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  • 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
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  • A61K31/00—Medicinal preparations containing organic active ingredients
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  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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  • A01K2207/00—Modified animals
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• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
  • A01K2227/106—Primate
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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  • A01K2267/00—Animals characterised by purpose
  • A01K2267/03—Animal model, e.g. for test or diseases
  • A01K2267/035—Animal model for multifactorial diseases
  • A01K2267/0387—Animal model for diseases of the immune system
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  • 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
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  • 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
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
  • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • 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 generally contemplates, in part, to non-human primate models for assessing the safety and efficacy of in vivo recombinant lentivirus gene therapies.
• the disclosure contemplates, in part, a method to reduce the number of B cells in a non-human primate (NHP) comprising: administering to the NHP, an amount of a recombinant HIV-1 derived lentivirus, wherein the recombinant lentivirus comprises (a) a viral envelope comprising (i) one or more mutated viral envelope glycoproteins that retain fusogenic activity and lack cognate receptor binding activity and (ii) one or more non-viral membrane-bound tropism polypeptides; and (b) a recombinant HIV-1 derived lentiviral vector comprising a polynucleotide encoding a promoter operably linked to a polynucleotide encoding an engineered antigen receptor; wherein the amount of recombinant lenti
• At least one of the one or more non-viral membranebound tropism polypeptides comprises an extracellular antigen targeting domain, a spacer domain, and a transmembrane domain.
• the extracellular antigen targeting domain binds an antigen expressed on an immune effector cell selected from the group consisting of: the alpha, beta, gamma, or delta chain of the T cell receptor, CD2, CD35, CD3s CD3y, CD4, CD8a, and CD8p.
• the extracellular antigen targeting domain comprises an anti-CD3s antibody or antigen binding fragment thereof.
• the engineered antigen receptor is selected from the group consisting of: a chimeric antigen receptor (CAR), a chimeric costimulatory receptor (CCR), an alpha-beta T cell receptor (aP TCR), a gamma delta T cell receptor (y5 TCR), a dimerizing agent regulated immunoreceptor complex (DARIC), a chimeric TGF-P receptor (CTBR), and a zetakine receptor.
• the immune effector cells comprise one or more of aP TCR T cells, y5 TCR T cells, natural killer (NK) cells, and natural killer T (NKT) cells.
• the viral envelope further comprises a secondary non- viral membrane bound tropism polypeptide selected from the group consisting of CD80, CD86, CD137L, OX40L, and ICOSL or variants thereof.
• the disclosure contemplates, in part, a method to reduce the number of B cells in a non-human primate (NHP) comprising: administering to the NHP, an amount of a recombinant HIV-1 derived lentivirus, wherein the recombinant lentivirus comprises (a) a viral envelope comprising (i) one or more mutated viral envelope glycoproteins that retain fusogenic activity and lack cognate receptor binding activity and (ii) a non-viral membrane-bound tropism polypeptide comprising an anti-CD3s antibody or antigen binding fragment thereof, a spacer domain, and a transmembrane domain, and optionally a secondary non-viral membrane bound tropism polypeptide selected from the group consisting of CD80, CD86, CD137L, OX40L, and ICOSL or variants thereof; and (b) a recombinant HIV-1 derived lentiviral vector comprising a polynucleotide encoding a promoter operably
• the one or more mutated viral envelope glycoproteins comprise a vesiculovirus envelope glycoprotein, one or more morbillivirus envelope glycoproteins or one or more henipavirus envelope glycoproteins.
• the vesiculovirus is selected from the group consisting of: vesicular stomatitis Alagoas virus (VSAV; Alagoas vesiculovirus), Carajas virus (CJSV; Carajas vesiculovirus), Chandipura virus (CHPV; Chandipura vesiculovirus), Cocal virus (COCV; Cocal vesiculovirus), vesicular stomatitis Indiana virus (VSIV; Indiana vesiculovirus), Isfahan virus (ISFV; Isfahan vesiculovirus), Maraba virus (MARAV; Maraba vesiculovirus), Morreton virus (MORV; Morreton vesiculovirus), vesicular stomatitis New Jersey virus (VSNJV; New Jersey vesiculovirus), and Piry virus (PIRYV; Piry vesiculovirus).
• VSAV vesicular stomatitis Al
• the vesiculovirus G protein is a COCV G glycoprotein (COCV-G) or a VSIV G glycoprotein (VSIV-G).
• the VSIV-G envelope protein comprises one or more amino acid substitutions selected from the group consisting of: K47A and R354A; K47A and R354G; K47A and R354F; K47A and R354Q; K47G and R354A; K47G and R354G; K47G and R354F; K47G and R354Q;K47F and R354A; K47F and R354G; K47F and R354F; K47F and R354Q; K47Q and R354A; K47Q and R354G; K47Q and R354F; and K47Q and R354A; K47Q and R354G; K47Q and R354F; and K47Q and R354Q.
• the vesiculovirus G protein is COCV-G.
• the COCV-G envelope protein comprises one or more amino acid substitutions at K47 and/or R354.
• the COCV-G envelope protein comprises one or more amino acid substitutions selected from the group consisting of: K47A and R354A; K47A and R354G; K47A and R354F; K47A and R354Q; K47G and R354A; K47G and R354G; K47G and R354F; K47G and R354Q;K47F and R354A; K47F and R354G; K47F and R354F; K47F and R354Q; K47Q and R354A; K47Q and R354G; K47Q and R354F; and K47Q and R354A; K47Q and R354G; K47Q and R354F; and K47Q and R354F; and K47Q and R354Q.
• the one or more morbillivirus envelope glycoproteins are measles virus F (MV-F) and measles virus H (MV-H).
• the MV-H protein comprises one or more amino acid substitutions selected from the group consisting of: Y481A, R533A, S548L, and F549S.
• the one or more henipavirus envelope glycoproteins are nipah virus F (NiV-F) and nipah virus G (NiV-G).
• the NiV-G protein comprises one or more amino acid substitutions at E501, W504, Q530, and E533.
• the spacer domain is hinge domain or stalk obtained or isolated from a polypeptide selected from the group consisting of CD3, CD4, CD8a, CD8P, CD28, or Glycophorin A; and/or wherein the transmembrane domain is isolated from a polypeptide selected from the group consisting of CD3, CD4, CD8a, CD8P, CD28, or Glycophorin A; and optionally, wherein the tropism polypeptide comprises a truncated intracellular domain isolated from a polypeptide selected from the group consisting of CD3, CD4, CD8a, CD8P, CD28, or Glycophorin A or a cytoplasmic tail of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or more.
• the viral envelope further comprises a secondary non- viral membrane bound tropism polypeptide selected from the group consisting of CD80, CD86, CD137L, OX40L, and ICOSL and optionally, comprises a C-terminal truncation that results in a cytoplasmic domain of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or more.
• a secondary non- viral membrane bound tropism polypeptide selected from the group consisting of CD80, CD86, CD137L, OX40L, and ICOSL and optionally, comprises a C-terminal truncation that results in a cytoplasmic domain of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or more.
• the engineered antigen receptor is a CAR comprising an extracellular antigen binding domain selected from the group consisting of: a receptor ectodomain, a ligand, or an antibody or antigen binding fragment thereof selected from the group consisting of: a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, a single chain Fv protein (scFv), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), a single-domain antibody (sdAb, a camelid VHH, Nanobody), and a centyrin.
• a Camel Ig a Llama Ig, an Al
• the engineered antigen receptor is a CAR comprising a hinge domain isolated or derived from a polypeptide selected from the group consisting of: CD4, CD8P, CD8a, CD28, CD134, CD137, CD152, CD278, IgGl, IgG2, IgG3, and IgG4.
• the engineered antigen receptor is a CAR comprising a primary signaling domain isolated or derived from a polypeptide selected from the group consisting of: FcRy, FcRp, CD3y, CD35, CD3s, CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
• engineered antigen receptor is a CAR comprising a costimulatory domains isolated or derived from a polypeptide selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD 11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD 134 (0X40), CD 137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein 10 (DAP10), Linker for activation of T-cells family member 1 (LAT), SH2 Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNFRS25, and zeta chain of T cell receptor associated protein kinase 70 (TLR1),
• the recombinant lentivirus is parenterally administered to the NHP.
• the recombinant lentivirus is intravascularly administered to the NHP.
• the recombinant lentivirus is intravenously or intraarterially administered to the NHP.
• 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more specimens are taken from the NHP, wherein one or more of the specimens is taken from the NHP every day, every other day, every three days, every four days, every five days, every six days, every 7 days, every eight days, every none days, or every ten or more days for a period of about 10 days to about 60 days or any intervening period of time.
• one or more specimens is taken from the NHP and the number of B cells is measured in the specimen.
• one or more specimens is taken from the NHP and the number of T cells is measured in the specimen. In some embodiments, one or more specimens is taken from the NHP and the number of CAR + T cells is measured in the specimen.
• one or more specimens is taken from the NHP and the vector copy number (VCN) of transduced T cells is measured in the one or more specimens.
• VCN vector copy number
• one or more specimens is taken from the NHP and the amount of one or more cytokines and/or chemokines is measured in the one or more specimens.
• one or more specimens is taken from the NHP and the amount of one or more cytokines or chemokines selected from the group consisting of CXCL11, GM-CSF, IFNy, IL-la, IL-ip, IL-IRA, IL-2, IL-5, IL-6, IL-8, IL-10, IL-12, IP- 10, MIP-la, and TNFa is measured in the one or more specimens.
• cytokines or chemokines selected from the group consisting of CXCL11, GM-CSF, IFNy, IL-la, IL-ip, IL-IRA, IL-2, IL-5, IL-6, IL-8, IL-10, IL-12, IP- 10, MIP-la, and TNFa is measured in the one or more specimens.
• the amount of recombinant lentivirus administered to the NHP does not cause a cytokine storm, cytokine release syndrome (CRS), or neurotoxicity in the NHP.
• CRS cytokine release syndrome
• the amount of recombinant lentivirus administered to the NHP decreases the number of B cells in a specimen at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more compared to the number of B cells in a specimen from an NHP that was not administered the recombinant lentivirus or compared to the number of B cells in a specimen from the NHP before the NHP was administered the recombinant lentivirus.
• composition comprising a pharmaceutically acceptable diluent and the recombinant lentivirus is administered to the NHP.
• the diluent is a pharmaceutically acceptable cell culture medium (e.g., DMEM), Dulbecco’s phosphate buffered saline (PBS), Ringer’s solution, 5% dextrose in water (D5W), or normal/physiologic saline (0.9% NaCl).
• DMEM DMEM
• Dulbecco phosphate buffered saline
• Ringer 5% dextrose in water
• D5W 5% dextrose in water
• normal/physiologic saline e.g., NaCl
• the amount of recombinant lenti virus administered to the NHP is about 1 x 10 6 TU/mL to about 1 x IO 10 TU/mL.
• the amount of recombinant lentivirus administered to the NHP is about 1 x 10 7 TU/mL to about 1 x 10 9 TU/mL.
• the amount of recombinant lentivirus administered to the NHP is about 1 x 10 7 TU/mL, 5 x 10 7 TU/mL, 1 x 10 8 TU/mL, 5 x 10 8 TU/mL, about 1 x 10 9 TU/mL, about 5 x 10 9 TU/mL, or aboutl x IO 10 TU/mL.
• the NHP is not administered a lymphodepleting chemotherapy prior to, during, or after administration of the recombinant lentivirus.
• the NHP is Macaca nemestrina.
• Figure 1 shows the effect on lymphocyte populations in a pig-tail macaque administered an in vivo HIV-1 derived lentivirus encoding an anti-CD20 CAR.
• the rightmost panel shows T cell expansion as a function of time after infusion; the leftmost panel shows B cell aplasia as a function of time after infusion.
• Figure 2 shows that the in vivo HIV-1 derived lentivirus encoding an anti-CD20 CAR did not increase cytokines/chemokines to levels associated with neurotoxicity in M. nemestrina.
• the gray boxes indicate cytokine levels associated with neurotoxicity.
