some embodiments, the second, third, and fourth TCR subunits are the same but different from the first TCR subunit.
  • the TAC-like chimeric receptor comprises: (a) an extracellular antigen binding domain comprising an antigen-binding fragment (e.g., scFv, sdAb) that specifically recognizes one or more epitopes of a target antigen; (b) an optional first linker; (c) an extracellular TCR binding domain that specifically recognizes the extracellular domain of a first TCR subunit; (d) an optional second linker; and (e) a full length second TCR subunit; wherein the first and second TCR subunits are both selected from the group consisting of TCRa, TCRP, TCRy, TCR5, CD3s, CD3y, and CD35.
  • the first and second TCR subunits are the same. In some embodiments, the first and second TCR subunits are different.
  • engineered receptors can be delivered in conjunction with the recombinant viruses described herein.
  • engineered receptors e.g., CAR, TCR, cTCR, TAC, and TAC-like engineered receptors
  • any other engineered receptor construct could be encoded for by the third nucleic acid disclosed herein.
  • C. Nucleic acids and vectors encoding the glycoprotein variant, envelope surfacebound targeting molecule, and heterologous molecule
  • the present disclosure provides nucleic acids and vectors for cloning, engineering, or producing any of the viral glycoprotein variants and recombinant viruses (e.g., lentivirus) described herein.
  • the two or more components necessary for producing a recombinant virus herein is on a single vector.
  • the two or more components (including the viral glycoprotein variant) necessary for producing a recombinant virus herein is split across two or more vectors, such as transfer vector, packaging vector(s), and envelope vector(s), in order to increase the safety and avoid generation of replication-competent recombinant virus, so that viral replication can proceed only when all necessary components are co-transfected into a cell line (e.g., a producer cell).
  • the transfer vector is replication incompetent and may contain a deletion in the 3 ’ LTR (e.g., removing the tat gene), rendering the recombinant virus “self-inactivating” (SIN) after integration.
  • a vector comprising a first nucleic acid encoding a viral glycoprotein variant, such as any of the viral glycoprotein variants described herein (e.g., any of the viral glycoprotein variants with reduced binding to LDL-R and comprising one or more deletions at amino acid positions equivalent to 141-158 and 271-292, or one or more substitutions at any of amino acid positions equivalent to 47, 143-148, 150-156, 158, 182, 209, 272, 278, 291 , 331 and 354 in reference to any of SEQ ID NOs: 1, 2, 39, 40, and 46, such as any of SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, 62-82, 86, 88-91, 96-104, 111-112, and 118-125).
  • a viral glycoprotein variant such as any of the viral glycoprotein variants described herein (e.g., any of the viral glycoprotein variants with reduced binding to LDL-R and comprising one or more deletions at amino acid
  • a vector comprising a second nucleic acid encoding an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on a target cell, such as envelope surface-bound anti-CD3 scFv.
  • the first nucleic acid encoding the viral glycoprotein variant and the second nucleic acid encoding the envelope surface-bound targeting molecule are present on two separate vectors (e.g., two envelope vectors).
  • the first nucleic acid encoding the viral glycoprotein variant and the second nucleic acid encoding the envelope surface-bound targeting molecule are on the same vector (e.g., envelope vector).
  • the first nucleic acid and the second nucleic acid can be under the control of a same promoter or different promoters, and the first nucleic acid can be upstream or downstream of the second nucleic acid.
  • the first nucleic acid and the second nucleic acid are under the control of a same promoter, and the first nucleic acid and second nucleic acid are connected to each other via a linking nucleic acid, such as IRES, or a nucleic acid sequence encoding a cleavable linker, such as a 2A peptide.
  • the promoter is a CMV promoter.
  • the 2A peptide is P2A, T2A, E2A, or F2A.
  • the 2A peptide is P2A.
  • a vector comprising, from 5’ to 3’ : (a) a promoter (e.g., CMV); (b) a first nucleic acid encoding a viral glycoprotein variant (e.g., any of the viral glycoprotein variants described herein, such as SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, 62-82, 86, 88-91, 96-104, 111-112, and 118-125); (c) a linking nucleic acid (e.g., IRES, or encoding a 2A peptide such as P2A); and (d) a second nucleic acid encoding an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv).
  • a promoter e.g., CMV
  • a first nucleic acid encoding a viral glycoprotein variant (e.g., any of the viral glycoprotein variants described herein, such as SEQ ID NOs: 4, 5, 8
  • a vector comprising, from 5’ to 3’: (a) a first promoter; (b) a first nucleic acid encoding a viral glycoprotein variant (e.g., any of the viral glycoprotein variants described herein (, such as SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, 62-82, 86, 88-91, 96-104, 111-112, and 118-125); (c) a second promoter; and (d) a second nucleic acid encoding an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv).
  • a viral glycoprotein variant e.g., any of the viral glycoprotein variants described herein (, such as SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, 62-82, 86, 88-91, 96-104, 111-112, and 118-125
  • a second promoter e.g., anti-CD3
  • a vector comprising, from 5’ to 3’: (a) a promoter (e.g., CMV); (b) a second nucleic acid encoding an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv); (c) a linking nucleic acid (e.g., IRES, or encoding a 2A peptide such as P2A); and (d) a first nucleic acid encoding a viral glycoprotein variant (e.g., any of the viral glycoprotein variants described herein, such as SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, 62-82, 86, 88-91, 96-104, 111-112, and 118-125).
  • a promoter e.g., CMV
  • a second nucleic acid encoding an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv)
  • a linking nucleic acid e.
  • a vector comprising, from 5’ to 3’: (a) a first promoter; (b) a second nucleic acid encoding an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv); (c) a second promoter; and (d) a first nucleic acid encoding a viral glycoprotein variant (e.g., any of the viral glycoprotein variants described herein, such as SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, 62-82, 86, 88-91, 96-104, 111-112, and 118- 125).
