EP4214242A1 - Vektorisierte antikörper für antivirale therapie - Google Patents

Vektorisierte antikörper für antivirale therapie

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Publication number
EP4214242A1
EP4214242A1 EP21789967.3A EP21789967A EP4214242A1 EP 4214242 A1 EP4214242 A1 EP 4214242A1 EP 21789967 A EP21789967 A EP 21789967A EP 4214242 A1 EP4214242 A1 EP 4214242A1
Authority
EP
European Patent Office
Prior art keywords
seq
aav
virus
mab
viral
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP21789967.3A
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English (en)
French (fr)
Inventor
Ye Liu
Joseph Bruder
Devin MCDOUGALD
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Regenxbio Inc
Original Assignee
Regenxbio Inc
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Filing date
Publication date
Application filed by Regenxbio Inc filed Critical Regenxbio Inc
Publication of EP4214242A1 publication Critical patent/EP4214242A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/10—Antioedematous agents; Diuretics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/007—Vectors comprising a special translation-regulating system cell or tissue specific

Definitions

  • compositions and methods are described for the systemic delivery of anti-viral fully human post-translationally modified (HuPTM) therapeutic monoclonal antibody (“mAb”) or the HuPTM antigen-binding fragment of an anti-viral therapeutic mAb — e.g., a fully human-glycosylated (HuGly) Fab of the anti-viral therapeutic mAb — to a human subject diagnosed with or at risk for a viral disease indicated for treatment or prophylaxis with the therapeutic mAb.
  • HumanPTM fully human post-translationally modified
  • mAb therapeutic monoclonal antibody
  • HuPTM antigen-binding fragment of an anti-viral therapeutic mAb e.g., a fully human-glycosylated (HuGly) Fab of the anti-viral therapeutic mAb
  • Outbreaks of infectious disease are a serious health, social and economic concern in many parts of the world, including, but not limited to diseases caused by respiratory viruses (e.g. influenza virus, rhinovirus, and coronaviruses (e.g. Middle East Respiratory Syndome coronavirus (MERS-CoV), sever acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)), arboviruses, hepatitis viruses (hepatitis C virus (HPC), hepatitis B virus (HBV)), herpesviruses (e.g.
  • respiratory viruses e.g. influenza virus, rhinovirus, and coronaviruses (e.g. Middle East Respiratory Syndome coronavirus (MERS-CoV), sever acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
  • arboviruses e.g. influenza
  • herpes simplex type I HSV I
  • herpes simplex type II HSV II
  • cytomegalus virus HCMV
  • Epstein-Barr virus EBV
  • papillomaviruses e.g. human papillomaviruse (HPV)
  • HPV human papillomaviruse
  • rhabdoviruses e.g. rabies virus (RBV)
  • RBV rabies virus
  • EBOV Zaire ebolavirus
  • retroviral viruses e.g. HIV
  • Gene therapy is being investigated as an alternative to deliver antibodies and peptides to treat or prevent a wide range of infectious diseases.
  • Gene therapy for infectious diseases may be accomplished introduction of genes encoding antibodies or other peptides or proteins designed to block or inhibit viral infection or the expression or function of viral gene products, such that the entry of the invectious agent into a host cell or replication of the infectious agent is blocked or limited.
  • HIV human immunodeficiency virus
  • gene therapies to infectious diseases, including but not limited to, compartmentalized augmentation of the immune response, in vivo antibody production against infectious agents, introduction of suicide genes, replacement of susceptibility genes, blocking gene expression (e.g. antisense, interfering peptides, and ribosome-mediated cleavage of RNA.
  • monoclonal antibodies are approved or in clinical development for the treatment of infectious diseases making it an attractive infectious-disease gene therapy strategy.
  • Therapeutic antibodies delivered by gene therapy have several advantages over inj ected or infused therapeutic antibodies that dissipate over time resulting in peak and trough levels. Sustained expression of the transgene product antibody, as opposed to injecting an antibody repeatedly, allows for a more consistent level of antibody to be present at the site of action, and is less risky and more convenient for patients, since fewer injections need to be made. Furthermore, antibodies expressed from transgenes are post-translationally modified in a different manner than those that are directly inj ected because of the different microenvironment present during and after translation.
  • compositions and methods for gene therapy designed to target the liver and generate a depot of transgenes for expression of the therapeutic antibodies that result in a therapeutic or prophylactic serum levels of the antibody within 20 days, 30 days, 40 days, 50 days, 60 days, or 90 days of administration of the rAAV composition.
  • Serum levels include 1.5 to 15 pg/ml antibody for antibodies that meet threshold viral binding and neutralization parameters.