• Figure 3 shows the effect on lymphocyte populations in a pig-tail macaque administered an in vivo HIV-1 derived lentivirus encoding an anti-CD20 CAR.
• the rightmost panel shows the vector copy number detected in T cells as a function of time after infusion; the leftmost panel shows B cell aplasia as a function of time after infusion.
• SEQ ID Nos: 1-10 set forth amino acid sequences of fusogens.
• SEQ ID NO: 11 sets forth the polynucleotide sequence encoding a CAR.
• SEQ ID NO 12 sets forth the amino acid sequences encoded by SEQ ID NO: 11.
• SEQ ID Nos: 13-92 set forth amino acid sequences of fusogens.
• SEQ ID Nos: 93-106 set forth amino acid sequences of linker polypeptides.
• SEQ ID Nos: 107-126 set forth amino acid sequences of viral self-cleaving polypeptides.
• X refers to any amino acid, a specified group of amino acids or the absence of an amino acid.
• amino acid position(s) of a fusogen is with reference to the fusogen lacking a signal sequence (i.e., the amino acid sequence after the signal peptide has been cleaved).
• Non-human primate animal models that can be used to assess the safety and efficacy of a lentiviral-based in vivo gene therapy designed for human applications.
• Non-human primate models present unique challenges to lentivirus-based in vivo gene therapies for oncology (e.g., chimeric antigen receptor T cell therapy) because they express an array of HIV post-entry mechanisms that restrict HIV-1 replication not present in human cells.
• CypA cyclophilin A
• CA HIV-1 capsid protein
• TAM5a tripartite-containing motif 5a
• Old World monkeys such as rhesus macaques (Macaco mulatto , cynomolgus macaques (Macaco fascicularis), and pig-tail macaques (Macaco nemestrind) express different combinations of, and different alleles of, TRIM5a, CypA, and TRIM5a- CypA fusion proteins (TRIMCyp) to regulate HIV-1 replication.
• Rhesus macaques express several variants of TRIM5a. These TRIM5a variants and CypA work together to restrict HIV-1 replication with varying efficiencies.
• TRIMCyp fusion proteins are also seen in some, but not all, populations of rhesus macaques, where it restricts HIV-2 but not HIV-1 replication. Cynomolgus macaques can express TRIM5a and different TRIM5a-CypA fusion proteins. Similar to rhesus macaques, TRIM5a and CypA work together to restrict HIV-1 replication. TRIMCyp fusion protein variants are expressed in cynomolgus macaques but only restrict HIV-2, not HIV-1, infection. In contrast, pig-tail macaques do not express TRIM5a mRNA. Instead, TRIM5 isoforms TRIM59 and TRIM5q associated with TRIMCyp are expressed.
• TRIMCyp restricts HIV-2 but not HIV-1 replication, rendering pig-tail macaques sensitive to HIV-1 infection due to the absence of TRIM5a and expression of a TRIMCyp that is not able to restrict HIV-1 replication.
• pig-tail macaques are used as the non-human primate model for the in vivo HIV-1 lentivirus-based gene therapies contemplated herein.
• the disclosure generally relates to developing in vivo lentivirus gene therapy models in non-human primates (NHPs).
• the disclosure contemplates, in part, a method of reducing B cells in an NHP using recombinant lentivirus.
• a method comprises administering a recombinant lentivirus derived from human immunodeficiency virus (HIV) type 1 (HIV-1) to a NHP, wherein the recombinant lentivirus comprises an envelope that has a component that mediates fusion and one or more components that drive targeting to an immune effector cell, and further comprises a vector that encodes an engineered antigen receptor that targets cells of the B cell lineage in the NHP.
• HIV human immunodeficiency virus
• a method comprises administering a recombinant lentivirus derived from HIV-1 that comprises a viral envelope that comprises one or more mutated viral envelope glycoproteins that retain fusogenic activity and lack cognate receptor binding activity, z.e., the ability to bind a cognate receptor on a cell, and one or more non-viral membrane-bound tropism polypeptides engineered to bind an immune effector cell, and a vector comprising encoding an engineered antigen receptor that targets cells of the B cell lineage in the NHP.
• the disclosure further contemplates a method to reduce the number of cells of the B cell lineage in an NHP that comprises administering an HIV-1 derived recombinant lentivirus that comprises a viral envelope comprising one or more mutated viral envelope glycoproteins that retain fusogenic activity and lack cognate receptor binding activity and a non-viral membrane-bound tropism polypeptide comprising an anti-CD3 antibody or antigen binding fragment thereof, a spacer domain, and a transmembrane domain; optionally, one or more other non-viral membrane bound tropism polypeptides that target an immune effector cell; and a recombinant lenti viral derived from HIV-1 that encodes a promoter operably linked to a polynucleotide encoding an engineered antigen receptor that targets cells of the B cell lineage in the NHP.
• the disclosure also contemplates a method to reduce the number of cells of the B cell lineage in an NHP that comprises administering an HIV-1 derived recombinant lentivirus that comprises a viral envelope comprising one or more mutated viral envelope glycoproteins that retain fusogenic activity and lack cognate receptor binding activity; a non-viral membrane-bound tropism polypeptide comprising an anti-CD3 antibody or antigen binding fragment thereof, a spacer domain, and a transmembrane domain; optionally, a secondary non-viral membrane bound tropism polypeptide selected from the group consisting of CD80, CD86, CD137L, OX40L, and ICOSL or variants thereof; and a recombinant HIV-derived lend viral vector comprising a polynucleotide encoding a promoter operably linked to a polynucleotide encoding a chimeric antigen receptor that binds an antigen expressed on a cell of the B cell lineage.
• Techniques for recombinant (z.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification and related techniques and procedures may be generally performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology as cited and discussed throughout the present specification.
• an element means one element or one or more elements.
• the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• a range e.g., 1 to 5, about 1 to 5, or about 1 to about 5, refers to each numerical value encompassed by the range.
• the range “1 to 5” is equivalent to the expression 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.
• the term “substantially” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• “substantially the same” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that produces an effect, e.g., a physiological effect, that is approximately the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• “enhance” or “promote,” or “increase” or “expand” refers generally to the ability of a recombinant lentivirus comprising a vector encoding an engineered antigen receptor contemplated herein to produce, elicit, or cause a greater effect compared to the effect prior to treatment or the effect caused by either vehicle or a control lentivirus.
• a measurable effect may include an increase in T cell activation, transduction, expansion, persistence, cytokine secretion, and/or an increase in the ability to reduce or decrease the numbers of target cells in a non-human primate.
• maintain or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” refers generally to the ability of a recombinant lentivirus contemplated herein to produce, elicit, or cause a substantially similar or comparable effect compared to the effect caused by either vehicle, a control lentivirus.
• the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
• a “recombinant HIV-1 lentivirus” refers to a recombinantly produced or non- naturally occurring lentivirus that contains a viral envelope; HIV-1 lentivirus structural proteins, matrix (MA), capsid (CA), nucleocapsid (NC), and p6 and enzymatic proteins, integrase (IN), protease (PR), and reverse transcriptase (RT); and further comprises a recombinant lend viral vector that contains HIV-1 genomic elements for packaging, reverse transcription and integration.
• MA matrix
• CA capsid
• NC nucleocapsid
• p6 and enzymatic proteins integrase
• integrase integrase
• PR protease
• RT reverse transcriptase
• B cells refers to cells that show at least one phenotypic, physiological, morphological, functional, or immunological characteristic of a B cell.
• B cells suitable for use in particular embodiments contemplated herein include but are not limited to B cells that express one or more of the following markers CDlc, CD2, CD5, CD6, CDlla, CDllc, CD14, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD35, CD37, CD38, CD39, CD40, CD43, CD45RA, CD45RB, CD45RO, CD49c, CD49d, CD51, CD57, CD62L, CD69, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD83, CD84, CD85, CD86, CD89, CD98, CD95, CD99, CD100, CD106, CD107a, CD
• binding domain refers to a domain that enables an engineered antigen receptor to specifically bind to a target antigen.
• the binding domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
• an “antibody” refers to a polypeptide that comprises at least a light chain immunoglobulin variable region and/or a heavy chain immunoglobulin variable region, which specifically recognizes and binds an epitope on an antigen.
• an “isolated antibody or antigen binding fragment thereof’ refers to an antibody or antigen binding fragment thereof that has been separated from its natural environment and/or that is derived from a natural, synthetic, semi-synthetic, or recombinant source.
• An “antigen binding fragment” or “antigen binding portion” refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen.
• antigen binding fragments suitable for incorporation into engineered antigen receptors contemplated herein include, but are not limited to, a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, a single chain Fv protein (“scFv”), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), a single-domain antibody (sdAb or Nanobody, e.g., a camelid VHH), a centyrin, other portions of full length antibodies sufficient for antigen binding, and combinations thereof.
• a Camel Ig a Llama Ig, an Alpaca Ig, Ig NAR
• a “heavy chain antibody” refers to an antibody that contains two heavy chain variable domains and no light chains;
• a “camelid antibody” refers to an antibody isolated from a Camel, Alpaca, or Llama that contains two heavy chain variable domains and no light chains.
• IgNAR immunoglobulin new antigen receptor
• VNAR variable new antigen receptor
• CNAR constant new antigen receptor
• a “single-chain Fv” or “scFv” antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain and in either orientation (e.g, VL-VH or VH-VL).
• 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.
• a “single domain antibody,” “sdAb,” or “nanobody” as used herein refers an antibody fragment that contains the smallest known antigen binding unit of the variable region of a heavy chain antibody, e.g., a camelid VHH or shark VNAR.
• a “humanized VHH” refers to a single domain non-human VHH that has undergone humanization to reduce potential immunogenicity of the antibody in human recipients.
• a “humanized VNAR” refers to a single domain non-human VNAR that has undergone humanization to reduce potential immunogenicity of the antibody in human recipients.
• Linker refers to a plurality of amino acid residues between various polypeptide domains added for appropriate spacing, conformation, and function.
• Linkers include a “variable region linking sequence,” an amino acid sequence that connects the VH and VL domains of an antibody or antigen binding fragment thereof and provides a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that comprises the same light and heavy chain variable regions.
• a linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids long.
• linkers include, but are not limited to glycine polymers; glycine-serine polymers; glycine-alanine polymers; alanine-serine polymers; GGG; DGGGS (SEQ ID NO: 93); TGEKP (SEQ ID NO: 94); GGRR (SEQ ID NO: 95); GGGGS (SEQ ID NO: 96); GGGGSGGGGS (SEQ ID NO: 97); GGGGSGGGGSGGGGS (SEQ ID NO: 98); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 99); GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 100) ; EGKSSGSGSESKVD (SEQ ID NO: 101); KESGSVSSEQLAQFRSLD (SEQ ID NO: 102); GGRRGGGS (SEQ ID NO: 103); LRQRDGERP (SEQ ID NO: 104); LRQKDGGGSERP (SEQ ID NO:
• a “hinge domain,” is a type of domain that may be present in an engineered antigen receptor.
• a hinge domain plays a role in positioning an antigen binding domain away from the effector cell surface to enable proper cell/cell contact, antigen binding and activation.
• a hinge domain is placed between a binding domain and a transmembrane domain (TM).
• TM transmembrane domain
• a hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
• a hinge domain may be altered by substituting one or more cysteine and/or proline residues in a naturally occurring immunoglobulin hinge domain with one or more other amino acid residues (e.g. , one or more serine residues).
• a “transmembrane domain” or “TM domain” refers to a hydrophobic portion of polypeptide that is disposed between an extracellular domain and an intracellular domain and anchors the polypeptide to the plasma membrane of the cell.
• a TM domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
• intracellular signaling domain refers to a portion of a polypeptide that participates in transducing the message of effective binding of a target antigen by a receptor expressed on an immune effector cell to the immune effector cell’s interior to an elicit effector function (an “effector function” refers to a specialized function of an immune effector cell), e.g, activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors, or other cellular responses elicited with antigen binding to the receptor expressed on the immune effector cell.