  • the first promoter can be the same or different from the second promoter.
  • a third nucleic acid encoding a heterologous molecule (e.g., heterologous protein or RNA).
  • the third nucleic acid encodes a heterologous protein, such as an engineered receptor (e.g., CAR).
  • the first, second, and third nucleic acids are on three vectors.
  • the first nucleic acid is on the same vector as the second nucleic acid (e.g., envelope vector), while the third nucleic acid is on a separate vector (e.g., transfer vector).
  • the third nucleic acid encoding the heterologous molecule comprises long terminal repeat (LTR) sequences flanking the transgene to enable integration into the target cell genome.
  • LTR long terminal repeat
  • the LTR (5’ LTR and/or 3’ LTR) is mutated (e.g., truncated, such as truncating U3) to generate self-inactivating viral vector.
  • the promoter is selected from the group consisting of a Rous Sarcoma Virus (RSV) promoter, a Simian Virus 40 (SV40) promoter, a cytomegalovirus immediate early gene promoter (CMV IE), a murine stem cell virus promoter (MCSV), an elongation factor 1 alpha promoter (EFl -a), a phosphoglycerate kinase- 1 (PGK) promoter, a ubiquitin-C (UBQ-C) promoter, a cytomegalovirus enhancer/chicken beta-actin (CAG) promoter, a polyoma enhancer/herpes simplex thymidine kinase (MCI) promoter, a beta actin (P-ACT) promoter, a “myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND)” promoter, an RSV Virus 40 (SV40
  • the transfer vector (e.g., carrying the third nucleic acid) can employ a promoter same as or different from the envelope vector (e.g., carrying the first and/or second nucleic acid) and/or packaging vector(s). See exemplary transfer vector promoters in Table C.
  • the third nucleic acid encoding the heterologous molecule e.g., heterologous protein or RNA
  • therapeutic protein e.g., an engineered receptor
  • a constitutive promoter e.g., constitutive promoters
  • constitutive promoters allow heterologous genes (also referred to as transgenes) to be expressed constitutively in the transduced target cells.
  • Exemplary promoters contemplated herein include, but are not limited to, CMV, hEFla, ubiquitin C promoter (UbiC), PGK, SV40, chicken P- Actin promoter coupled with CMV early enhancer (CAGG), an RSV promoter, a polyoma enhancer/herpes simplex thymidine kinase (MCI) promoter, a P-ACT promoter, and an MND promoter.
  • CMV CMV
  • hEFla ubiquitin C promoter
  • PGK ubiquitin C promoter
  • SV40 chicken P- Actin promoter coupled with CMV early enhancer (CAGG)
  • CAGG CMV early enhancer
  • RSV promoter CMV early enhancer
  • MCI polyoma enhancer/herpes simplex thymidine kinase
  • P-ACT P-ACT promoter
  • MND promoter MND promoter
  • hEFla promoter not only induced the highest level of transgene expression, but was also optimally maintained in the CD4 and CD8 human T cells (Molecular Therapy, 17(8): 1453- 1464 (2009)).
  • the third nucleic acid encoding the heterologous molecule e.g., heterologous protein or RNA
  • therapeutic protein e.g., an engineered receptor
  • an inducible promoter belongs to the category of regulated promoters.
  • the inducible promoter can be induced by one or more conditions, such as a physical condition, microenvironment of the transduced target cell (e.g., T cell), or the physiological state of the transduced target cell, an inducer (z.e., an inducing agent), or a combination thereof.
  • the inducing condition does not induce the expression of endogenous genes in the transduced target cell, and/or in the individual that receives the pharmaceutical composition.
  • the inducing condition is selected from the group consisting of: inducer, irradiation (such as ionizing radiation, light), temperature (such as heat), redox state, tumor environment, and the activation state of the transduced target cell.
  • the vector(s) also contains a selectable marker gene or a reporter gene to select cells expressing the heterologous molecule (e.g., heterologous protein or RNA) described herein, e.g., therapeutic protein (e.g., an engineered receptor) from the population of target cells transfected through recombinant viruses (e.g., pseudotyped LVVs).
  • a selectable marker gene or a reporter gene to select cells expressing the heterologous molecule (e.g., heterologous protein or RNA) described herein, e.g., therapeutic protein (e.g., an engineered receptor) from the population of target cells transfected through recombinant viruses (e.g., pseudotyped LVVs).
  • exemplary selectable markers can include, but are not limited to, peptide protein tags (e.g., FLAG-tag), fluorescent tags, and antibiotic resistance cassettes. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences to enable expression in the target cells.
  • the vector may contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid sequences.
  • Genes necessary to produce a recombinant virus can be split across two or more vectors to increase recombinant virus safety.
  • Recombinant virus is typically produced using an envelope vector encoding a viral glycoprotein, packaging vector(s) encoding viral structural proteins, and a transfer vector encoding the transgene of interest.
  • the genes required produce a recombinant virus include gag, pol, rev and optionally tat (depending on the vectors used). gag encodes for viral structural proteins, pol encodes for enzymes involved in reverse transcription and genome integration, while rev and tat encode for gene regulatory proteins.
  • the transfer plasmid comprises a 5’ LTR fused to a heterologous promoter
  • Tat is no longer required, as expression of the transgene is Tat-independent.
  • Promoters may be chosen for each vector based on the desired application, such as depending on the cell type to be transduced (e.g., primary cells vs. cell lines, see Table C), or the gene to be expressed (e.g., a dual promoter for co-expressing two transgenes).