  • compositions and methods are described for the systemic delivery of an anti-viral HuPTM mAh or an anti-viral HuPTM antigen-binding fragment of a therapeutic mAb (for example, a fully human-glycosylated Fab (HuGlyFab) of a therapeutic mAb), and/or, an anti-viral peptide to a patient (human subject) diagnosed with an infectious disease or condition indicated for treatment with the therapeutic anti-viral mAb and/or peptide or at risk for infection with a viral pathogen for the prevention or reduction in incidence of infection by the viral pathogen.
  • a therapeutic mAb for example, a fully human-glycosylated Fab (HuGlyFab) of a therapeutic mAb
  • an anti-viral peptide to a patient (human subject) diagnosed with an infectious disease or condition indicated for treatment with the therapeutic anti-viral mAb and/or peptide or at risk for infection with a viral pathogen for the prevention or reduction in incidence of infection by the viral pathogen.
  • Such antigen-binding fragments of therapeutic mAbs include a Fab, F(ab')2, or scFv (single-chain variable fragment) (collectively referred to herein as “antigen-binding fragment”).
  • “HuPTM Fab” as used herein may include other antigen binding fragments of a mAb.
  • full-length mAbs can be used. Delivery may be advantageously accomplished via gene therapy — e.g.
  • a viral vector or other DNA expression construct encoding a therapeutic anti-viral mAb or its antigenbinding fragment (or a hyperglycosylated derivative of either) or anti-viral peptide to a patient (human subject) diagnosed with a condition indicated for treatment with the therapeutic anti-viral peptide or mAb — to create a permanent depot in liver, or in alternative embodiments, muscle, of the patient that continuously supplies the HuPTM mAb or antigen-binding fragment of the therapeutic mAb, e.g., a human-glycosylated transgene product, or peptide, to the circulation of the subject where the mAb or antigen-binding fragment thereof or peptide exerts its therapeutic or prophylactive effect.
  • a viral vector or other DNA expression construct encoding a therapeutic anti-viral mAb or its antigenbinding fragment (or a hyperglycosylated derivative of either) or anti-viral peptide to create a permanent depot in liver, or in alternative embodiments,
  • gene therapy vectors particularly recombinant adeno-associated virus vectors (rAAV) gene therapy vectors, which when administered to a human subject result in expression of an anti-viral antibody or peptide to achieve a maximum or steady state serum concentration (for example, 20, 30, 40, 50, 60 or 90 days after administration) of about 1.5 pg/mL to about 15 pg/mL (including about 2 pg/mL to about 10 pg/mL, about 3 pg/mL to about 12 pg/mL, about 5 pg/mL to about 15 pg/mL, 5 pg/mL to about 10 pg/mL, or about 10 pg/mL to about 15 pg/mL anti-viral antibody, when the antibody binds to its target virus, or epitope thereof, for example, in an antibody binding assay (e.g.
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • IC50 in at least the nanomolar range, and, in particular embodiments, picomolar range.
  • Dosages include 1E11 to 1E14 vector genomes per kilogram body weight (vg/kg) administered, particularly, parenterally, including intravenously.
  • Dosages result in sufficient copy number of viral genomes incorporated into liver cells, for example, from at least 10, 20, 50, 60 or 80 vector genome copies (or vector genomes, vg) per diploid genome (vg/dg) in liver tissue and up to 100, 150, 200, 500 or 100 vg/dg in liver tissue by 30, 60, 90 or 100 days or one year after administration.
  • the administration is a single administration.
  • the dosage achieves the therapeutic or prophylactive serum levels of the anti-viral antibody while minimizing or avoiding adverse effects such as transaminitis and/or the presence of anti-drug antibodies.
  • the recombinant vector used for delivering the transgene includes non -replicating recombinant adeno-associated virus vectors (“rAAV”).
  • rAAV non -replicating recombinant adeno-associated virus vectors
  • the AAV type has a tropism for liver and/or muscle cells, or example AAV8 subtype of AAV.
  • other viral vectors may be used, including but not limited to lentiviral vectors; vaccinia viral vectors, or non-viral expression vectors referred to as “naked DNA” constructs.
  • Expression of the transgene can be controlled by constitutive or tissue-specific expression control elements, particularly elements that are liver and/or muscle specific control elements, for example one or more elements of Table 1.
  • the HuPTM mAb or HuPTM antigen-binding fragment encoded by the transgene can include, but is not limited to, a full-length or an antigen-binding fragment of a therapeutic antibody that binds to and neutralizes a respiratory virus (e.g.
  • influenza virus e.g., Middle East Respiratory Syndome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-Co-V), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
  • MERS-CoV Middle East Respiratory Syndome coronavirus
  • SARS-Co-V severe acute respiratory syndrome coronavirus
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • an arbovirus e.g., Middle East Respiratory Syndome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-Co-V), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
  • an arbovirus e.g., a hepatitis virus (hepatitis C virus (HPC), hepatitis B virus (HBV)), herpesvirus (e.g. herpes simplex type I (HSV I), herpes simplex type II (HSV
  • the HuPTM mAb or HuPTM antigen-binding fragment encoded by the transgene can include, but is not limited to, a full-length or an antigen-binding fragment of a therapeutic antibody that binds to one or more strains of SARS-CoV-2.