• “Intracellular signaling domains” include the polypeptide domain or functional fragment thereof, which transduces the effector function signal and that directs the cell to perform a specialized function.
• the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transducing effector function signal.
• T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR e.g., a TCR/CD3 complex) and costimulatory signaling domains that act in an antigen-independent manner to provide a secondary or costimulatory signal.
• a “primary signaling domain” refers to a signaling domain that regulates the primary activation of a TCR complex either in a stimulatory way, or in an inhibitory way.
• Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.
• costimulatory signaling domain refers to an intracellular signaling domain of a co- stimulatory molecule.
• Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen.
• a “tropism polypeptide” refers to a polypeptide that binds one or more antigens on a target host cell, e.g., an immune effector cell.
• a tropism polypeptide comprises an extracellular antigen targeting domain, a spacer domain, and a transmembrane domain.
• non- viral membrane bound tropism polypeptide refers to a polypeptide that binds one or more antigens on a target host cell, e.g., an immune effector cell; that is not native to, or derived from, either in whole or in part, a virus; and that is attached to a lipid bilayer, cell membrane, or viral envelope.
• a non- viral membrane bound tropism polypeptide comprises an extracellular antigen targeting domain, a spacer domain, and a transmembrane domain.
• extracellular antigen targeting domain refers to a polypeptide that enables a tropism polypeptide or non- viral membrane-bound tropism polypeptide to specifically bind to a antigen on a target host cell, e.g., an immune effector cell.
• the extracellular antigen targeting domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
• a “spacer domain,” refers to a polypeptide disposed between an extracellular antigen targeting domain and a transmembrane domain.
• a spacer domain positions the extracellular antigen targeting domain away from the viral envelope surface to enable proper virus/target cell contact and binding.
• a spacer domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
• Illustrative examples of spacer domains include but are not limited to hinge or stalk domains from IgGl, IgG2, IgG4, CD2, CD3, CD4, CD8a, CD8P, and CD28 and polypeptide linkers of similar amino acid composition and lengths.
• lentivirus-based gene therapies are a new class of gene therapies that do not have well established non-human animal models. Although rodent models are effective surrogates exploring pathways and disease mechanisms and identifying potential treatment targets they are not as useful for evaluating drug safety/toxicity.
• Non-human primates (NHPs) most closely resemble humans, regarding lifespan, size, telomere length, immune system characteristics, and genetic heterogeneity, conferring high predictive value for the safety/toxicity of cell therapies.
• NHPs can also be used to evaluate the efficacy of a platform in vivo lentivirus- based CAR T therapy that targets B cells.
• the present inventors have established an effective NHP model to evaluate the safety and efficacy of an in vivo lentivirus-based gene therapy platform.
• a method to evaluate an in vivo lentivirus-based gene therapy platform in an NHP comprises administering an amount of a recombinant lentivirus contemplated herein to the NHP.
• the term “amount” as used herein refers to “an amount effective,” “an amount sufficient,” “an effective amount,” or “a sufficient amount” of recombinant lentivirus contemplated herein to achieve a beneficial or desired effect, including a decrease in the number of B cells or B cell aplasia in the NHP.
• the effective amount of recombinant lentivirus is administered to the NHP and causes a desired effect without causing measurable toxicity.
• a method to reduce the number of target cells in a NHP comprises administering an amount of a recombinant lentivirus comprising a lentiviral vector encoding an engineered antigen receptor that binds an antigen expressed on a target cell.
• the recombinant lentivirus and lentiviral vector are derived from HIV-1 and are administered to a pig-tail macaque.
• a method to reduce the number of B cells in an NHP comprises administering a recombinant lentivirus comprising a viral envelope that comprises one or more mutated viral envelope glycoproteins that retain fusogenic activity and lack cognate receptor binding activity and one or more non- viral membrane-bound tropism polypeptides, and a recombinant lentiviral vector comprising a polynucleotide encoding a promoter operably linked to a polynucleotide encoding an engineered antigen receptor that binds an antigen expressed on the B cell.
• the amount of recombinant lentivirus administered to the NHP is sufficient to transduce a population of immune effector cells sufficient to reduce the number of B cells in the NHP.
• the number of B cells is reduced in the absence of toxicity, e.g., cytokine release syndrome (CRS), or neurotoxicity.
• the recombinant lentivirus and lentiviral vector are derived from HIV-1 and are administered to a pig-tail macaque.
• a method to reduce the number of B cells in an NHP comprises administering a recombinant HIV-1 derived lentivirus comprising (i) a viral envelope that comprises one or more mutated viral envelope glycoproteins that retain fusogenic activity and lack cognate receptor binding activity; a non- viral membrane-bound tropism polypeptide comprising an extracellular antigen targeting domain that binds an antigen expressed on an immune effector cell, a spacer domain, and a transmembrane domain; optionally a secondary non- viral membrane bound tropism polypeptide that binds an antigen expressed on an immune effector cell; and (ii) a recombinant lentiviral vector comprising a polynucleotide encoding a promoter operably linked to a polynucleotide encoding an engineered antigen receptor that binds an antigen expressed on the B cell.
• the amount of recombinant lentivirus administered to the NHP is sufficient to transduce a population of immune effector cells, e.g., T cells, sufficient to reduce the number of B cells in the NHP.
• the number of B cells is reduced in the absence of toxicity, e.g., cytokine release syndrome (CRS), or neurotoxicity.
• the recombinant lentivirus and lentiviral vector are derived from HIV-1 and are administered to a pig-tail macaque.
• a method to reduce the number of B cells in an NHP comprises administering a recombinant HIV-1 derived lentivirus comprising (i) a viral envelope that comprises one or more mutated viral envelope glycoproteins that retain fusogenic activity and lack cognate receptor binding activity; a non-viral membrane-bound tropism polypeptide comprising an extracellular antigen targeting domain comprising an antibody or antigen binding fragment thereof that binds an antigen expressed on a T cell, e.g., CD3, a spacer domain, and a transmembrane domain; a secondary non-viral membrane bound tropism polypeptide that binds an antigen expressed on a T cell cell, e.g., CD80, CD86, CD137L, OX40L, and ICOSL or variants thereof; and (ii) a recombinant HIV-1 derived lentiviral vector comprising a polynucleotide
• the amount of recombinant lentivirus administered to the NHP is sufficient to transduce a population of T cells, sufficient to reduce the number of B cells in the NHP.
• the number of B cells is reduced in the absence of toxicity, e.g., cytokine release syndrome (CRS), or neurotoxicity.
• CRS cytokine release syndrome
• a composition comprising a recombinant lentivirus contemplated herein is parenterally administered to an NHP.
• parenteral methods of administration include but are not limited to intravascular (intravenous or intraarterial), intraosseous, intraperitoneal, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, intramuscular, and intramedullary.
• a composition comprising a recombinant lentivirus contemplated herein is intravascularly administered to an NHP.
• a composition comprising a recombinant lentivirus contemplated herein is intraarterially administered to an NHP.
• a composition comprising a recombinant lentivirus contemplated herein is intravenously administered to an NHP.
• composition comprising a recombinant lentivirus contemplated herein is parenterally administered to an NHP at an amount of about 1 x 10 6 TU/mL to about 1 x 10 10 TU/mL, about 1 x 10 7 TU/mL to about 1 x 10 10 TU/mL, about 1 x 10 8 TU/mL to about 1 x 10 10 TU/mL, about 1 x 10 6 TU/mL to about 1 x 10 9 TU/mL, about 1 x 10 6 TU/mL to about 1 x 10 8 TU/mL, or about 1 x 10 7 TU/mL to about 1 x 10 9 TU/mL.
• composition comprising a recombinant lentivirus contemplated herein is parenterally administered to an NHP at an amount of about 1 x 10 6 TU/mL, about 5 x 10 6 TU/mL, about 1 x 10 7 TU/mL, 5 x 10 7 TU/mL, 1 x 10 8 TU/mL, 5 x
• 10 8 TU/mL about 1 x 10 9 TU/mL, about 5 x 10 9 TU/mL, or 1 x IO 10 TU/mL about 1 x 10 7 TU/mL, 5 x 10 7 TU/mL, 1 x 10 8 TU/mL, 5 x 10 8 TU/mL, about 1 x 10 9 TU/mL, about 5 x
• composition comprising a recombinant lentivirus contemplated herein is parenterally administered to an NHP at a rate of dose of about 5 mL/kg to about 15 mL/kg, about 6 mL/kg to about 14 mL/kg, about 7 mL/kg to about 13 mL/kg, about 8 mL/kg to about 12 mL/kg, or about 9 mL/kg to about 11 mL/kg.
• composition comprising a recombinant lentivirus contemplated herein is parenterally administered to an NHP at a rate of dose of about 5 mL/kg, about 6 mL/kg, about 7 mL/kg, about 8 mL/kg, about 9 mL/kg, about 10 mL/kg, about 11 mL/kg, about 12 mL/kg, about 13 mL/kg, about 14 mL/kg, or about 15 mL/kg.
• one or more specimens are taken from the NHP and analyzed.
• Specimens include but are not limited to saliva, blood, plasma, blood cells, lymph, lymph nodes, cerebral spinal fluid, urine, stool, bone, bone marrow, brain, liver, spleen, thymus, stomach, colon, kidneys, heart, lungs, muscle, and other organ tissues.
• a specimen may be analyzed by any method known in the art including but not limited to PCR, qPCR, RT- qPCR, northern blot, southern blot, immunohistochemistry (IHC), flow cytometry, and the like.
• a specimen may be analyzed to measure the amounts and types of cells present including but not limited to the amount of target cells and the amount of genetically modified cells, the amount of cytokines produced, the vector copy number and expansion of the genetically modified cells.
• Specimens may also be used to assess the specificity of the gene therapy by measuring the biodistribution of the recombinant lentivirus in off-target and on-target cells and tissues.
• 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 more specimens are taken from the NHP.
• one or more specimens is taken every day, every other day, every three days, every four days, every five days, every six days, every 7 days, every eight days, every none days, or every ten or more days for a period of about 10 days, about 15 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 36 days, about 37 days, about 38 days, about 39 days, about 40 days, about 41 days, about 42 days, about 43 days, about 44 days, about 45 days, about 46 days, about 47 days, about 48 days, about 49 days, about 50 days, about 51 days, about 52 days, about 53 days, about 54 days, about 55 days, about 56 days, about 57 days, about 58 days, about 59 days, or about 60 days.
• the number of B cells or the extent of B cell aplasia in an NHP administered a recombinant lentivirus contemplated herein is measured in one or more specimens taken from the NHP.
• the number and/or phenotype of T cells in an NHP administered a recombinant lentivirus contemplated herein is measured in one or more specimens taken from the NHP.
• the number of CAR + T cells in an NHP administered a recombinant lentivirus contemplated herein is measured in one or more specimens taken from the NHP.
• the VCN of CAR + T cells (transduced T cells) in an NHP administered a recombinant lentivirus contemplated herein is measured in one or more specimens taken from the NHP.
• one or more cytokines in an NHP administered a recombinant lentivirus contemplated herein is measured in one or more specimens taken from the NHP.
• Illustrative examples of cytokines measured in the one or more specimens include but are not limited to CXCL11, GM-CSF, IFNy, IL- la, IL-ip, IL- IRA, IL-2, IL-5, IL-6, IL-8, IL-10, IL-12, IP-10, MIP-la, and TNFa.
• an NHP is administered a composition comprising a recombinant lentivirus contemplated herein and the number of B cells is decreased in the NHP compared to the number of B cells in the NHP before administration of the composition comprising the recombinant lentivirus.
• the decrease in the number of B cells in the NHP is measurable one, two, three, four, or five days after the administration of the recombinant lentivirus to the NHP.
• the number of B cells in a blood specimen decreases by at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more compared to the number of B cells in a blood specimen before administration of the recombinant lentivirus to the NHP.
• the number of B cells in a blood specimen decreases by at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more within one, two, three, four, or five days after administration compared to the number of B cells in a blood specimen before administration of the recombinant lentivirus to the NHP.