  • the envelope vector may encode a viral glycoprotein, such as any of the viral glycoprotein variants described herein (e.g., any of the viral glycoprotein variants with reduced binding to LDL-R and comprising one or more deletions at amino acid positions equivalent to 141-158 and 271-292, or one or more substitutions at any of amino acid positions equivalent to 143-148, 150-156, 158, 182, 209, 272, 278, and 291 in reference to any of SEQ ID NOs: 1, 2, 39, 40, and 46, such as any of SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, 62-82, 86, 88- 91, 96-104, 111-112, and 118-125).
  • a viral glycoprotein such as any of the viral glycoprotein variants described herein (e.g., any of the viral glycoprotein variants with reduced binding to LDL-R and comprising one or more deletions at amino acid positions equivalent to 141-158 and 271-292, or one or more substitutions at
  • the envelope vector may comprise a first nucleic acid encoding a viral glycoprotein or variant thereof (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G variant).
  • the envelope vector may further comprise a second nucleic acid encoding an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on a target cell (e.g., anti-CD3 scFv).
  • the first and second nucleic acids are under control of a single promoter, and the first and second nucleic acids are connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A).
  • a linking nucleic acid sequence such as IRES
  • a linking nucleic acid encoding a cleavable linker e.g., 2A peptide, such as P2A, T2A.
  • the first and second nucleic acids are under control of different promoters.
  • the first and second nucleic acids are on separate envelope vectors.
  • Packaging vector(s) can encode one or more packaging proteins to produce any of the recombinant viruses described herein.
  • at least one packaging vector is used to produce the recombinant virus.
  • the single packaging vector may contain nucleic acid encoding packaging proteins Gag, Pol, Rev, and Tat.
  • the packaging vector comprises a gag gene, a pol gene, a tat gene, and a rev gene.
  • two or more packaging vectors are used to produce Gag, Pol, Rev. For example, one packaging vector comprises gag and pol genes, another packaging vector comprises rev.
  • the gag gene, pol gene, and Rev response element are on one vector, and the rev gene is on another vector.
  • One or more nucleic acids encoding the packaging proteins can be under the control of a single promoter (e.g., packaging genes on a same vector) or different promoters (e.g., packaging genes on a same vector or different vectors).
  • the one or more nucleic acids encoding the packaging proteins may be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A).
  • a linking nucleic acid sequence such as IRES
  • a linking nucleic acid encoding a cleavable linker e.g., 2A peptide, such as P2A, T2A.
  • the transfer vector comprises the heterologous nucleic acid to be delivered and integrated into the genome of the transduced target cell (e.g., T cell).
  • the transfer vector may comprise a chimeric 5’ LTR, a heterologous nucleic acid of interest (e.g., encoding for a heterologous molecule, such as an engineered receptor), and a 3’ LTR.
  • the chimeric 5’ LTR may comprise a portion of the 5’ LTR fused to a promoter. Suitable promoters can include promoters derived from CMV, MSCV, EFl -a, PGK, RSV, and UbC.
  • the transfer vector comprises a chimeric 5’ LTR
  • the 3’ LTR may be deleted or truncated to remove the tat gene.
  • a recombinant virus comprising any of the viral glycoprotein variants described herein (e.g., any of the viral glycoprotein variants with reduced binding to LDL-R and comprising one or more deletions at amino acid positions equivalent to 141-158 and 271-292, or one or more substitutions at any of amino acid positions equivalent to 47, 143-148, 150-156, 158, 182, 209, 272, 278, 291, 331 and 354 in reference to any of SEQ ID NOs: 1, 2, 39, 40, and 46, such as any of SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, 62-82, 86, 88-91, 96-104, 111-112, and 118-125), the method comprising introducing a first nucleic acid encoding the viral glycoprotein variant and one or more packaging vectors comprising one or more nucleic acids encoding one or more packaging proteins into a
  • the method further comprises introducing a second nucleic acid encoding any of the envelope surface-bound targeting molecules described herein (e.g., anti-CD3 scFv).
  • the first nucleic acid and the second nucleic acid are on the same vector (e.g., envelope vector).
  • the first nucleic acid and the second nucleic acid are on different vectors.
  • the one or more packaging vectors, the first nucleic acid, and/or the second nucleic acid are introduced into the producer cell simultaneously or sequentially, in any order.
  • the method further comprises isolating the enveloped (e.g., pseudotyped) recombinant virus.
  • the methods further comprise transfecting or transducing a producer cell line with a third nucleic acid encoding a heterologous molecule (e.g., heterologous protein or RNA, such as therapeutic protein or RNA) that is to be delivered to the target cell.
  • a heterologous molecule e.g., heterologous protein or RNA, such as therapeutic protein or RNA
  • the third nucleic acid encodes an engineered receptor (e.g., a CAR).
  • the third nucleic acid is on a different vector from the first and/or second nucleic acids.
  • the one or more vectors e.g., envelope vector
  • the vector e.g., transfer vector
  • the third nucleic acid are introduced into a producer cell simultaneously.
  • the one or more vectors comprising the first and second nucleic acids and the vector comprising the third nucleic acid are introduced into a producer cell sequentially, in any order.
  • one or more packaging vectors can be introduced into the producer cell simultaneously or sequentially (in any order) with the first, second, and/or third nucleic acids.
  • the first nucleic acid encoding the viral glycoprotein variant and the second nucleic acid encoding the envelope surface-bound targeting molecule are on the same vector, while the third nucleic acid encoding the heterologous molecule (e.g., heterologous protein or RNA) is on a different vector.
  • the vector comprising the first and second nucleic acids can be introduced into a producer cell with the vector comprising the third nucleic acid simultaneously, or sequentially, in any order.
  • one or more packaging vectors can be introduced into the producer cell simultaneously or sequentially (in any order) with the first, second, and/or third nucleic acids.
  • the recombinant virus provided for herein can be produced or made by, for example, culturing the producer cell under conditions sufficient to make the recombinant virus.
  • the producer cell already comprises one or more nucleic acids that encode the components to produce the recombinant virus. These can be structural or non- structural viral components or proteins.