  • artificial peptide-based viral inhibitors that exhibit antiviral activity against one or more viral human pathogens are also provided.
  • the anti-viral peptide is expressed from an engineered vector.
  • the anti-viral peptide encoded by the nucleic acid of recombinant expressed coding for said anti-viral peptide can include, but is not limited to an anti-viral peptide that binds to respiratory viruses (e.g. influenza virus, rhinovirus, and coronaviruses (e.g.
  • MERS-CoV Middle East Respiratory Syndome coronavirus
  • SARS-Co-V severe acute respiratory syndrome coronavirus
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • arboviruses arboviruses
  • hepatitis viruses hepatitis C virus (HPC), hepatitis B virus (HBV)
  • herpesviruses e.g. herpes simplex type I (HSV I), herpes simplex type II (HSV II) cytomegalus virus (CMV), Epstein- Barr virus (EBV)
  • papillomaviruses e.g.
  • the anti-viral peptide nucleic acid of recombinant expressed coding for said anti-viral peptide can include, but is not limited to, an anti-viral that binds to one or more strains of SARS-CoV-2.
  • the anti-viral peptides may be used as a therapeutic in the treatment of COVID-19 or a prophylactic measure in previously infected individuals after acute exposure.
  • the anti-viral peptide can be expressed from a DNA construct comprising a nucleic acid sequence encoding for the anti-viral peptide, operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells or human muscle cells.
  • an anti-viral peptide and a HuPTM mAb or HuPTM antigen-binding fragment are expressed from a single construct.
  • Gene therapy constructs for the therapeutic antibodies are designed such that both the heavy and light chains are expressed.
  • the coding sequences for the heavy and light chains can be engineered in a single construct in which the heavy and light chains are separated by a cleavable linker or IRES so that separate heavy and light chain polypeptides are expressed.
  • the linker is a Furin-T2A linker (SEQ ID NOS; 198 or 199).
  • the coding sequences encode for a Fab or F(ab’)2, scFv, or nanobody (Nb).
  • the full length heavy and light chains of the antibody are expressed.
  • the constructs express an scFv in which the heavy and light chain variable domains are connected via a flexible, non- cleavable linker.
  • the construct expresses, from the N-terminus, NH 2 -V L - linker-V H -COOH or NH2-V H -linker-V L -COOH.
  • the construct expresses a single antibody fragment consisting of a single monomeric variable antibody domain (“nanobody”).
  • the single antibody fragment is a heavy chain variable domain or a light chain variable domain.
  • the construct expresses, from the N-terminus to the C-terminus, NH2- leader or localization sequence- VH-COOH.
  • the construct expresses, from the N-terminus to the C-terminus, NH2 -leader or localization sequence- VL-COOH.
  • antibodies expressed from transgenes in vivo are not likely to contain degradation products associated with antibodies produced by recombinant technologies, such as protein aggregation and protein oxidation. Aggregation is an issue associated with protein production and storage due to high protein concentration, surface interaction with manufacturing equipment and containers, and purification with certain buffer systems. These conditions, which promote aggregation, do not exist in transgene expression in gene therapy.
  • Oxidation such as methionine, tryptophan, and histidine oxidation
  • Oxidation is also associated with protein production and storage, and is caused by stressed cell culture conditions, metal and air contact, and impurities in buffers and excipients.
  • the proteins expressed from transgenes in vivo may also oxidize in a stressed condition.
  • humans, and many other organisms are equipped with an antioxidation defense system, which not only reduces the oxidation stress, but sometimes also repairs and/or reverses the oxidation.
  • proteins produced in vivo are not likely to be in an oxidized form. Both aggregation and oxidation could affect the potency, pharmacokinetics (clearance), and immunogenicity.
  • HuPTM mAb or HuPTM Fab in liver cells of the human subject should result in a “biobetter” molecule for the treatment of disease accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding a full-length HuPTM mAb or HuPTM Fab of a therapeutic mAb to a patient (human subject) diagnosed with a disease indication for that mAb, to create a permanent depot in the subject that continuously supplies the human-glycosylated, sulfated transgene product produced by the subject’s transduced cells.
  • the cDNA construct for the HuPTMmAb or HuPTM Fab should include a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced human cells.
  • the full-length HuTPM mAb or HuPTM Fab can be produced in human cell lines by recombinant DNA technology, and the glycoprotein can be administered to patients.
  • Combination therapies involving systemic delivery of the full-length HuPTM anti-viral mAb or HuPTM anti-viral Fab to the patient accompanied by administration of other available treatments are encompassed by the methods provided herein.
  • the additional treatments may be administered before, concurrently or subsequent to the gene therapy treatment.