• an NHP administered a recombinant lentivirus contemplated herein is not administered a lymphodepleting chemotherapy prior to, during, or after administration of the recombinant lentivirus.
• a composition comprises a recombinant lentivirus and a pharmaceutically acceptable carrier suitable for enteral or parenteral, e.g., intravascular (intravenous or intraarterial), intraosseous, intraperitoneal, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, intramuscular, and intramedullary, administration and formulation.
• a pharmaceutically acceptable carrier suitable for enteral or parenteral, e.g., intravascular (intravenous or intraarterial), intraosseous, intraperitoneal, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, intramuscular, and intramedullary, administration and formulation.
• lentivirus refers to a complex retrovirus. Among retroviruses, lentiviruses are the most efficient at transducing resting or growth- arrested cells.
• Illustrative lentiviruses suitable for deriving or engineering recombinant lentiviruses contemplated in particular embodiments herein include, but are not limited to human immunodeficiency virus (HIV) including HIV type 1 (HIV-1) and HIV type 2 (HIV-2); visna-maedi vims (VMV); caprine arthritis-encephalitis vims (CAEV); equine infectious anemia vims (EIAV); feline immunodeficiency vims (FIV); bovine immune deficiency vims (BIV); and simian immunodeficiency vims (SIV).
• HBV human immunodeficiency virus
• HIV-1 HIV type 1
• HIV-2 HIV type 2
• VMV visna-maedi vims
• CAEV caprine arthritis-encephalitis vims
• EIAV equine infectious anemia vims
• FIV feline immunodeficiency vims
• BIV bovine
• a recombinant lentivims is derived or engineered from an HIV-1 lentivims.
• a recombinant lentivims comprises an envelope engineered to target and fuse with an immune effector cell.
• Lentiviruses comprise a lentiviral vector and an outer surface, a lipid bilayer, cell membrane, or viral envelope that mediates vims - cell attachment and fusion.
• the outer surface can be pseudotyped or engineered to express one or more non- viral membranebound tropism polypeptides that enable selective and/or specific binding of the vims to the desired cell type.
• the outer surface can also be engineered to express a viral polypeptide that mediates vims - cell fusion but that lacks the ability to bind its cognate receptor on the cell surface of both target and non-target cells.
• the lentivims comprises a viral envelope that specifically binds and fuses to a desired target cell but not a non-target cell.
• a recombinant lentivims comprises an outer surface, a lipid bilayer, cell membrane, or viral envelope comprising one or more mutated viral envelope glycoproteins that mediate fusion of the viral particle with the target host cell but that do not bind its cognate receptor expressed on the target host cell or other non-target cells, and one or more non-viral membrane-bound tropism polypeptides that bind an antigen expressed on the target host cell, e.g. , an immune effector cell.
• one or more mutated viral envelope glycoproteins comprise a vesiculovirus envelope glycoprotein or one or more morbillivirus envelope glycoproteins or henipavirus envelope glycoproteins.
• a mutated viral envelope glycoprotein comprises a heterologous signal peptide that is eventually cleaved after translocation to the ER membrane.
• vesiculoviruses suitable for use in particular embodiments from which G glycoproteins can be isolated include, but are not limited to vesicular stomatitis Alagoas virus (VSAV; Alagoas vesiculovirus), Carajas virus (CJSV; Carajas vesiculovirus), Chandipura virus (CHPV; Chandipura vesiculovirus), Cocal virus (COCV; Cocal vesiculovirus), vesicular stomatitis Indiana vims (VSIV, f.k.a.
• VSAV vesicular stomatitis Alagoas virus
• CJSV Carajas virus
• CHPV Chandipura virus
• COCV Cocal virus
• vesicular stomatitis Indiana vims VSIV, f.k.a.
• VSV Indiana vesiculovirus
• Isfahan vims ISFV; Isfahan vesiculovirus
• Maraba vims MARAV; Maraba vesiculovirus
• Morreton vims MORV; Morreton vesiculovims
• VSNJV vesicular stomatitis New Jersey vims
• Piry vims PIRYV; Piry vesiculovims.
• a vesiculovims G envelope protein comprises one or more amino acid substitutions that that enable the polypeptide to mediate fusion of the viral particle and the target host cell but that ablate the polypeptide’s ability to bind its cognate receptor expressed on the target host cell, e.g. , LDL-R.
• a vesiculovims G envelope protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 15-322 disclosed in U.S. Patent Application No.
• a vesiculovims G envelope protein comprises amino acid substitution at positions 47 and/or 354 (or 358 in some strains, e.g., CJSV, VSNJV) with reference to the vesiculovims G envelope protein lacking a signal peptide.
• a vesiculovims is vesicular stomatitis Indiana vims (VSIV).
• a mutated viral envelope glycoprotein is derived from a VSIV envelope glycoprotein (e.g., SEQ ID NO: 1: KFnVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQA DGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPG FPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHN STTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKA CKMQYCKHWGVREPSGVWFEMADKDEFAAARFPECPEGSSISAPSQTSVDVSEI QDVERIEDYSECQETWSKIRAGEPISPVDESYEAPK
• a mutated viral envelope glycoprotein is derived from a VSIV-G polypeptide set forth in SEQ ID NO: 1 comprising L47I and/or H80Q amino acid substitutions, such substitutions being present in naturally occurring variants of VSIV.
• a mutated VSIV-G envelope protein comprises one or more of: one or more amino acid substitutions at H8, N9, Q10, K47, K50, A51, S183, S179, N180, 1182, M184, Y209, T214, 1347, T350, T352, E353, and R354 (substitution with any amino acid; a conservation substitution; a disruptive substitution; substitution with D, E, A, G, F, or Q; or substitution with A, G, F, or Q); an insertion of TT between N9 and Q10, an insertion of GGS between H8 and N9, an insertion of GGS between N9 and Q10, an insertion of TT between N208 and Y209, an insertion of GGS between P46 and K47, and an insertion of GGS between N208 and Y209; or a deletion of residues 1-8.
• a VSIV-G envelope protein comprises one or more amino acid substitutions at H8, K47, Y209, and R354.
• a VSIV-G envelope protein comprises one or more amino acid substitutions at H8, K47, Y209, and R354, wherein each substitution can be with A, G, F, or Q.
• Amino acid positions are with reference to a VSIV-G envelope protein that lacks a signal peptide, e.g., SEQ ID NO: 1.
• a mutated viral envelope glycoprotein is derived from a VSIV-G polypeptide set forth in SEQ ID NO: 1 comprising L47I and/or H80Q amino acid substitutions, such substitutions present in naturally occurring variants of VSIV.
• a mutated VSIV-G polypeptide comprises one or more amino acid substitutions at K47, 1182, and/or R354 (substitution with any amino acid; a conservation substitution; a disruptive substitution; substitution with D, E, A, G, F, or Q; or substitution with A, G, F, or Q).
• a mutated VSIV-G polypeptide comprises amino acid substitutions at K47, 1182, or R354; K47 and 1182; K47 and R354; 1182 and R354; or at K47, 1182, and R354 of SEQ ID NO: 1.
• a mutated VSIV-G polypeptide comprises one or more of the following amino acid substitutions: K47A, K47Q, I182E, I182D, R354A, and/or R354Q.
• a mutated VSIV-G polypeptide comprises the following amino acid substitutions: K47A, K47Q, I182E, I182D, R354A, or R354Q; K47A and I182E; K47A and I182D; K47Q and I182E; K47Q and I182D; I182E and R354A; I182E and R354Q; I182D and R354A; I182D and R354Q; K47A and R354A; K47A and R354Q; K47Q and R354A; K47Q and R354Q; K47Q and R354Q; K47A, I182E, and R354A; K47A, I182D, and
• a VSIV-G envelope protein comprises one or more amino acid substitutions at H8, K47, Y209, and R354.
• a VSIV- G envelope protein comprises one or more amino acid substitutions at H8, K47, Y209, and R354, wherein each substitution can be with A, G, F, or Q.
• a VSIV-G envelope protein comprises one or more amino acid substitutions at K47 and R354 (with reference to a VSIV-G envelope protein that lacks a signal peptide, e.g., SEQ ID NO: 1).
• a VSIV-G envelope protein comprises one or more amino acid substitutions selected from the group consisting of: K47A and R354A; K47A and R354G; K47A and R354F; K47A and R354Q; K47G and R354A; K47G and R354G; K47G and R354F; K47G and R354Q;K47F and R354A; K47F and R354G; K47F and R354F; K47F and R354Q; K47Q and R354A; K47Q and R354G; K47Q and R354F; and K47Q and R354A; K47Q and R354G; K47Q and R354F; and K47Q and R354Q.
• a VSIV-G envelope comprises the amino acid substitutions K47Q or K47A and R354A or R354Q. Amino acid positions are with reference to a VSIV-G envelope protein that lacks a signal peptide.
• a mutated VSIV-G envelope protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13-76 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical thereto that mediates fusion of the viral particle and a cell but that substantially ablates or ablates the polypeptide’s ability to bind its cognate receptor expressed on a cell, e.g., LDL-R.
• a mutated VSIV-G envelope protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 61-76 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical thereto that mediates fusion of the viral particle and a cell but that substantially ablates or ablates the polypeptide’s ability to bind its cognate receptor expressed on a cell, e.g., LDL- R.
• a mutated VSIV-G envelope protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 61, 65, 69, and 73 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical thereto that mediates fusion of the viral particle and a cell but that substantially ablates or ablates the polypeptide’s ability to bind its cognate receptor expressed on a cell, e.g., LDL-R.
• Table 2
• a vesiculovirus is cocal virus (COCV).
• COCV-G COCV envelope glycoprotein
• SEQ ID NO: 3 KFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDEEGITMKVKMPKTHKAIQA DGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGF PPQNCGYATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHN STVWYSDYKVTGLCDATLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKV CKMNYCKHAGVRLPSGVWFEFVDQDVYAAAKLPECPVGATISAPTQTSVDVSLI LDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNP
• a COCV-G envelope protein comprises one or more amino acid substitutions at K47 and/or R354.
• a COCV-G envelope protein comprises one or more amino acid substitutions at K47 and/or R354, wherein each amino acid can be substituted with A, G, F, or Q.
• a COCV-G envelope protein comprises one or more amino acid substitutions at K47 and/or R354 (with reference to a COCV-G envelope protein that lacks a signal peptide, e.g., SEQ ID NO: 3).
• a COCV-G envelope protein comprises one or more amino acid substitutions selected from the group consisting of: K47A and R354A; K47A and R354G; K47A and R354F; K47A and R354Q; K47G and R354A; K47G and R354G; K47G and R354F; K47G and R354Q;K47F and R354A; K47F and R354G; K47F and R354F; K47F and R354Q; K47Q and R354A; K47Q and R354G; K47Q and R354F; and K47Q and R354A; K47Q and R354G; K47Q and R354F; and K47Q and R354Q.
• a mutated COCV-G envelope protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 77-92 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical thereto that mediates fusion of the viral particle and a cell but that substantially ablates or ablates the polypeptide’s ability to bind its cognate receptor expressed on a cell, e.g., LDL-R.
• a mutated COCV-G envelope protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 89-92 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical thereto that mediates fusion of the viral particle and a cell but that substantially ablates or ablates the polypeptide’s ability to bind its cognate receptor expressed on a cell, e.g., LDL- R.
• a mutated VSIV-G envelope protein comprises an amino acid sequence set forth in SEQ ID NOs: 89 or 92 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical thereto that mediates fusion of the viral particle and a cell but that substantially ablates or ablates the polypeptide’s ability to bind its cognate receptor expressed on a cell, e.g., LDL-R.
• one or more mutated morbillivirus envelope glycoproteins are derived from measles virus F (MV-F) and measles virus H (MV-H).
• a recombinant particle comprises one or more measles virus viral envelope glycoproteins modified to lack cell binding activity and retain fusogenic activity.
• a recombinant particle comprises a modified MV-F glycoprotein and an MV-H glycoprotein modified to lack cell binding activity and retain fusogenic activity.