  • Producer cell lines are known in the art and can be modified with the molecules of interest to produce the recombinant virus of interest. Any suitable producer cell line known in the art can be used, such as HEK293, 293 T, HeLa, D-17, MDCK, BHK and Cf2Th cells. In some embodiments, the producer cell line is 293T.
  • an engineered cell e.g., engineered CAR-T cell
  • a target cell e.g., T cell
  • a recombinant virus comprises a heterologous nucleic acid (e.g., encoding an engineered receptor, such as CAR) to be delivered into the target cell and any of the viral glycoprotein variants described herein (e.g., any of the viral glycoprotein variants with reduced binding to LDL-R and comprising one or more deletions at amino acid positions equivalent to 141-158 and 271-292, or one or more substitutions at any of amino acid positions equivalent to 47, 143- 148, 150-156, 158, 182, 209, 272, 278, 291, 331 and 354 in reference to any of SEQ ID NOs: 1, 2, 39, 40, and 46, such as any of SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, 62-82,
  • the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on the target cell (e.g., anti-CD3 scFv).
  • a heterologous nucleic acid into a target cell comprising contacting the target cell with a recombinant virus, wherein the recombinant virus comprises the heterologous nucleic acid to be delivered into the target cell and any of the viral glycoprotein variants described herein (e.g., any of the viral glycoprotein variants with reduced binding to LDL-R and comprising one or more deletions at amino acid positions equivalent to 141-158 and 271-292, or one or more substitutions at any of amino acid positions equivalent to 47, 143-148, 150-156, 158, 182, 209, 272, 278, 291, 331 and 354 in reference to any of SEQ ID NOs: 1, 2, 39, 40, and 46, such as any of SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, 62-82, 86, 88-91, 96-104, 111-112, and 118-125).
  • the viral glycoprotein variants described herein e.g., any of the viral glycoprotein
  • the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on a target cell (e.g., anti-CD3 scFv).
  • a method of specifically delivering a heterologous nucleic acid (e.g., encoding a CAR) into a target cell (e.g., T cell) comprising contacting the target cell with a recombinant virus (e.g., lentivirus), wherein the recombinant virus comprises: i) the heterologous nucleic acid to be delivered into the target cell, ii) an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on the target cell (e.g., anti-CD3 scFv), and iii) a viral glycoprotein variant with reduced binding to LDL-R (e.g., any of the viral glycoprotein variants described herein, such as any of SEQ ID NOs: 4, 5, 8
  • the recombinant virus comprising the viral glycoprotein variant and envelope surface-bound targeting molecule has improved specificity and transduction efficiency for the target cell relative to a recombinant virus comprising a corresponding reference (e.g., wildtype) viral glycoprotein.
  • the envelope surfacebound targeting molecule activates the transduced target cell simultaneously with transduction.
  • a disease or disorder e.g., cancer, such as CD19 + /CD20 + cancer, or an autoimmune disease
  • an individual e.g., human
  • administering to the individual a therapeutically acceptable amount of any of the recombinant viruses described herein (e.g., comprising a third nucleic acid encoding an anti-CD20+CD19 CAR) or a pharmaceutical composition thereof.
  • the recombinant virus comprises a heterologous nucleic acid encoding a therapeutic protein (e.g., CAR) or RNA that treats the disease or disorder.
  • a method of treating a disease or disorder e.g., cancer, such as CD19 + /CD20 + cancer, or an autoimmune disease
  • a disease or disorder e.g., cancer, such as CD19 + /CD20 + cancer, or an autoimmune disease
  • an individual e.g., human
  • administering comprising administering to the individual an effective amount of a recombinant virus (e.g.
  • a lentivirus or a pharmaceutical composition comprising thereof, wherein the recombinant virus comprises a heterologous nucleic acid encoding a therapeutic protein (e.g., an engineered receptor, such as a CAR, e.g., anti-CD20+CD19 CAR) or RNA that treats the disease or disorder, wherein the recombinant virus is pseudotyped with a viral glycoprotein variant with reduced binding to LDL-R (e.g. any of the viral glycoprotein variants described herein, such as any of SEQ ID NOs: 4, 5, 8-10, 12-34, 47-52, 54-59, 62-82, 86, 88-91, 96-104, 111-112, and 118-125).
  • a therapeutic protein e.g., an engineered receptor, such as a CAR, e.g., anti-CD20+CD19 CAR
  • RNA that treats the disease or disorder
  • the recombinant virus is pseudotyped with a viral glycoprotein variant with
  • the recombinant virus further comprises an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) that specifically recognizes a cell surface protein on a target cell (e.g., T cell).
  • a target cell e.g., T cell
  • the target cell is the disease cell (e.g., cancer cell).
  • the target cell is an immune effector cell (e.g., T cell, NK cell).
  • a disease or disorder e.g., cancer, such as CD19 + /CD20 + cancer, or an autoimmune disease
  • the transduced cells are autologous cells (e.g., autologous CAR-T).
  • the transduced cells are allogeneic cells (e.g., allogeneic CAR-T).
  • the individual, to whom the transduced cells (e.g., CAR-T) or recombinant viruses are administered is a primate, such as a human, monkey, gorilla, chimpanzee, etc.
  • the individual is a human.
  • the individual can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric individuals.
  • the individual is an animal, including but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, chinchillas, cats, dogs, birds, horses, donkeys, cows, goats, sheep, deer, monkeys, apes, etc.
  • the individual is a livestock.
  • the individual is a companion animal.
  • the individual is a validated animal model for disease, cellular immunotherapy, and/or for assessing toxic outcomes.
  • the disease or disorder is an infectious disease, an autoimmune disease, an inflammatory disease, a respiratory disease, a neurological disease, a muscle disease, a cardiovascular disease, a gastrointestinal disease, a pulmonary disease, a metabolic disease, or a cancer.