  • Such additional treatments can include but are not limited to co-therapy with the therapeutic mAh, including delivering one of more different (including 2, 3, 4 or 5 or more) antibodies against the target virus by gene therapy to deliver an anti-viral cocktail of antibodies.
  • kits for manufacturing the viral vectors particularly the AAV based viral vectors.
  • methods of producing recombinant AAVs comprising culturing a host cell containing an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding a therapeutic antibody operably linked to expression control elements that will control expression of the transgene in human cells; a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans; sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid proteins; and recovering recombinant AAV encapsidating the artificial genome from the cell culture.
  • compositions comprising rAAV vectors which comprise an optimized expression cassette containing a liver-specific promoter and a codon optimized and CpG depleted transgene with a modified furin-T2A processing signal that express a transgene, for example, heavy and light chains of an anti-viral therapeutic antibody.
  • rAAV vector encoding the heavy and light chains of an anti-viral antibody, or antigen binding fragment thereof, wherein each of the heavy and light chains are linked to a signal peptide and operably linked to a regulatory element that promotes expression in the liver such that a therapeutically effective amount of the antibody is produced into the serum.
  • the method results in a Cmin serum concentration in the human subject, by at least 20 days, 30 days or 40 days after administration, of at least 1.0 pg/ml, 1.1 pg/ml, 1.2 pg/ml, 1.3 pg/ml, 1.4 pg/ml, 1.5 pg/ml, 1.6 pg/ml, 2.0 pg/ml, 2.5 pg/ml, 3.0 pg/ml, 4.0 pg/ml, 5.0 pg/ml, or 6.0 pg/ml.
  • the serum concentration of the antibody is sufficient to prevent (including to reduce the incidence of by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%) viral infection or the symptoms of (including to ameliorate the symptoms of) viral infection.
  • Viral infection may be detected by standard diagnostic tests such as testing a biological sample, for example sputum or nasal swab, for viral nucleic acid by PCR or other assay or for viral antigen.
  • the methods of the invention result in sufficient transgene product in the serum to treat or prevent the viral infection and to ameliorate viral symptoms, such as, for example, cough, rhinitis, fever, headache, body aches, respiratory distress, pneumonia, etc.
  • a pharmaceutical composition for treating or preventing infection by a viral pathogen in a human subject in need thereof comprising an adeno-associated virus (AAV) vector having:
  • an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a heavy chain and a light chain of a substantially full-length or full-length mAb, or an antigen binding fragment thereof, which mAb has a KD for said virus or an epitope thereof of 1 to 10,000 pM and a neutralization potency (IC50) of 1 to 1000 pM in an assay for neutralization of said viral pathogen, operably linked to one or more regulatory sequences that promote expression of the transgene in human liver cells; wherein said AAV vector is formulated for intravenous administration to said human subject such that within 20 days days after said administration, the anti-viral mAb is present at a serum concentration of 1.5 pg/ml to 15 pg/ml in said human subject.
  • ITRs AAV inverted terminal repeats
  • the pharmaceutical composition of paragraph 1 wherein the viral capsid is at least 95% identical to the amino acid sequence of an AAV3B, AAV5, AAV7 (SEQ ID NO: 1), AAV8 (SEQ ID NO:2), AAV9 (SEQ ID NO:3), AAVrhlO (SEQ ID NO4), AAVrh46 (SEQ ID NO:5), AAVrh73 (SEQ ID NO: 6), AAVS3 (SEQ ID NO: 8), AAV-LK03 (SEQ ID NO: 7), AAVrh8, AAV64R1, or AAVhu37.
  • the regulator sequence comprises an ApoE.hAAT (SEQ ID NO:21) regulatory sequence, a LSPX1 promoter (SEQ ID NOV), a LSPX2 promoter (SEQ ID NO: 10), a LTP1 promoter (SEQ ID NO: 11), a LTP2 (SEQ ID NO: 12) promoter, CRE selected from Table 16, CRE (Table 16).hAAT or a LTP3 (SEQ ID NO: 13) promoter.
  • the pharmaceutical composition of paragraph 8 wherein said signal sequence comprises or consists ofMYRMQLLLLIALSLALVTNS (SEQ ID NO:50) or a signal sequence selected from Table 2.
  • the pharmaceutical composition of any of paragraphs 1 to 10 which is administered at a dosage of 1E11 to 1E14 vg/kg.
  • the pharmaceutical composition of any of paragraphs 1 to 11 wherein said administration results in a 10-100 vector genome per decagram of liver tissue at 100 days after administration.
  • said viral pathogen is an influenza virus, rhinovirus, coronavirus (including SARS-CoV2, SARS-CoV or MERS-CoV), arbovirus, hepatitis virus (including, hepatitis C virus (HPC), hepatitis B virus (HBV)), herpesvirus (including herpes simplex type I (HSV I) and herpes simplex type II (HSV II)) cytomegalus virus (CMV), Epstein-Barr virus (EBV)), papillomavirus (including, human papillomavirus (HPV)), rhabdovirus, Zaire ebolavirus (EBOV), lyssaviruses (e.g.