• one or more mutated morbillivirus envelope glycoproteins are derived from measles virus F (MV-F) polypeptide (e.g., SEQ ID NO: 5: QIHWGNLSKIGVVGIGSASYKVMTRSSHQSLVIKLMPNITLLNNCTRVEIAEYRRL LRTVLEPIRDALNAMTQNIRPVQSVASSRRHKRFAGVVLAGAALGVATAAQITA GIALHQSMLNSQAIDNLRASLETTNQAIEAIRQAGQEMILAVQGVQDYINNELIPS MNQLSCDLIGQKLGLKLLRYYTEILSLFGPSLRDPISAEISIQALSYALGGDINKVLE KLGYSGGDLLGILESRGIKARITHVDTESYFIVLSIAYPTLSEIKGVIVHRLEGVSYN IGSQEWYTTVPKYVATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLR GSTKSCARTLVSGSFGNRF
• the MV-H polypeptide comprises one or more amino acid substitutions at positions Y463, R515, S530, and F531 of a MV-H polypeptide (e.g., SEQ ID NO 6).
• the MV-H polypeptide comprises one or more of the amino acid substitutions Y463A, R515A, S530L, and F531S in an MV-H polypeptide (e.g., SEQ ID NO: 7:
• one or more mutated henipaviruses envelope glycoproteins are derived from nipah virus F (NiV-F) and nipah vims G (NiV-G).
• a recombinant particle comprises one or more nipah vims viral envelope glycoproteins modified to lack cell binding activity and retain fusogenic activity.
• a recombinant particle comprises a modified NiV-F glycoprotein and a NiV-G glycoprotein modified to lack cell binding activity and retain fusogenic activity.
• one or more mutated henipavirus envelope glycoproteins are derived from a nipah virus F (NiV-F) polypeptide (e.g., SEQ ID NO: 8: LHYEKLSKIGLVKGVTRKYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTR LNGILTPIKGALEIYKNNTHDLVGDVRLAGVIMAGVAIGIATAAQITAGVALYEA MKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCK QTELSLDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYA TEDFDDLLESDSITGQIIYVDLSSYYUVRVYFPILTEIQQAYIQELLPVSFNNDNSEW ISIVPNFILVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPREL VVSSHVPRF
• the NiV-G polypeptide comprises one or more amino acid substitutions at positions E468, W471, Q497, and E500 of an NiV-G polypeptide (e.g., SEQ ID NO: 9).
• the NiV-G polypeptide comprises one or more of the amino acid substitutions E468A, W471A, Q497A, and E500A in an NiV-G polypeptide (e.g., SEQ ID NO: 10: MKKINEGLLDSKILSAFNTVIALLGSIVUVMNIMIIQNYTRSTDNQAVIKDALQGIQ QQIKGLADKIGTEIGPKVSLIDTSSTITIPANIGLLGSKISQSTASINENVNEKCKFTL PPLKIHECNISCPNPLPFREYRPQTEGVSNLVGLPNNICLQKTSNQILKPKLISYTLP VVGQSGTCITDPLLAMDEGYFAYSHLERIGSCSRGVSKQRIIGVGEVLDRGDEVPS LFMTNVW
• a recombinant lentivirus contemplated herein comprises an outer surface, a lipid bilayer, cell membrane, or viral envelope comprising or expressing one or more non- viral membrane bound tropism polypeptides that are engineered to specifically bind a target host cell.
• a recombinant lentivirus contemplated herein comprises a viral envelope comprising or expressing a primary non- viral membrane bound tropism polypeptide and a secondary non- viral membrane bound tropism polypeptide that are both engineered to specifically bind a target host cell.
• a non- viral membrane bound tropism polypeptide comprises an extracellular antigen targeting domain, a spacer domain, and a transmembrane domain.
• a non- viral membrane bound tropism polypeptide comprises from N-terminus to C-terminus, an extracellular antigen targeting domain, a spacer domain, and a transmembrane domain.
• a non- viral membrane bound tropism polypeptide comprises an extracellular antigen targeting domain, a spacer domain, a transmembrane domain, and a cytoplasmic tail (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or more).
• a non- viral membrane-bound tropism polypeptide comprises an extracellular antigen targeting domain that binds an antigen expressed on a target host cell.
• a non-viral membrane-bound tropism polypeptide comprises an extracellular antigen targeting domain that binds an antigen expressed on an immune effector cell.
• a non-viral membrane- bound tropism polypeptide comprises an extracellular antigen targeting domain that comprises an antibody or antigen binding fragment thereof that binds an antigen expressed on the surface of an immune effector cell.
• a non- viral membrane-bound tropism polypeptide comprises an extracellular antigen targeting domain that comprises an antibody or antigen binding fragment selected from the group consisting of: a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, a single chain Fv protein (scFv), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), a singledomain antibody (sdAb, a camelid VHH, Nanobody), and a centyrin.
• an antibody or antigen binding fragment selected from the group consisting of: a Camel Ig, a Llama Ig, an Alpaca Ig, I
• a non- viral membrane-bound tropism polypeptide comprises an extracellular antigen targeting domain that comprises an scFv or one or more VHHs that bind an antigen expressed on an immune effector cell.
• a non- viral membrane-bound tropism polypeptide comprises an extracellular antigen targeting domain that binds the alpha, beta, gamma, or delta chain of the T cell receptor, CD2, CD35, CD3s CD3y, CD4, CD8a, and CD8p.
• the extracellular antigen targeting domain comprises an scFv, a murine scFv, a humanized scFv, or a human scFv or one or more VHHs that binds the alpha, beta, gamma, or delta chain of the T cell receptor, CD2, CD35, CD3s CD3y, CD4, CD8a, and CD8p.
• a non- viral membrane-bound tropism polypeptide comprises an anti-CD3s antibody or antigen binding fragment thereof.
• anti-CD3s antibodies or antigen binding fragments thereof suitable for using in particular embodiments include scFvs or other antigen binding fragments isolated from clone 10D12 (Miltenyi), clone REA994 (Miltenyi), CD3-1 (Mabtech), and SP34-2 (BD Biosciences), and variants thereof that bind NHP CD3s having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
• a non- viral membrane-bound tropism polypeptide comprises an anti-CD3s scFv, a spacer domain, a transmembrane domain, and a cytoplasmic tail (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or more).
• a non- viral membrane-bound tropism polypeptide comprises an extracellular antigen targeting domain; spacer domain comprising a hinge domain or stalk obtained or isolated from a polypeptide selected from the group consisting of CD3, CD4, CD8a, CD8P, CD28, or Glycophorin A; and/or a transmembrane domain isolated from a polypeptide selected from the group consisting of CD3, CD4, CD8a, CD8P, CD28, or Glycophorin A; and optionally, a truncated intracellular domain isolated from a polypeptide selected from the group consisting of CD3, CD4, CD8a, CD8P, CD28, or Glycophorin A or a cytoplasmic tail of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or more.
• a non- viral membrane-bound tropism polypeptide comprises an extracellular antigen targeting domain that binds the alpha, beta, gamma, or delta chain of the T cell receptor, CD2, CD35, CD3s CD3y, CD4, CD8a, and CD8P; spacer domain comprising a hinge domain or stalk obtained or isolated from a polypeptide selected from the group consisting of CD3, CD4, CD8a, CD8P, CD28, or Glycophorin A; and/or a transmembrane domain isolated from a polypeptide selected from the group consisting of CD3, CD4, CD8a, CD8P, CD28, or Glycophorin A; and optionally, a truncated intracellular domain isolated from a polypeptide selected from the group consisting of CD3, CD4, CD8a, CD8P, CD28, or Glycophorin A or a cytoplasmic tail of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or more.
• a recombinant lentivirus comprises an envelope that comprises (a) one or more mutated viral envelope glycoproteins that mediate fusion of the viral particle with the target host cell but that do not bind its cognate receptor expressed on the target host cell or other non-target cells; (b) a non- viral membrane-bound tropism polypeptide comprises an extracellular antigen targeting domain that binds the alpha, beta, gamma, or delta chain of the T cell receptor, CD2, CD35, CD3s CD3y, CD4, CD8a, and CD8P; spacer domain comprising a hinge domain or stalk obtained or isolated from a polypeptide selected from the group consisting of CD3, CD4, CD8a, CD8P, CD28, or Glycophorin A; and/or a transmembrane domain isolated from a polypeptide selected from the group consisting of CD3, CD4, CD8a, CD8P, CD28, or Glycophorin A; and optionally, a truncated intra
• a secondary non- viral membrane bound tropism polypeptide comprises an extracellular antigen targeting domain that binds a T cell surface antigen including but not limited to the alpha or beta chains of a TCR, CD28, CD134 (0X40), CD137 (4-1BB), and CD278 (ICOS).
• a secondary non- viral membrane bound tropism polypeptide comprises an extracellular antigen targeting domain comprising an antibody or antigen binding fragment thereof that specifically binds a T cell surface antigen including but not limited to the alpha or beta chains of a TCR, CD28, CD134 (0X40), CD137 (4- 1BB), and CD278 (ICOS).
• a secondary non- viral membrane bound tropism polypeptide comprises all or part of a co- stimulatory molecule including but not limited to CD80, CD86, OX40L, 4-1BBL, and ICOSL or a functional fragment thereof.
• the functional fragment is a CD80, CD86, OX40L, 4-1BBL, and ICOSL comprising a C- terminal truncation that results in a cytoplasmic domain of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or more.
• a recombinant lentivirus comprises an envelope that comprises (a) one or more mutated viral envelope glycoproteins that mediate fusion of the viral particle with the target host cell but that do not bind its cognate receptor expressed on the target host cell or other non-target cells; (b) a non- viral membrane-bound tropism polypeptide comprises an extracellular antigen targeting domain that binds the alpha, beta, gamma, or delta chain of the T cell receptor, CD2, CD35, CD3s CD3y, CD4, CD8a, and CD8P, a spacer domain, and a transmembrane domain; and further comprises a secondary non- viral membrane bound tropism polypeptide comprising all or part of a co- stimulatory molecule including but not limited to CD80, CD86, OX40L, 4-1BBL, and ICOSL or a functional fragment thereof, e.g., a CD80, CD86, OX40L, 4-1BBL, and ICOSL, comprising
• Lentiviral vectors are useful tools to deliver genetic material to cells.
• a “lentiviral vector” is a nucleic acid molecule derived from a lentiviral genome that is used to transfer or deliver another nucleic acid from a recombinant lentivirus into a cell and/or into the cell’s genome.
• a recombinant lentiviral vector is based on or derived from a lentivirus genome that has been engineered to remove lentiviral accessory proteins but leave elements intact for packaging, reverse transcription and integration.
• Recombinant lentiviruses contemplated herein comprise genomes that lack one or more lentiviral accessory genes, e.g., the genes env, vif, vpr, vpu and nef, thereby increasing the safety of the lentiviruses.
• a recombinant lentivirus contains two copies of a lentiviral vector, a genomic RNA comprising backbone sequences derived from a lentivirus genome.
• lentiviral vector sequences can be used, or combined and numerous substitutions and alterations in certain of the lentiviral vector sequences may be accommodated without impairing the ability of a vector to package polynucleotides into a recombinant lentivirus or lentiviral particle and to transfer the polynucleotide into a host cell.
• Illustrative examples of lentiviral vectors suitable for use in particular embodiments contemplated herein include but are not limited to those described in Naldini et al., (1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Pat. Nos. 6,013,516; and U.S. Pat. No. 5,994,136, each of which are incorporated herein by reference in their entireties.
• a lentiviral vector contemplated herein comprises one or more LTRs, and one or more, or all, of the following accessory elements: a Psi ( ) packaging signal, a cPPT/FLAP, an export element, poly (A) sequences, and may optionally comprise a WPRE or HPRE, an insulator element, a selectable marker, and/or a cell suicide gene.
• a lentiviral vector integrates into the host cell genome.
• a lentiviral vector is integration defective, episomal, and does not integrate in the host cell genome.