  • the disease or disorder is a cancer.
  • the types of cancers to be treated include, but are not limited to, carcinoma, blastoma, sarcoma, certain leukemia or lymphoid malignancies, benign and malignant tumors, malignancies (e.g., sarcomas, carcinomas, and melanomas), breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, T cell related cancer or B cell related cancer.
  • the cancers may be non-solid tumors (such as hematological tumors) or solid tumors.
  • Adult tumors/cancers and pediatric tumors/cancers are also included.
  • the disease or disorder is a solid tumor.
  • Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas).
  • solid tumors such as sarcomas and carcinomas
  • solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular
  • the disease or disorder is a liquid tumor.
  • Liquid tumors are cancers that can affect the blood, bone marrow or lymphatic system. Examples of liquid tumors include leukemias, lymphomas, and multiple myelomas, and myeloproliferative neoplasms.
  • the disease or disorder is a B cell proliferative disease, such as B cell-related cancer.
  • the B cell proliferative disease is associated with increased expression of an antigen on the surface of a B cell, including but are not limited to, CD10, CD19, CD20, CD22, CD79b, and PD-L1.
  • the B cell related cancer is, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, low grade/follicular NHL, small lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, lymphocyte predominant Hodgkin's disease (LPHD), leukemia, hairy cell leukemia (HCL),
  • the disease or disorder is an autoimmune disease.
  • autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, diabetes (type I), bullous epidermal detachment, epididymitis, glomerulonephritis, Graves' disease, Guillain- Guillain-Barr syndrome, hypodermic disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatism fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren syndrome (Sjogren's syndrome), spinal arthritis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative
  • Addison's disease
  • the individual has not received prior treatment with another therapeutic agent prior to administration of the transduced cells (e.g., CAR-T) or recombinant virus described herein or pharmaceutical composition thereof.
  • the individual has been treated with a therapeutic agent targeting the disease or disorder (e.g., cancer or autoimmune disease) prior to administration of the transduced cells or recombinant virus described herein or pharmaceutical composition thereof.
  • the individual is refractory or non-responsive to the other therapeutic agent.
  • the individual has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT.
  • HSCT hematopoietic stem cell transplantation
  • the administration of the transduced cells or recombinant virus described herein or pharmaceutical composition thereof effectively treats the individual despite the individual having become resistant to another therapy.
  • the transduced cells or recombinant virus described herein or pharmaceutical composition thereof is administered to an individual having a disease or disorder before the administration of another treatment or therapeutic agent.
  • the transduced cells or recombinant virus described herein or pharmaceutical composition thereof is administered to an individual prophylactically to prevent a disease or disorder.
  • the appropriate dosage may depend on the type of disease to be treated, the severity and course of the disease, whether the transduced cells (e.g., CAR-T) or recombinant virus or pharmaceutical composition thereof is administered for preventive or therapeutic purposes, previous therapy, the individual’s clinical history and response to the treatment, and the discretion of the attending physician.
  • the recombinant virus or pharmaceutical composition thereof is suitably administered to the individual at one time or over a series of treatments.
  • the transduced cells e.g., CAR-T
  • the transduced cells e.g., CAR-T
  • recombinant virus described herein or pharmaceutical composition thereof is administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody, an engineered cell, or an agent, such as a cytotoxic or therapeutic agent.
  • another therapeutic intervention such as an antibody, an engineered cell, or an agent, such as a cytotoxic or therapeutic agent.
  • the transduced cells or recombinant virus described herein or pharmaceutical composition thereof is co-administered with another therapy sufficiently close in time such that the transduced cells recombinant virus described herein or pharmaceutical composition thereof enhances the effect of one or more additional therapeutic agents, or vice versa.
  • the transduced cells or recombinant virus described herein or pharmaceutical composition thereof is administered prior to the one or more additional therapeutic agents. In some embodiments, the transduced cells or recombinant virus described herein or pharmaceutical composition thereof is administered after the one or more additional therapeutic agents.
  • compositions comprising recombinant viruses
  • the transduced cells e.g., CAR-T
  • recombinant viruses described herein can comprise a pharmaceutical composition, and for example include a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising: i) any of the transduced cells or recombinant viruses described herein, and ii) optionally a pharmaceutically acceptable excipient.
  • excipient means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, water, saline, dextrose, glycerol, or the like and combinations thereof. The choice of excipient may be determined in part by the particular cell and/or by the method of administration. Accordingly, there are a variety of suitable compositions.
  • each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • Sterile injectable solutions can be prepared by incorporating the recombinant virus described herein or pharmaceutical composition thereof in a solvent, such as in a mixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.
  • a suitable carrier such as a suitable carrier, diluent, or excipient
  • the compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired.
  • auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired.
  • acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.
  • one or more pharmaceutically acceptable surface-active agents surfactant
  • buffers isotonicity agents
  • salts amino acids, sugars, stabilizers and/or antioxidant are used in the formulation.
  • Suitable pharmaceutically acceptable surfactants comprise but are not limited to polyethylen-sorbitan-fatty acid esters, polyethylene-polypropylene glycols, polyoxyethylenestearates and sodium dodecyl sulphates. Buffers may be used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Suitable buffers comprise but are not limited to histidine-buffers, citrate-buffers, succinate-buffers, acetate- buffers and phosphate-buffers.
  • Isotonicity agents are used to provide an isotonic formulation.
  • An isotonic formulation is liquid, or liquid reconstituted from a solid form, e.g. a lyophilized form and denotes a solution having the same tonicity as some other solution with which it is compared, such as physiologic salt solution and the blood serum.
  • Suitable isotonicity agents comprise but are not limited to salts, including but not limited to sodium chloride (NaCl) or potassium chloride, sugars including but not limited to glucose, sucrose, trehalose or and any component from the group of amino acids, sugars, salts and combinations thereof.