  • rabies virus (RBV)
  • a retrovirus including, HIV
  • the pharmaceutical composition of paragraph 13 wherein the viral pathogen is SARS-CoV-2.
  • the pharmaceutical composition of paragraph 14, wherein the anti-viral mAb is CC12.1, CC12.23, JS016, LY-CoV555, VIR-7831, TY027, BRII-96, BRII-98, CT-P59, SCTA01, STI1499, AZD8895, AZS1061, SAB-185, S309, VHH72, VHH55, CR3022, HOM, P2B-2F6, B38, H4, EY6A, CAI, CB6, C105, CV30, COVA2-39, COVA2-04, F26G19, ADI-55689, ADI- 56046, BGB-DXP593, 311mab31B5, 311mab32D4, AZD1061 (cilgavimab), or AZD7442 (tixage
  • the pharmaceutical composition of any of paragraphs 1 to 17 wherein the neutralization assay is a VS V pseudoparticle neutralization assay.
  • a composition comprising an adeno-associated virus (AAV) vector having: a. a viral AAV capsid, that is optionally at least 95% identical to the amino acid sequence of an AAV3B, AAV5, AAV7 (SEQ ID NO:1), AAV8 (SEQ ID NO:2), AAV9 (SEQ ID NO:3), AAVrhlO (SEQ ID NO:4), AAVrh46 (SEQ ID NO:5), AAVrh73 (SEQ ID NO:5), AAVS3 (SEQ ID NO:8), AAV-LK03 (SEQ ID NO:7), AAVrh8, AAV64R1, or AAVhu37; and b.
  • AAV adeno-associated virus
  • an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a heavy and a light chain of a substantially full-length or full-length mAb, which mAb has a Kd for a viral pathogen of 1 to 10,000 pM and a neutralization potency of 1 to 1000 pM in a neutralization assay for neutralization of said viral pathogen, operably linked to one or more regulatory sequences that promote expression of the transgene in human liver cells; c. wherein the transgene encodes a signal sequence at the N-terminus of the heavy chain and the light chain of said mAb that directs secretion and post translational modification of said mAb in liver cells.
  • ITRs AAV inverted terminal repeats
  • composition of paragraph 20 wherein the transgene comprises a Furin/2A linker between the nucleotide sequences coding for the heavy and light chains of said mAb.
  • composition of paragraph 21 wherein the nucleic acid encoding a Furin 2A linker is incorporated into the expression cassette in between the nucleotide sequences encoding the heavy and light chain sequences, resulting in a construct with the structure: Signal sequence - Heavy chain - Furin site - 2A site - Signal sequence - Light chain - PolyA.
  • said signal sequence comprises or consists of MYRMQLLLLIALSLALVTNS (SEQ ID NO:50) or a signal sequence selected from Tables 2 or 3.
  • said viral pathogen is influenza virus, rhinovirus, coronavirus (including SARS-CoV-2, SARS-CoV or MERS CoV), arbovirus, hepatitis virus (including, hepatitis C virus (HPC), hepatitis B virus (HBV)), herpesvirus (including herpes simplex type I (HSV I) and herpes simplex type II (HSV II)) cytomegalus virus (CMV), Epstein-Barr virus (EBV)), papillomavirus (including, human papillomavirus (HPV)), rhabdovirus, Zaire ebolavirus (EBOV), lyssaviruses (e.g.
  • rabies virus (RBV)
  • a retrovirus including, HIV
  • the composition of paragraph 25 wherein the viral pathogen is SARS-CoV-2.
  • the composition of paragraph 26, wherein the anti-viral mAb is CC12.1, CC 12.23, LY-CoV555, JS016, VIR-7831, TY027, BRII-96, BRII-98, CT-P59, SCTA01, STI1499, AZD8895, AZS1061, SAB-185, S309, VHH72, VHH55, CR3022, H014, P2B-2F6, B38, H4, EY6A, CAI, CB6, C105, CV30, COVA2-39, COVA2-04, F26G19, ADI-55689, ADI-56046, 311mab31B5, 311mab32D4, BGB-DXP593, AZD1061 (cilgavimab), or AZD7442 (tixagevima
  • the composition of any of paragraphs 28 or 29 wherein the neutralization assay is a VSV pseudoparticle neutralization assay.
  • a method of treating or reducing the incidence of a viral infection in a human subject in need thereof comprising: administering to the liver of said subject a therapeutically effective amount of a recombinant nucleotide expression vector comprising a transgene encoding a substantially full-length or full-length anti-viral mAb, which mAb has a Kd for said viral pathogen of 1 to 10,000 pM and a neutralization potency of 1 to 1000 pM in a neutralization assay for neutralization of said viral pathogen, operably linked to one or more regulatory sequences that promote expression of the transgene in human liver cells, so that a depot is formed that releases a HuPTM form of said anti-virus mAb such that within 20, 30 or 40 days after said administration, the anti-viral mAb is present at a serum concentration of 1.5 pg/ml to 15 pg/ml in said human subject.