• integration defective lentivirus or “IDLV” refers to a lentivirus having an integrase that lacks the capacity to integrate the viral vector into the host cell genome.
• HIV-1 integrase suitable to reduce integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, DI 161, D116A, N120G, N120I, N120E, E152G, E152A, K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199C, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221L, W235F, W235E, K236S, K236A, K246A, G247W, D253A,
• an HIV-1 integrase deficient integrase comprises a D64V, D16H, DI 16A, E152G, or E152A mutation; D64V, DI 16A, and E152G mutations; D64V, DI 16A, and E152A mutations; or a D64V mutation.
• a “long terminal repeat” or “LTR” in its natural unmodified sequence context is a direct repeat and contains U3, R and U5 regions domains located at the ends of the lentiviral genome. LTRs generally provide functions fundamental to lentiviral gene expression and replication.
• the U3 region contains the enhancer and promoter elements to initiate transcription of the viral genome;
• the R region contains a trans-activation responsive (TAR) element that mediates activation of transcription through its binding to the Tat viral protein and a polyadenylation sequence; and the U5 region and adjacent primer binding site (PBS) play important roles in initiating reverse transcription.
• TAR trans-activation responsive
• PBS primer binding site
• a “FLAP element” or “cPPT/FLAP” refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a lentivirus, e.g., HIV-1 or HIV-2.
• a lentivirus e.g., HIV-1 or HIV-2.
• Suitable FLAP elements are described in U.S. Pat No. 6,682,907 and in Zennou, et al., 2000, Cell, 101:173.
• a “packaging signal” or “packaging sequence” refers to Psi
• RNA export element refers to a cis-acting post-transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell.
• RNA export elements include, but are not limited to, the human immunodeficiency vims (HIV-1) rev response element (RRE) (see e.g., Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423.
• heterologous sequences in viral vectors may be increased by incorporating posttranscriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vectors.
• posttranscriptional regulatory elements can increase expression of a heterologous nucleic acid, e.g., woodchuck hepatitis vims posttranscriptional regulatory element (WPRE; Zufferey et al., J. Virol. 73: 2886, 1999); the posttranscriptional regulatory element present in hepatitis B vims (HPRE) (Huang et al., Mol. Cell. Biol. 13:7476-7486, 1993); and the like (Liu et al., Genes Dev. 9:1766, 1995).
• WPRE woodchuck hepatitis vims posttranscriptional regulatory element
• HPRE posttranscriptional regulatory element present in hepatitis B vims
• Lentiviral vectors preferably contain several safety enhancements as a result of modifying the LTRs.
• “Self-inactivating” (SIN) vectors refer to lentiviral vectors in which a deletion in the U3 region of the 3' LTR to prevent viral transcription beyond the first round of viral replication. HIV-1 based lentivectors can tolerate significant U3 deletions, including the removal of the LTR TATA box (e.g., deletions from -418 to -18), without significant reductions in vector titers.
• An additional safety enhancement is provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during production of viral particles.
• heterologous promoters examples include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex vims (HSV) (thymidine kinase) promoters.
• SV40 viral simian virus 40
• CMV cytomegalovirus
• MoMLV Moloney murine leukemia virus
• RSV Rous sarcoma virus
• HSV herpes simplex vims
• an HIV-1 based lentiviral vector (z.e., a lentiviral vector derived from HIV-1) comprises a polynucleotide comprising or encoding a promoter operably linked to a polynucleotide encoding an engineered antigen receptor.
• a recombinant lentivirus contemplated herein comprises a lentiviral vector comprising a polynucleotide encoding a promoter operably linked to a polynucleotide encoding one or more engineered antigen receptors.
• Engineered antigen receptors include but are not limited to a chimeric antigen receptor (CAR), a chimeric costimulatory receptor (CCR), an alpha-beta T cell receptor (aP TCR), a gamma delta T cell receptor (y5 TCR), a dimerizing agent regulated immunoreceptor complex (DARIC), a chimeric TGF-P receptor (CTBR), and a zetakine receptor.
• CAR chimeric antigen receptor
• CCR chimeric costimulatory receptor
• aP TCR alpha-beta T cell receptor
• y5 TCR gamma delta T cell receptor
• DARIC dimerizing agent regulated immunoreceptor complex
• the engineered antigen receptor is designed redirect an immune effector cell to bind or target an antigen on a target cell, e.g., a B cell.
• the engineered antigen receptor comprises an extracellular antigen binding domain that binds to alpha folate receptor (FRa), avP6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD16, CD 19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), C- type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T cell lymphoma-associated antigen 1 (FRa), avP
• the engineered antigen receptor comprises an extracellular antigen binding domain that binds to a B cell antigen selected from the group consisting of CDlc, CD2, CD5, CD6, CDlla, CDllc, CD14, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD35, CD37, CD38, CD39, CD40, CD43, CD45RA, CD45RB, CD45RO, CD49c, CD49d, CD51, CD57, CD62L, CD69, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD83, CD84, CD85, CD86, CD89, CD98, CD95, CD99, CD100, CD106, CD107a, CDwlO8, CDwl21b (IL-1RII), CDwl24 (IL-4R), CDwl27 (IL-7R), CD184, CD185, CD194, CD
• the engineered antigen receptor comprises an extracellular antigen binding domain that binds to BCMA, CD19, CD20, CD22, CD38, CD79A, CD79B, and GPCR5D.
• a lentiviral vector comprises a polynucleotide encoding a promoter operably linked to a polynucleotide encoding a TCR.
• Naturally occurring TCRs comprise two subunits, an alpha chain and a beta chain subunit (aPTCR), or a gamma chain and a delta chain subunit (ySTCR), each of which is a unique protein produced by a recombination event in each T cell’s genome.
• the TCR is an aPTCR.
• the TCR is a ySTCR.
• polynucleotides encoding TCRs are preferably isolated from their natural context in a (naturally-occurring) chromosome of a T cell and incorporated into a lentiviral vector contemplated herein.
• the TCR is an exogenous TCR because it is introduced into immune effector cells that do not normally express the particular TCR.
• the essential aspect of the TCRs is that it has high avidity for a tumor antigen presented by a major histocompatibility complex (MHC) or similar immunological component.
• MHC major histocompatibility complex
• CARs are engineered to bind target antigens in an MHC independent manner.
• a TCR can be expressed with additional polypeptides attached to the aminoterminal or carboxyl-terminal portion of the alpha chain or beta chain of a TCR, or of the gamma chain or delta chain of a TCR so long as the attached additional polypeptide does not interfere with the ability of the chains to form a functional T cell receptor and recognize antigen.
• Antigens that are recognized by the engineered TCRs contemplated in particular embodiments include, but are not limited to B cell antigens contemplated herein.
• a lentiviral vector comprises a polynucleotide encoding a promoter operably linked to a polynucleotide encoding a chimeric antigen receptor (CAR).
• CARs combine antibody-based specificity to a desired antigen with a T cell receptor-activating intracellular domain to generate a chimeric to redirect immune effector cell specificity in a major histocompatibility (MHC) independent manner.
• MHC major histocompatibility
• CARs exploit cell- specific targeting abilities of monoclonal antibodies, soluble ligands or cell specific coreceptors and thereby triggering proliferation of immune effector cells, cytokine production, phagocytosis or production of molecules that can mediate cell death of the target antigen expressing cell.
• a CAR comprises an extracellular antigen binding domain that binds to a specific target antigen, a hinge domain, a transmembrane domain and one or more intracellular signaling domains.
• the present disclosure contemplates all permutations of CAR architectures, from first generation CARs to third generation CARs.
• a first-generation CAR comprises an extracellular antigen binding domain, a hinge domain, a transmembrane domain, and a primary signaling domain
• a second-generation CAR comprises an extracellular antigen binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and a primary signaling domain
• a third-generation CAR comprises an extracellular antigen binding domain, a hinge domain, a transmembrane domain, two costimulatory signaling domains and a primary signaling domain.
• an antigen binding domain includes any naturally occurring, synthetic, semisynthetic, or recombinantly produced binding partner for a biological molecule of interest.
• the extracellular binding domain comprises an antibody or antigen binding fragment thereof.
• the extracellular binding domain comprises an antibody or antigen binding fragment thereof is selected from the group consisting of: a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, an single chain Fv protein (“scFv”), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single-domain antibody (sdAb, a camelid VHH, Nanobody).
• the CAR comprises an extracellular domain that binds an antigen selected from the group consisting of: CDlc, CD2, CD5, CD6, CD1 la, CD11c, CD14, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD35, CD37, CD38, CD39, CD40, CD43, CD45RA, CD45RB, CD45RO, CD49c, CD49d, CD51, CD57, CD62L, CD69, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD83, CD84, CD85, CD86, CD89, CD98, CD95, CD99, CD100, CD106, CD107a, CDwlO8, CDwl21b (IL-1RII), CDwl24 (IL-4R), CDwl27 (IL- 7R), CD184, CD185, CD194, CD196, CD197, CD269, CD40,
• HVEM CD272
• BTLA-4 CD273
• PD-L1 CD274
• CD276 TACI
• CD278 BAFF-R
• CD279 CD300
• CMRF35 CCR10
• K light chain X light chain
• IL- 10R IL-11R
• IL-13Ral LTK
• Sca-1 SlamF6, and TNFRS25.
• the CAR comprises an extracellular antigen binding domain that binds to BCMA, CD19, CD20, CD22, CD38, CD79A, CD79B, and GPCR5D.
• the CAR comprises a LEU 16 scFv directed against
• a CAR comprises a hinge domain.
• the hinge domain comprises the CH2 and CH3 domains of IgGl, IgG4, or IgD.
• Illustrative hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD28, CD8a, and CD4, which may be wild-type hinge regions from these molecules or may be altered.
• the transmembrane (TM) domain of the CAR fuses the extracellular binding portion and intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell.
• the TM domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
• Illustrative TM domains may be derived from (z.e., comprise) at least the transmembrane region(s) of the alpha, beta, gamma, or delta chain of the T-cell receptor, CD35, CD3s, CD3y, CD3 ⁇ , CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PD-1.
• a CAR contemplated herein comprises a TM domain and a short oligo- or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length that links the TM domain and the adjacent intracellular signaling domain of the CAR.
• a glycine-serine linker provides a particularly suitable linker.
• a CAR comprises an intracellular signaling domain that comprises one or more “co- stimulatory signaling domains” and a “primary signaling domain.”
• Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.
• IT AM containing primary signaling domains suitable for use in CARs contemplated in particular embodiments include those derived from FcRy, FcRp, CD3y, CD35, CD3s, CD3( ⁇ , CD22, CD79a, CD79b, and CD66d.
• a CAR comprises a CD3( ⁇ primary signaling domain and one or more costimulatory signaling domains.
• co- stimulatory domains suitable for use in CARs contemplated in particular embodiments include those isolated from TERI, TER2, TER3, TER4, TER5, TER6, TER7, TER8, TER9, TER10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD 134 (0X40), CD 137 (4- IBB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70.
• a CAR comprises one or more co- stimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3 ⁇ primary signaling domain.
• a CAR comprises: an extracellular domain that binds to
• the CAR comprises a polynucleotide sequence set forth in SEQ ID NO: 11 and an amino acid sequence set forth in SEQ ID NO: 12.
• a lentiviral vector comprising a polynucleotide encoding s CAR comprises a polynucleotide sequence encoding a signal peptide but that when the CAR is processed and translocated to the ER membrane the signal peptide is cleaved such that the CAR is expressed on a transduced immune effector cell without the signal peptide.
• Chimeric costimulatory receptors are molecules that combine antibodybased specificity to a desired antigen with a T cell receptor-costimulatory domain but that lack a primary signaling domain. CCRs redirect immune effector cell specificity in an MHC independent manner and enhance the immune effector cell response in the presence of a CAR.