  • isotonicity agents are generally used in a total amount of about 5 mM to about 350 mM.
  • Non-limiting examples of salts include salts of any combinations of the cations sodium potassium, calcium or magnesium with anions chloride, phosphate, citrate, succinate, sulphate or mixtures thereof.
  • Non-limiting examples of amino acids comprise arginine, glycine, ornithine, lysine, histidine, glutamic acid, asparagic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophane, methionine, serine, proline.
  • Non-limiting examples of sugars include trehalose, sucrose, mannitol, sorbitol, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine (also referred to as “meglumine”), galactosamine and neuraminic acid and combinations thereof.
  • Non-limiting examples of stabilizer includes amino acids and sugars as described above as well as commercially available cyclodextrins and dextrans of any kind and molecular weight as known in the art.
  • Non-limiting examples of antioxidants include excipients such as methionine, benzylalcohol or any other excipient used to minimize oxidation.
  • the transduced cells e.g., CAR-T
  • recombinant virus described herein or pharmaceutical composition thereof can also contain preservatives to prevent the growth of microorganisms.
  • Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride.
  • a mixture of two or more preservatives is used.
  • the preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition.
  • the pharmaceutical composition is formulated to have a pH in the range of about 4.5 to about 9.0, including for example pH ranges of about any one of 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0.
  • the pharmaceutical composition can also be made to be isotonic with blood by the addition of a suitable tonicity modifier, such as glycerol.
  • transduced cells e.g., CAR-T
  • recombinant virus described herein or pharmaceutical composition thereof are preferably sterile.
  • the pharmaceutical composition may be rendered sterile by filtration through sterile filtration membranes.
  • Various additives which enhance the stability and sterility of the transduced cells or recombinant virus described herein or pharmaceutical compositions thereof including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
  • antimicrobial preservatives for example, parabens, chlorobutanol, phenol, and sorbic acid.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum mono stearate and gelatin.
  • any suitable methods for the administration of the transduced cells or recombinant viruses can be used herein.
  • the route of administration is in accordance with any known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intratumoral, intraarterial, or intralesional routes.
  • the compositions are injected directly into a site of a local disease site in the individual, a lymph node, an organ, a tumor, and the like.
  • the transduced cells (e.g., CAR-T) or recombinant virus described herein or pharmaceutical composition thereof is suitable for administration to a human.
  • the pharmaceutical composition is suitable for administration to a human by parenteral administration.
  • the transduced cells or recombinant virus described herein or pharmaceutical composition thereof comprising a carrier is suitable for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.
  • a pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, including pharmaceutically acceptable cell culture media.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions and sterile powders for the preparation of sterile injectable solutions.
  • the use of such media and agents for pharmaceutically active substances is well known in the art.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a condition requiring only the addition of the sterile liquid excipient methods of treatment, methods of administration, and dosage regimens described herein for inj ection, immediately prior to use.
  • the recombinant virus described herein or pharmaceutical composition thereof is contained in a single-use vial, such as a single-use sealed vial.
  • the transduced cells or recombinant virus described herein or pharmaceutical composition thereof is contained in a multi-use vial. In some embodiments, the transduced cells or recombinant virus described herein or pharmaceutical composition thereof is contained in bulk in a container. In some embodiments, the transduced cells or recombinant virus described herein or pharmaceutical composition thereof is cryopreserved.
  • the pharmaceutical composition comprising the transduced cells or recombinant virus must meet certain standards for administration to an individual. Methods are known in the art to assess the appearance, identity, purity, safety, and/or potency of pharmaceutical compositions.
  • the pharmaceutical composition is substantially free of extraneous protein capable of producing allergenic effects, such as proteins of an animal source used in cell culture, and free of any viral pathogens.
  • substantially free is less than about any of 10%, 5%, 1%, 0.1%, 0.01%, 0.001%, 1 ppm or less of total volume or weight of the pharmaceutical composition. Preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Division of Biological Standards.
  • This Example describes generation and characterization of viral glycoprotein variants with reduced binding to LDL-R, which may be used in LVVs to reduce non-specific transduction.
  • VSV-G has a 16-amino acid signal peptide (MKCLLYLAFLFIGVNC; SEQ ID NO: 36)
  • MARAV-G has a 16-amino acid signal peptide (MLRLFLFCFLALGAHS; SEQ ID NO: 38)
  • COV-G has a 17-amino acid signal peptide (MNFLLLTFIVLPLCSHA; SEQ ID NO: 35).
  • COV-G (SEQ ID NO: 1) shares about 74% amino acid similarity with VSV syn-G (SEQ ID NO: 46)
  • MARAV-G (SEQ ID NO: 40) shares about 80% amino acid similarity with VSV syn-G (SEQ ID NO: 46) (FIG. 1).
  • AlphaFold2 is a computational tool that accurately predicts protein structures at a level comparable to experimental methods like X-ray crystallography. AlphaFold2 can also be used to predict protein interactions and may provide valuable input to guide experimental efforts.
  • VSV-G Based on the interaction region predicted by AlphaFol d2, two deletions and several point mutations were designed. Some known substitutions in VSV-G, such as H8A (equivalent to Q8A in COV-G), I182E (equivalent to V182E in COV-G, A182E in MARAV-G) and Y209A (equivalent to Y209A in COV-G), were designed and tested in COV-G as well (see Table 1). Known double mutant K47Q+R354A was also included as a positive control.
  • Lentiviruses encoding an anti-CD20+CD19 dual CAR and pseudotyped with each of these mutant COV-G were produced. Briefly, envelope vectors encoding the COV-G variant, transfer vectors encoding FL AG-tagged anti-CD20+CD19 dual CAR, and Gag/Pol and Rev helper plasmids were introduced into Lenti-X 293T cells to allow lentivirus packaging. Following transfection, virus was harvested and concentrated from the Lenti-X 293 T cell supernatant. Virus titer (RNA copies/mL) were quantified using Lenti-XTM qRT-PCR Titration Kit (cat # 631235).