  • the viral capsid is at least 95% identical to the amino acid sequence of an AAV3B, AAV5, AAV7 (SEQ ID NO:1), AAV8 (SEQ ID NO:2), AAV9 (SEQ ID NO:3), AAVrhlO (SEQ ID NO:4), AAVrh46 (SEQ ID NO:5), AAVrh73 (SEQ ID NO:6), AAVS3 (SEQ ID NO:8), AAV-LK03 (SEQ ID NO:7), AAVrh8, AAV64R1, or AAVhu37.
  • the regulator sequence comprises or consists of an ApoE.hAAT (SEQ ID NO:21) regulatory sequence, a LSPX1 promoter (SEQ ID NOV), a LSPX2 promoter (SEQ ID NO: 10), a LTP1 promoter (SEQ ID NO: 11), a LTP2 (SEQ ID NO: 12) promoter, CRE selected from Table 16, CRE (Table 16).hAAT or a LTP3 (SEQ ID NO: 13) promoter.
  • transgene comprises a Furin/2A linker between the nucleotide sequences coding for the heavy and light chains of said mAb.
  • Furin 2A linker is a Furin/T2A linker having the amino acid sequence RKRR(GSG)APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NOS: 103 or 104).
  • the transgene encodes a signal sequence at the N-terminus of the heavy chain and the light chain of said antigen-binding fragment that directs secretion and post translational modification in said human liver cells.
  • said signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO:50) or a signal sequence selected from Tables 2 or 3.
  • transgene has the structure: Signal sequence- Heavy chain - Furin site - 2A site - Signal sequence- Light chain - PolyA.
  • the mAb is a hyperglycosylated mutant or wherein the Fc polypeptide of the mAb is glycosylated or aglycosylated.
  • the mAb contains an alpha 2,6-sialylated glycan.
  • any of paragraphs 31 to 43 wherein the mAb contains a tyrosine sulfation The method of any of paragraphs 31 to 44 in which production of said HuPTM form of said mAb or antigen-binding fragment thereof is confirmed by transducing human liver cells in culture with said recombinant nucleotide expression vector and expressing said mAb or antigen-binding fragment thereof.
  • the method of any of paragraphs 31 to 45 wherein the recombinant expression vector is AAV8 or AAVS3.
  • the anti-anti-viral mAh is CC12.1, CC12.23, JS016, LY- CoV555, VIR-7831, TY027, BRII-96, BRII-98, CT-P59, SCTA01, STI1499, AZD8895, AZS1061, SAB-185, S309, VHH72, VHH55, CR3022, HOM, P2B-2F6, B38, H4, EY6A, CAI, CB6, C105, CV30, COVA2-39, COVA2-04, F26G19, ADI-55689, ADI-56046, BGB-DXP593, 311mab31B5, 311mab32D4, or AZD1061 (cilgavimab), or AZD7442 (tixagevimab) or a combination thereof.
  • said virus is an influenza virus, rhinovirus, coronavirus, arbovirus, hepatitis virus (including, hepatitis C virus (HPC), hepatitis B virus (HBV)), herpesvirus (including herpes simplex type I (HSV I) and herpes simplex type II (HSV II)) cytomegalus virus (CMV), Epstein-Barr virus (EBV)), papillomavirus (including, human papillomavirus (HPV)), rhabdovirus, Zaire ebolavirus (EBOV), lyssaviruses (e.g. rabies virus (RBV)), or a retrovirus (including, HIV).
  • hepatitis virus including, hepatitis C virus (HPC), hepatitis B virus (HBV)
  • herpesvirus including herpes simplex type I (HSV I) and herpes simplex type II (HSV II)
  • CMV cytomegalus virus
  • a method of producing recombinant AAVs comprising:
  • trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and an AAV capsid protein operably linked to expression control elements that drive expression of the AAV rep and the AAV capsid protein in the host cell in culture and supply the AAV rep and the AAV capsid protein in trans, wherein the capsid has liver tropism;
  • transgene encodes a substantially full-length or full- length mAb or antigen binding fragment that comprises the heavy and light chain variable domains of an antibody against a respiratory virus, including influenza virus, rhinovirus, coronavirus (including MERS-Co,V, SARS-CoV and SARS-CoV-2), arbovirus, hepatitis virus (including, hepatitis C virus (HPC), hepatitis B virus (HBV)), herpesvirus (including herpes simplex type I (HSV I) and herpes simplex type II (HSV II)) cytomegalus virus (CMV), Epstein- Barr virus (EB V)), papillomavirus (including, human papillomavirus (HPV)), rhabdovirus, Zaire ebolavirus (EBOV), lyssaviruses (e.g.