• a lentiviral vector comprises a polynucleotide encoding a promoter operably linked to a polynucleotide encoding a CAR that comprises a first costimulatory domain and a primary signaling domain; a ribosomal skip or self-cleaving viral peptide; and a CCR comprises a second costimulatory domain, wherein the first and second costimulatory domains are different from each other and synergize to immune effector cell proliferation, persistence, cytokine production, and/or phagocytosis or production of molecules that can mediate cell death of the target antigen expressing cell.
• a CCR that comprises an extracellular antigen binding domain comprising an antibody or antigen binding fragment thereof; a hinge domain; a transmembrane domain; and a costimulatory signaling domains.
• a CCR that comprises an extracellular antigen binding domain comprising an antibody or antigen binding fragment thereof that binds a B cell antigen that is different from the B cell antigen bound by a CAR.
• a CCR comprises an extracellular antigen binding domain that binds BCMA, CD19, CD20, CD22, CD38, CD79A, CD79B, and GPCR5D; a hinge domain selected from the group consisting of a CD4 hinge, a CD8P hinge, a CD8a hinge, a CD28 hinge, a CD 134 hinge, a CD 137 hinge, a CD 152 hinge, an IgGl hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge; a transmembrane domain isolated or derived from a polypeptide selected from the group consisting of an alpha, beta, gamma, or delta chain of the T-cell receptor, CD35, CD3s, CD3y, CD3 ⁇ , CD4, CD5, CD8a, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD 154, CD278, am
• a lentiviral vector comprises a polynucleotide encoding a promoter operably linked to a polynucleotide encoding a DARIC.
• a DARIC provides a binding component and a signaling component that are each expressed as separate fusion proteins comprising an extracellular multimerization domain.
• the multimerization domains of the components associate by binding to a bridging factor (see U.S. Pat. Appl. No. 2016/0311901, which is incorporated herein by reference in its entirety).
• a bridging factor leads to formation of the DARIC system components, formation of the DARIC complex does not produce significant signaling on its own.
• a DARIC binding component comprises an antigen binding domain that binds to an antigen selected from the group consisting of: CDlc, CD2, CD5, CD6, CD1 la, CD11c, CD14, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD35, CD37, CD38, CD39, CD40, CD43, CD45RA, CD45RB, CD45RO, CD49c, CD49d, CD51, CD57, CD62L, CD69, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD83, CD84, CD85, CD86, CD89, CD98, CD95, CD99, CD100, CD106, CD107a, CDwl
• the DARIC comprises an extracellular antigen binding domain that binds to BCMA, CD19, CD20, CD22, CD38, CD79A, CD79B, and GPCR5D.
• the DARIC binding component comprises a LEU 16 scFv directed against CD20.
• Polypeptides, fusion polypeptides, and polypeptide variants are contemplated herein.
• Exemplary polypeptides include, but not limited to, mutated viral envelope glycoproteins, non-viral membrane-bound tropism polypeptides, CAR polypeptides, CCR polypeptides, DARIC binding and signaling components, CTBRs, zetakines, fusion polypeptides and fragments thereof, e.g., SEQ ID NOs: 1-10, and 12-126.
• Polypeptide,” “peptide,” and “protein” are used interchangeably, unless specified to the contrary, and according to conventional meaning, i.e., as a sequence of amino acids.
• Polypeptides are not limited to a specific length, e.g., they may comprise a full-length protein sequence, a fragment of a full-length protein, or a fusion protein, and may include post-translational modifications, e.g., glycosylations, acetylations, phosphorylations and the like.
• isolated peptide or an “isolated polypeptide” is a synthetic polypeptide, a semi-synthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.
• Polypeptides include “polypeptide variants.” Polypeptide variants may differ from a naturally occurring polypeptide in one or more amino acid substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated. In particular embodiments, polypeptides include polypeptide variants having at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity to any reference sequence contemplated herein, typically where the variant maintains at least one biological activity of the reference sequence.
• a polypeptide variant is a viral envelope glycoprotein that has been modified to preserve its fusogenic activity and disable its binding activity to its cognate receptor expressed on a cell.
• a polypeptide variant is a viral envelope glycoprotein that has at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity to a reference sequence and that has been modified to preserve its fusogenic activity and disable its binding activity to its cognate receptor expressed on a cell.
• Polypeptides variants include biologically active “polypeptide fragments.”
• biologically active polypeptide fragments include but are not limited to signal peptides, binding domains, hinges, transmembrane domains, intracellular domains, and the like.
• biologically active fragment or minimal biologically active fragment refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the naturally occurring polypeptide activity.
• a biologically active fragment is a polypeptide comprising an N-terminal and/or C-terminal truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.
• a mutated viral envelope glycoprotein or non- viral membrane-bound tropism polypeptide comprises a truncation of the cytoplasmic domain that results in a cytoplasmic tail or stub of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or more.
• Polypeptides contemplated in particular embodiments comprise a signal peptide.
• Signal peptides are often present at the N-terminus of polypeptides and assist in the trafficking, processing and maturation of the expressed polypeptide.
• the signal peptide is usually cleaved in the ER and the mature polypeptide comprises an amino acid sequence lacking the signal peptide.
• Illustrative examples of signal peptides suitable for use in particular DARIC signaling components include but are not limited to an IgGl heavy chain signal polypeptide, an IgK light chain signal polypeptide, a CD8a signal polypeptide, or a human GM-CSF receptor alpha signal polypeptide.
• a polypeptide comprises GM-CSFa signal polypeptide.
• polypeptides contemplated herein may comprise one or more amino acids denoted as “X” or “X n ” wherein n is an integer that denotes the particular X amino acid. “X” if present in an amino acid SEQ ID NO, refers to any one or more amino acids or particular amino acids if disclosed.
• polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
• amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (Proc. Natl. Acad. Sci. USA. 82: 488-492. (1985)), Kunkel et al., (Methods in Enzymol, 154: 367- 382. (1987)), U.S. Pat. No. 4,873,192, Watson, J. D.
• a polypeptide variant comprises one or more conservative substitutions.
• a “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties.
• a conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
• Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
• polypeptide sequences encoding them can be separated by an IRES sequence as disclosed elsewhere herein or a polypeptide cleavage signal.
• two or more polypeptides can be expressed as a fusion protein that comprises one or more polypeptide cleavage signals between the polypeptides.
• Exemplary polypeptide cleavage signals include, but are not limited to, protease cleavage sites, nuclease cleavage sites and ribosomal skipping polypeptides or self-cleaving viral polypeptides (see, e.g., in Ryan etal., 1997. J. Gener. Virol. 78, 699-722; deFelipe and Ryan, 2004. Traffic, 5(8); 616-26; and Scymczak et al. (2004) Nature Biotech. 5, 589-594).
• Exemplary protease cleavage sites include, but are not limited to the cleavage sites of potyvirus NIa proteases (e.g, tobacco etch virus protease), poty virus HC proteases, potyvirus PI (P35) proteases, byovirus NIa proteases, byovirus RNA-2-encoded proteases, aphthovirus L proteases, enterovirus 2A proteases, rhinovirus 2 A proteases, picoma 3C proteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase.
• potyvirus NIa proteases e.g, tobacco etch virus protease
• potyvirus PI (P35) proteases by
• ribosomal skipping polypeptides include but are not limited to: a viral 2A peptide or sequence (Donnelly et ah, 2001. J. Gen. Virol. 82: 1027- 1041).
• the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.
• the viral 2A peptide is selected from the group consisting of: a foot-and-mouth disease virus (FMDV) 2A peptide, an equine rhinitis A vims (ERAV) 2A peptide, a Thosea asigna vims (TaV) 2A peptide, a porcine teschovims-1 (PTV-1) 2A peptide, a Theilovims 2A peptide, and an encephalomyocarditis vims 2A peptide.
• FMDV foot-and-mouth disease virus
• EAV equine rhinitis A vims
• TaV Thosea asigna vims
• PTV-1 porcine teschovims-1
• viral 2A sequences include, but are not limited to: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 107); ATNFSLLKQAGDVEENPGP (SEQ ID NO: 108); LLKQAGDVEENPGP (SEQ ID NO: 109); GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 110); EGRGSLLTCGDVEENPGP (SEQ ID NO: 111); LLTCGDVEENPGP (SEQ ID NO: 112);
• GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 113);
• QCTNYALLKLAGDVESNPGP (SEQ ID NO: 114); LLKLAGDVESNPGP (SEQ ID NO: 115); GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 116);
• VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 117); LLNFDLLKLAGDVESNPGP (SEQ ID NO: 118); TLNFDLLKLAGDVESNPGP (SEQ ID NO: 119);
• NFDLLKLAGDVESNPGP (SEQ ID NO: 120); QLLNFDLLKLAGDVESNPGP (SEQ ID NO: 121); APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 122);
• VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT (SEQ ID NO: 123); LNFDLLKLAGDVESNPGP (SEQ ID NO: 124);
• LLAIHPTEARHKQKIV APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 125); and EARHKQKIV APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 126).
• a lentiviral vector comprises or encodes a promoter operably linked to a polynucleotide encoding a fusion protein.
• Polynucleotides encoding mutated viral envelope glycoproteins, non-viral membrane-bound tropism polypeptides, CAR polypeptides, CCR polypeptides, DARIC binding and signaling components, CTBRs, zetakines, fusion polypeptides and variants thereof are contemplated herein.
• polynucleotide or “nucleic acid” refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA/RNA hybrids. Polynucleotides may be single- stranded or double-stranded and either recombinant, synthetic, or isolated.
• Polynucleotides include, but are not limited to: premessenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, circular RNA (circRNA), synthetic RNA, genomic RNA (viral genomic RNA), genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.
• pre-mRNA premessenger RNA
• mRNA messenger RNA
• RNA circular RNA
• synthetic RNA synthetic RNA
• genomic RNA viral genomic RNA
• genomic DNA gDNA
• PCR amplified DNA complementary DNA
• cDNA complementary DNA
• synthetic DNA or recombinant DNA.
• Polynucleotides refer to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide, as well as all intermediate lengths.
• intermediate lengths means any length between the quoted values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc., 151, 152, 153, etc., 201, 202, 203, etc.
• polynucleotides or variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence.
• isolated polynucleotide refers to a polynucleotide that has been purified from the sequences which flank it in a naturally-occurring state.
• an isolated polynucleotide is a synthetic polynucleotide, a semi- synthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source, or other polynucleotide that does not exist in nature and that has been made by the hand of man.
• a polynucleotide comprises or encodes a lentiviral vector. In various embodiments, a polynucleotide comprises a genomic RNA encoding a lentiviral vector.
• polynucleotides include, but are not limited to, polynucleotides encoding polypeptides set forth in SEQ ID NOs: 1-10 and 12-126, and SEQ ID NO: 11.
• polynucleotides may be codon-optimized.
• codon-optimized refers to substituting codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability and/or activity of the polypeptide.
• Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, (x) systematic variation of codon sets for each amino acid, and/or (xi) isolated removal of spurious translation initiation sites.
• sequence identity or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide- by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
• polynucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity to any of the reference sequences described herein.
• a vector may comprise a polynucleotide comprising or encoding one or more exogenous, endogenous, or heterologous expression control sequences operably linked to a polynucleotide encoding one or more polypeptides contemplated herein.
• “Expression control sequences,” “control elements,” or “regulatory sequences” present in a vector are those non-translated regions of the vector including but not limited to an origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgamo sequence or Kozak sequence) introns, a poly adenylation sequence, 5' and 3' untranslated regions, all of which interact with host cellular proteins to carry out transcription and translation.
• promoter refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds.
• An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter.
• promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and/or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide.
• operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
• the term refers to a functional linkage between an expression control sequence (such as a promoter, and/or enhancer) and a second polynucleotide sequence encoding a polypeptide, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
• Illustrative expression control sequences suitable for use in particular embodiments include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian vims 40 (SV40) (e.g., early or late), a Moloney murine leukemia vims (MoMLV) LTR promoter, a Rous sarcoma vims (RSV) LTR, a herpes simplex vims (HSV) (thymidine kinase) promoter, H5, P7.5, and Pl 1 promoters from vaccinia vims, an elongation factor 1 - alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5)
• vectors comprise a poly adenylation sequence 3' to a sequence to be transcribed and/or expressed.