  • LV pseudotyped LVs were used to transduce 25,000 Jurkat cells (a CD3 + T cell line).
  • LV pseudotyped with COV-G WT was used as positive control.
  • LV pseudotyped with K47Q and R354A double mutations in COV-G (referred to as COVGmut) were used as benchmark control for impaired ability to interact with LDL-R (see US20200216502A1, the content of which is incorporated herein by reference in its entirety).
  • the COV-G variants were assessed by performing a transduction efficiency assay using LVs pseudotyped with the COV-G variants.
  • Envelope vectors encoding the COV-G variants, an N-terminal FLAG-tagged anti-CD20+CD19 dual CAR transfer vector, and packaging vectors were packaged in 293T cells and physical virus titer was determined by PCR.
  • Low dose LVV (25 LVV copies per cell) and high dose LVV (125 LVV copies per cell) were transduced in Jurkat T cells (immortalized T lymphocyte cell line) and Nalm6 B cells (B cell precursor leukemia cell line) without any transduction enhancer.
  • the two deletion variants (COVG-ml and C0VG-m2) and several point mutant variants (I272E (C0VG-m5), I278E (C0VG-m6), and I291E (C0VG-m7)) greatly reduced transduction of pseudotyped LVVs in both Jurkat T cells and Nalm6 B cells.
  • I272E (C0VG-m5), I278E (C0VG-m6), I291E (C0VG-m7), and V182E (C0VG-m9) single mutations decreased the transduction efficiency of pseudotyped LVVs even more than the double point mutant control K47Q+R354A (COVG-mut), for both Jurkat T cells and Nalm6 B cells (FIG. 2).
  • COV-G variant-pseudotyped LVVs were packaged with transfer vector encoding anti-CD20+CD19 dual CAR and used to transduce Jurkat and Nalm6 cells. 3 days later, CAR (Flag+) expression was detected by flow cytometry.
  • C0VG-ml9 I144A
  • COVG-m20 D145A
  • C0VG-m21 S146A
  • COVG-m23 F148A
  • COVG-m27 C153A
  • C0VG-m31 C158A
  • Other mutations such as C0VG-ml8 (W143A), COVG-m22 (Q147A), COVG-m24 (N150A), COVG-m25 (G151A), COVG-m26 (K152A), COVG-m28 (E154A), and COVG-m29 (T155A), partially reduced transduction efficiency (FIG. 3).
  • COV-G variants generated herein particularly those comprising amino acid deletions at positions 141-158 or 271-292 relative to SEQ ID NO: 1 (SEQ ID NO: 4-5), or point mutations at any of positions 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 158, 182, 272, 278, and 291 relative to SEQ ID NO: 1 (SEQ ID NOs: 8-10, 12, 62-75), showed reduced transduction efficiency when pseudotyped with LVVs on both Jurkat T cells and Nalm6 B cells, which are reflective of reduced glycoprotein binding to LDL-R.
  • Example 3 Previously identified C0VG-m9 (V182E) COVG mutant was used as a reference. LV pseudotyped with COVG WT was used as positive control while non-transduced Jurkat cells were used as negative control. 4-10 days after transduction, the percentage of transgene (VHH- expressing Tandem CAR) positive Jurkat cells was determined by flow cytometry (FIG. 4). CAR positive values between 2-20% were used to determine infectious titer and expressed as transducing units (TU)/mL.
  • MARAV-G (MARAVG) variants were constructed and assessed by performing a transduction efficiency assay using LVs pseudotyped with the MARAV-G (MARAVG) variants.
  • Lentiviruses encoding a tandem anti-CD19/CD20 CAR were produced by pseudotyping LVs with each of these MARAVG mutant along with Gag/Pol and Rev helper plasmids. These LVs were used to transduce Jurkat (CD3+ T cell line) cells, and infectious titer was determined between day 4 and 10 post-transduction.
  • 31 MARAVG mutants were designed by substituting single amino acids at positions Q226, K47, A182, 1331, or R354 of the wild-type MARAVG (MARAVG WT) sequence (Uniprot ID: F8SPF4). Wild type VSVG (Uniprot ID: P0C2X0), and COVG (Uniprot ID: 056677) were used as reference. A serial dilution of each of these LVs were used to transduce 25,000 Jurkat (CD3+ T cell line) cells. LV pseudotyped with MARAVG WT was used as positive control while non-transduced Jurkat cells were used as negative control.
  • Example 3 Characteristics of LVVs pseudotyped with viral glycoprotein variants and envelope surface-bound targeting molecule (e.g., anti-CD3 antibody)
  • an envelope vector encoding both viral glycoprotein variants and an envelope surface-bound agent for cell specific targeting (such as anti-CD3, CD5, CD2 or CD7 antibodies) was generated.
  • Anti-CD3 scFv targeting CD3 + T cells was introduced on the envelope surface of the LVV by anchoring anti-CD3 scFv with a CD8-derived transmembrane domain.
  • Transduction of target cells is achieved in two steps: (1) target cell binding via selected scFv and other components; and (2) fusion to the endosome membrane by the glycoprotein variants with reduced LDL-R binding. This enables the targeting of specific target (e.g., hematological) cell populations such as hematopoietic stem cells, T cells, or NK cells in vivo.
  • specific target e.g., hematological
  • envelope vectors encoding envelope surface-bound anti-CD3 scFv and COV-G variants (or MARAV-G variants) connected via a 2A (e.g., T2A) sequence were designed (see Table 4), and LVV transduction was tested in human PBMCs.