  • a respiratory virus including influenza virus, rhinovirus, coronavirus (including MERS-Co,V, SARS-CoV and SARS-Co
  • rabies virus (RBV)
  • a retrovirus including, HIV
  • a host cell containing: a. an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises comprising a transgene encoding a substantially full-length or full-length anti-viral mAb, which mAb has a KD for said viral pathogen of 1 to 10,000 pM and a neutralization potency (IC50) of 1 to 1000 pM in a neutralization assay for neutralization of said viral pathogen, operably linked to one or more regulatory sequences that promote expression of the transgene in human liver cells; b.
  • an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises comprising a transgene encoding a substantially full-length or full-length anti-viral mAb, which mAb has a KD for said viral pathogen of 1 to 10,000 pM and a neutralization potency (IC50) of 1 to 1000 pM in a neutralization
  • trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and an AAV capsid protein operably linked to expression control elements that drive expression of the AAV rep and the AAV capsid protein in the host cell in culture and supply the AAV rep and the AAV capsid protein in trans, wherein the capsid has liver tropism; c. sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid protein.
  • transgene encodes a substantially full-length or full- length mAb or antigen binding fragment that comprises the heavy and light chain variable domains of an antibody against an influenza virus, rhinovirus, coronavirus (including MERS CoV, SARS CoV and SARS CoV-2), arbovirus, hepatitis virus (including, hepatitis C virus (HPC), hepatitis B virus (HBV)), herpesvirus (including herpes simplex type I (HSV I) and herpes simplex type II (HSV II)) cytomegalus virus (CMV), Epstein-Barr virus (EBV)), papillomavirus (including, human papillomavirus (HPV)), rhabdovirus, Zaire ebolavirus (EBOV), lyssaviruses (e.g.
  • rabies virus (RBV)
  • a retrovirus including, HIV
  • the host cell of paragraph 61 wherein the antibody is against SARS-CoV-2.
  • AAV capsid protein is an AAV8, AAVrh46, AAVrh73, AAVS3, or AAV-LK03 capsid protein.
  • FIGS. 1A-1C A schematic of an rAAV vector genome construct containing an expression cassette encoding the heavy and light chains of a therapeutic mAb separated by a Furin-2A linker, operably linked to a liver-specific enhancer and/or promoter, controlled by expression elements, flanked by the AAV ITRs.
  • the transgene can comprise nucleotide sequences encoding the heavy and light chains of the Fab portion or the full-length heavy (CHI plus hinge) and light chains with Fc regions.
  • FIG. 2 The amino acid sequence of a transgene construct for the Fab region of an exemplary therapeutic antibody to SARS-CoV-2 (CC12.1). Glutamine glycosylation sites; asparaginal (N) glycosylation sites, non-consensus asparaginal (N) glycosylation sites; and tyrosine-O-sulfation sites are as indicated in the legend. Complementarity-determining regions (CDR) are underscored. The heavy chain hinge regions are shown in italic.
  • FIGS. 3A and B Amino acid sequence alignment of the amino acid sequences of the heavy (FIG. 3A) and light (FIG. 3B), respectively, in order of appearance) chain Fab portions of the therapeutic antibodies disclosed herein. Positions that may be substituted to produce hyperglycosylated variants of the Fab regions are highlighted. One substitutions that should result in hyperglycosylation of the Fab region by human cells are annotated above the amino acid residue positions.
  • FIG. 5 Glycans that can be attached to HuGlyFab regions of full length mAbs or the antigen-binding domains. (Adapted from Bondt et al., 2014, Mol & Cell Proteomics 13.1 : 3029-3039).
  • FIG. 6 Clustal Multiple Sequence Alignment of constant heavy chain regions (CH2 and CH3) of IgGl (SEQ ID NO: 141), IgG2 (SEQ ID NO: 142), and IgG4 (SEQ ID NO: 143).
  • the hinge region from residue 219 to residue 230 of the heavy chain, is shown in italics.
  • the numbering of the amino acids is in EU-format.
  • FIGS. 7A-7D A. Schematic showing the genome configuration of recombinant AAV8 and AAV9 vectors for expression of lanadelumab.
  • the expression cassette utilizes the CAG promoter (SEQ ID NO: 36) to drive expression of a human antibody that binds to and inhibits for example, plasma kallikrein (pKal).
  • Amutant IL2 signal sequence mIL2, SEQ ID NO:50
  • the furin-F2A sequence SEQ ID NO: 106 drives the cleavage of the polyprotein into heavy and light chain components.
  • Top panels demonstrate reporter transgene (eGFP) expression following transfection of different plasmid quantities (4 pg-nontransfected).
  • Bottom left panel depicts lanadelumab expression in the cell lysate while the bottom right panel detects plasmid expressed lanadelumab secreted into the cell supernatant.