• polyadenylation (or poly A) site denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II.
• Polyadenylation signals can promote mRNA stability by addition of a polyA tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency. Cleavage and polyadenylation are directed by a poly(A) signal in the RNA.
• the core poly(A) signal for mammalian pre-mRNAs has two recognition elements flanking a cleavage-polyadenylation site. Typically, an almost invariant AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5' cleavage product.
• the core poly(A) signal is an ideal polyA signal (e.g, AATAAA, ATTAAA, AGTAAA).
• the poly(A) sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbit P-globin polyA sequence (rPgpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art.
• the poly (A) sequence is synthetic.
• Recombinant lentiviruses contemplated herein are engineered to bind and transduce a cell.
• a recombinant lentivirus contemplated herein is engineered to bind and transduce immune effector cells.
• Cells may be non-genetically modified to express one or more of the polypeptides contemplated herein, or in particular preferred embodiments, cells may be genetically modified to express one or more of the polypeptides contemplated herein.
• the term “genetically engineered” or “genetically modified” refers to the addition of extra genetic material in the form of DNA or RNA into the total genetic material in a cell.
• the terms, “genetically modified cells” or “modified cells” are used interchangeably in particular embodiments.
• a recombinant lentivirus comprises a viral envelope comprising one or more mutated viral envelope glycoproteins that mediate virus-target cell fusion and one or more non- viral membrane-bound tropism polypeptides that bind an antigen expressed on a target cell, e.g., an immune effector cell.
• an “immune effector cell” is any cell of the immune system that has one or more effector functions e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC).
• Illustrative types of immune effector cells contemplated in particular embodiments include, without limitation, T lymphocytes, dendritic cells (DC), Treg cells, natural killer (NK) cells, natural killer T (NKT) cells, and macrophages.
• T cell or “T lymphocyte” are art-recognized and are intended, in particular embodiments, to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, and/or activated T lymphocytes.
• T lymphocytes suitable for use in particular embodiments include but not limited to cytotoxic T cells (CTLs; CD8 + T cells), TILs, helper T cells (HTLs; CD4 + T cells), CD4 + CD8 + T cells, CD4' CD8" T cells, or any other subset of T cells that has an effector function.
• CTLs cytotoxic T cells
• TILs TILs
• HTLs helper T cells
• CD4 + CD8 + T cells CD4 + CD8 + T cells
• CD4' CD8" T cells or any other subset of T cells that has an effector function.
• the cells comprise «P T cells.
• the cells comprise y5 T cells.
• a recombinant lentivirus comprises a viral envelope comprising one or more mutated viral envelope glycoproteins that mediate virus-target cell fusion and one or more non- viral membrane-bound tropism polypeptides that bind one or more antigens expressed on an immune effector cell, wherein the antigen is selected from the group consisting of: CD35, CD3s, CD3y, CD4, CD8a, CD8P, the alpha or beta chains of a TCR, CD28, CD134 (0X40), CD137 (4-1BB), and CD278 (ICOS).
• the antigen is selected from the group consisting of: CD35, CD3s, CD3y, CD4, CD8a, CD8P, the alpha or beta chains of a TCR, CD28, CD134 (0X40), CD137 (4-1BB), and CD278 (ICOS).
• immune effector cells include natural killer (NK) cells.
• NK cells do not express T cell antigen receptors (TCR), CD3 or surface immunoglobulins (Ig) B cell receptor, but usually express the surface markers CD 16 (FcyRIII) and CD56 in humans.
• a recombinant lentivirus comprises a viral envelope comprising one or more mutated viral envelope glycoproteins that mediate virus-target cell fusion and one or more non- viral membrane-bound tropism polypeptides that bind an antigen on an immune effector cell, wherein the antigen is CD 16 (FcyRIII) and/or CD56.
• immune effector cells include natural killer T (NKT) cells.
• NKT natural killer T
• a progenitor of an immune effector cell is transduced with a recombinant lentivirus contemplated herein and is subsequently induced to differentiate, or differentiates, into one or more immune effector cells.
• progenitors of immune effectors cells include hematopoietic stem cells (HSCs) contained within the CD34 + population of cells derived from cord blood, bone marrow or mobilized peripheral blood which naturally differentiate into mature immune effector cells, or which can be induced to differentiate into mature immune effector cells.
• HSCs hematopoietic stem cells
• a recombinant lentivirus comprises a viral envelope comprising one or more mutated viral envelope glycoproteins that mediate virus-target cell fusion and one or more non- viral membrane-bound tropism polypeptides that bind an antigen on a hematopoietic stem cell, wherein the antigen is selected from the group consisting of: CD7, CD33, CD34, CD45, CD49f, CD90, CD98, CD110, CD117, CD123, CD133, CD184, CD201, FMS-like tyrosine kinase 3 (FLT3), and thrombopoietin receptor.
• FLT3 FMS-like tyrosine kinase 3
• Formulations and compositions contemplated herein comprise a recombinant lentivirus and/or immune effector cells modified ex vivo formulated in pharmaceutically acceptable or physiologically-acceptable compositions for administration to a cell, tissue, organ, or an animal, either alone, or in combination with one or more other modalities of therapy.
• a composition comprises a recombinant lentivirus comprising one or more mutated viral envelope glycoproteins that mediate virus-target cell fusion, one or more non- viral membrane-bound tropism polypeptides, and a lentiviral vector comprising a polynucleotide comprising or encoding a promoter operably linked to a polynucleotide encoding an engineered antigen receptor, e.g., a CAR.
• a lentiviral vector comprising a polynucleotide comprising or encoding a promoter operably linked to a polynucleotide encoding an engineered antigen receptor, e.g., a CAR.
• the composition is a pharmaceutical composition.
• a “pharmaceutical composition” refers to a composition formulated in pharmaceutically-acceptable or physiologically - acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.
• “Pharmaceutically acceptable” refers to molecular entities and compositions that do not produce excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio when administered to a human.
• a composition comprises a pharmaceutically acceptable carrier and a recombinant lentivirus contemplated herein.
• pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient, or vehicle and the like with which a recombinant lentivirus is physiologically compatible with administration to a human, including but not limited to pharmaceutically acceptable cell culture media, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), and normal/physiologic saline (0.9% NaCl).
• PBS Dulbecco's phosphate buffered saline
• D5W 5% dextrose in water
• normal/physiologic saline (0.9% NaCl
• a composition is substantially free of mycoplasma, endotoxin, and microbial contamination.
• substantially free with respect to endotoxin is meant that there is less endotoxin per dose of cells than is allowed by the FDA for a biologic, which is a total endotoxin of 5 EU/kg body weight per day, which for an average 70 kg person is 350 EU per total dose of cells.
• compositions contemplated herein contain about 0.5 EU/mL to about 5.0 EU/mL, or about 0.5 EU/mL, 1.0 EU/mL, 1.5 EU/mL, 2.0 EU/mL, 2.5 EU/mL, 3.0 EU/mL, 3.5 EU/mL, 4.0 EU/mL, 4.5 EU/mL, or 5.0 EU/mL.
• NTP non-human primate
• the model was interrogated using a recombinant HIV-1 based lentivirus comprising a lentiviral vector encoding an MNDU3 promoter operably linked to an anti- CD20 CAR, e.g., SEQ ID NOs: 11, 12.
• HEK 293 cells were transiently transfected with plasmids encoding HIV-1 gag and pol; HIV-1 REV; a mutated VSIV-G protein comprising K47Q and R354A mutations; a primary non- viral membrane bound tropism polypeptide comprising an anti-CD3s antibody that bind M.
• nemestrina CD3s e.g., SP34- 2, and a CD8a hinge and transmembrane domain
• a secondary non- viral membrane bound tropism polypeptide comprising a CD80 polypeptide comprising a C-terminal truncation
• Transfected cells were cultured and lentivirus containing supernatant was harvested, and lentivirus was purified, titered, formulated in Dulbecco’s modified eagle’s medium (DMEM) without phenol red and stored in frozen aliquots.
• DMEM Dulbecco’s modified eagle’s medium
• Blood samples were drawn from a pig-tail macaque prior to intravenous infusion of recombinant lentivirus to establish baseline blood cell counts and to establish baseline cytokine levels.
• the pig-tail macaque was administered 5.0 x 10 8 TU/kg of the lentivirus at about 10 mL/kg for about 30 minutes.
• red blood cells were lysed using ammonium-chloride-potassium (ACK) buffer (ThermoFisher A 1049201), stained with ghost DyeTM Red, followed by staining with cell surface antibodies (see table below), fixed and signal acquired.
• ACK ammonium-chloride-potassium
• FIG. 1 shows data indicating that the in vivo HIV-1 derived lend virus was functional and efficacious in M. nemestrina.
• Figure 1 shows that the recombinant HIV-1 derived lentivirus encoding the anti-CD20 CAR transduced T cells and led to T cell expansion.
• Figure 1 left panel, shows that T cells transduced with the HIV-1 derived lentivirus encoding the anti-CD20 CAR potently reduced B cells and mediated durable B cell aplasia.
• cytokine and chemokine levels in serum were analyzed using the Meso Scale Discovery (MSD) UPLEX electrochemiluminescent sandwich immunoassay. Two panels of cytokines and chemokines were measured for each sample and each sample was analyzed in technical duplicates. Multispot assay plates were coated with human biotinylated capture antibodies (antibodies were validated as cross-reactive with pig-tail macaques) prior to addition of serum samples, followed by conjugated detection antibodies, per manufacturer’s instructions. Levels of cytokines and chemokines were measured using MESO QuickPlex SQ 120MM machine and analyzed via MSD Discovery Workbench software. Standard curves were generated using lyophilized proteins of known concentrations provided by MSD and prepared following manufacturer’s instructions.
• MSD Meso Scale Discovery
• Figure 2 shows that the in vivo HIV-1 derived lentivirus was safe at 5 x 10 8 TU/kg and did not increase cytokine/chemokines to levels associated with neurotoxicity in M. nemestrina.
• a lower dose of lentivirus was used in the Macaca nemestrina model established in Example 1.
• Blood samples were drawn from a pig-tail macaque prior to intravenous infusion of recombinant lentivirus to establish baseline blood cell counts and to establish baseline cytokine levels.
• the pig-tail macaque was administered 5.0 x 10 7 TU/kg of the lentivirus manufactured in Example 1 at about 10 mL/kg for about 30 minutes.
• lymphoid cell populations were monitored by flow cytometry. Briefly, red blood cells were lysed using ammonium-chloride-potassium (ACK) buffer (ThermoFisher A 1049201), stained with ghost DyeTM Red, followed by staining with cell surface antibodies (see table below), fixed and signal acquired.
• ACK ammonium-chloride-potassium
• Figure 3 shows data indicating that the in vivo HIV-1 derived lentivirus was functional and efficacious in M. nemestrina.
• Figure 3 shows the vector copy number (VCN) in T cells transduced with the HIV-1 derived lentivirus encoding the anti- CD20 CAR.
• Figure 3 left panel, shows T cells transduced with the HIV-1 derived lentivirus encoding the anti-CD20 CAR potently reduced B cells and mediated durable B cell aplasia.
• VCN vector copy number
• cytokine and chemokine levels in serum will be analyzed using the Meso Scale Discovery (MSD) UPLEX electrochemiluminescent sandwich immunoassay. Two panels of cytokines and chemokines will be measured for each sample and each sample is analyzed in technical duplicates. Multispot assay plates will be coated with human biotinylated capture antibodies (antibodies were validated as cross-reactive with pig-tail macaques) prior to addition of serum samples, followed by conjugated detection antibodies, per manufacturer’s instructions. Levels of cytokines and chemokines will be measured using MESO QuickPlex SQ 120MM machine and analyzed via MSD Discovery Workbench software. Standard curves will be generated using lyophilized proteins of known concentrations provided by MSD and prepared following manufacturer’ s instructions.
• MSD Meso Scale Discovery

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