  • the envelope surface-bound anti-CD3 scFv comprises a CD8-derived transmembrane domain in order to be anchored on the envelope surface.
  • Envelope vectors encoding the envelope surface-bound anti-CD3 scFv and COV-G variant (or MARAV-G variant), transfer vectors encoding an N-terminal FLAG-tagged anti- CD20+CD19 dual CAR, and packaging vectors were introduced into 293T cells to allow lentivirus packaging, and physical virus titer was determined by PCR. Low dose (25 LVV copies per cell) and high dose (125 LVV copies per cell) of packaged lentiviruses were transfected in human resting PBMCs without any transduction enhancer. Transduction efficiency was assessed by flow cytometry on day 3 using anti-FLAG antibodies for CAR expression (Flag + ) and anti-CD25 (lymphocyte activation marker) antibodies for target cell activation.
  • envelope surface-bound anti-CD3 scFv can greatly compensate for the transduction efficiency of LVVs pseudotyped with glycoproteins variants (e.g., COVG-mut, C0VG-m5, C0VG-m7) with reduced LDL-R binding in primary T cells.
  • glycoproteins variants e.g., COVG-mut, C0VG-m5, C0VG-m7
  • inclusion of envelope surface-bound anti-CD3 scFv increased CAR transduction from 2.16% for LVVs pseudotyped with COVG-mut to 8.05% for LVVs pseudotyped with anti-CD3 scFv and COVG-mut.
  • envelope surface-bound anti-CD3 scFv increased CAR transduction from 3.62% for LVVs pseudotyped with C0VG-m5 to 17.5% for LVVs pseudotyped with anti-CD3 scFv and C0VG-m5, which is even higher than the transduction rate of 13.1% for LVVs pseudotyped with the wildtype COVG control (FIG. 8).
  • a similar effect was seen for LVVs pseudotyped with envelope surface-bound anti-CD3 scFv and C0VG-m7.
  • LVVs pseudotyped with envelope surface-bound anti-CD3 scFv and C0VG-m9 showed a slightly higher transduction rate than LVVs pseudotyped with C0VG-m9.
  • envelope surface-bound anti-CD3 scFv greatly increased CD25 activation marker on T cells transduced with LVVs pseudotyped with envelope surface-bound anti-CD3 scFv and all tested COV-G variants (FIG. 9).
  • the transduced T cells were exposed to B cells (CD19 + , CD20 + ), and B cell numbers were measured by flow cytometry using an anti-CD19 antibody.
  • B cells CD19 + , CD20 +
  • B cell numbers were measured by flow cytometry using an anti-CD19 antibody.
  • primary B cells were depleted in all T cell treatment groups transduced with LVVs pseudotyped with envelope surface-bound anti-CD3 scFv and COV-G variants (FIG. 10), demonstrating functional CAR was expressed on these T cells.
  • T cells transduced with LVVs pseudotyped with anti-CD3 scFv and C0VG-m5 or anti-CD3 scFv and C0VG-m7 showed stronger CAR-specific B-cell killing compared to not only LVVs pseudotyped with C0VG-m5 or C0VG-m7, respectively, but also LVVs pseudotyped with the double point mutant variant COVG-mut.
  • LVs pseudotyped with each of the 6 “impaired” mutant COVG were produced, with Gag/Pol and Rev helper plasmids, along with envelop vector(s) encoding the mutant COVG and an envelope surfacebound anti-CD3 scFv (FIG. 11).
  • Previously identified C0VG-m9 (V182E) COVG mutant was used as a reference. These LVs were used to transduce Jurkat cells, and infectious titer was determined 4-10 days after transduction.
  • LVs pseudotyped with each of the 20 LOF mutant MARAVG (MG-5 was excluded as all the other AK47 mutants had ⁇ l-log lower infectious titer) were produced, with Gag/Pol and Rev helper plasmids, along with envelop vector(s) encoding the mutant MARAVG and an envelope surface-bound anti-CD3 scFv (FIG. 13).
  • These LVs were used to transduce Jurkat cells, and infectious titer was determined 4-10 days after transduction.
  • 18 novel “impaired” mutant MARAVG were identified whose fusogenic potential was restored by the anti-CD3 scFv to selectively transduce CD3+ Jurkat cells (FIG. 14).
  • LVs were also used to evaluate transduction of non-activated human PBMCs from two human donors, which demonstrated that “impaired” mutant MARAVG pseudotyped LVs with envelope surface-bound anti-CD3 scFv transduced human PBMCs with varying efficacy (FIG. 15). Briefly, PBMCs from two human donors were thawed and recovered by culturing overnight at 37°C in T cell medium. Next day, PBMCs were counted, plated and subsequently incubated with serial dilutions of LVs for four hours. Medium was changed four hours after incubation, and thereafter every 48-72 hours.
  • LVs pseudotyped with AA182/ or AI331 MARAVG mutants have less loss of infectivity (1- log) than AK47 and AR354 MARAVG mutants (1-2 log) (FIG. 16 and Table 6).
  • AA182”, “AI331”, “AK47” or “AR354” in FIG. 16 represents the MARAVG variant comprising the specific mutation at amino acid position A182, 1331, K47, or R354 (shown in FIG. 16).

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Abstract

La présente divulgation concerne des variants de glycoprotéines virales à liaison réduite au récepteur de lipoprotéines de faible densité (LDL-R) par rapport à une glycoprotéine virale de référence. Des virus recombinants pseudotypés avec des variants de glycoprotéines virales décrits ici et, éventuellement, une molécule de ciblage liée à la surface de l'enveloppe (par exemple, un scFv anti-CD3), des procédés pour leur production et des procédés pour leur utilisation sont également fournis.
PCT/US2025/027177 2024-05-01 2025-04-30 Variants de glycoprotéines virales et leurs utilisations Pending WO2025231185A1 (fr)

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