  • IV intravenous
  • IM intramuscular
  • AAV9 vectors (2ell gc) were injected either IV or IM and serum antibody levels were determined by ELISA at day 7 (D7), day 21 (D21), day 35 (D35), and day 49 (D49).
  • FIG. 10 depicts the expression of the monoclonal antibody lanadelumab (Mabl) in C2C12 muscle cells upon transduction of the cells with different cis plasmids expressing lanadelumab under the control of different regulatory elements: CAG (SEQ ID NO:36), LMTP6 (SEQ ID NO: 14), and ApoE.hAAT (SEQ ID NO:21).
  • CAG SEQ ID NO:36
  • LMTP6 SEQ ID NO: 14
  • ApoE.hAAT SEQ ID NO:21.
  • the cells were treated with FITC conjugated anti-Fc (IgG) antibody.
  • DAPI staining is shown to confirm confluency and viability of the cells under all conditions tested.
  • FIGS 11A and 11B A Serum expression levels (pg/ml) of lanadelumab upon intravenous injection of C/57BL6 mice with 2.5xl0 12 vg/kg of AAV8 vectors encoding a lanadelumab regulated by different liver-specific, liver-tandem and liver-muscle regulatory elements (see Table 1).
  • CAG SEQ ID NO:36
  • TBG SEQ ID NO:40
  • FIGS. 13A-13D A. Serum anti-kallikrein (pKal) (lanadelumab) antibody concentration following AAV8 delivery. Animals received bilateral injections of 5xl0 10 vg/kg into the GA muscle. Serum was collected biweekly and vectorized antibody concentration was quantified with ELISA. B. Vector genome quantification from relevant tissues with digital droplet PCR (ddPCR). C. Comparison of vector gene expression from liver. Data represent relative fold gene expression as quantified by the AACT method. D. Comparison of AAV transgene expression from tissues using digital droplet PCR (ddPCR). Anti-pKal antibody mRNA copies were normalized to GAPDH mRNA copies across tissues. Data are represented as mean ⁇ SEM. Statistical significance was determined using a one-way ANOVA followed by Tukey’s HSD post-test. *P ⁇ 0.05, **P ⁇ 0.01.
  • FIG. 14 Antibody concentrations in the serum of wild type mice treated with AAV8. Lanadelumab vectors produced with different BV/Sf9 production systems compared to an HEK system. C57BL/6 mice were intravenously injected with vectors at a dose of 2.5xl0 12 vg/kg.
  • FIGS. 15A-15F A and B show the pKal titration curve and signal-to-noise ratios for indicated pKal concentrations.
  • C Two pKal concentrations (6.25nM and 12.5nM) were used to measure the suppressive range of lanadelumab (compared to non-specific human IgG control antibody) in an antibody-dose response.
  • FIG. 19 A schematic of an rAAV vector genome construct containing an expression cassette encoding eGFP + 10-basepair barcode, operably linked to a liver-specific CRE selected from Table 16 and a hAAT promoter, controlled by expression elements, flanked by the AAV ITRs.
  • compositions and methods are described for the systemic delivery of a fully human post-translationally modified (HuPTM) therapeutic monoclonal antibody (mAb) or a HuPTM antigenbinding fragment of a therapeutic mAb (for example, a fully human-glycosylated Fab (HuGlyFab) of a therapeutic mAb) to a patient (human subject) diagnosed with a viral infectious disease or condition or at risk for infection with a viral pathogen indicated for treatment with the therapeutic mAb.
  • HumanPTM fully human post-translationally modified
  • mAb therapeutic monoclonal antibody
  • HuPTM antigenbinding fragment of a therapeutic mAb for example, a fully human-glycosylated Fab (HuGlyFab) of a therapeutic mAb
  • Delivery may be advantageously accomplished via gene therapy — e.g., by administering a viral vector or other DNA expression construct encoding a therapeutic mAb or its antigen-binding fragment (or a hyperglycosylated derivative of either) to a patient (human subject) diagnosed with a condition indicated for treatment with the therapeutic mAb or at risk for infection with the virus agains which the therapeutic mAb is directed — to create a permanent depot in a tissue or organ of the patient, particularly liver and/or muscle that continuously supplies the HuPTM mAb or antigen-binding fragment of the therapeutic mAb, e.g., a human-glycosylated transgene product, into the circulation of the subject to where the mAb or antigen-binding fragment there of exerts its therapeutic effect, including prevention of viral infection.
  • a viral vector or other DNA expression construct encoding a therapeutic mAb or its antigen-binding fragment (or a hyperglycosylated derivative of either) to a patient (human subject) diagnosed with
  • the HuPTM mAb or HuPTM antigen-binding fragment encoded by the transgene is a full-length or an antigen-binding fragment of a HuPTM mAb or HuPTM or peptide that targets a virus, prevents or reduces the incidence of viral infection, and/or manages, reduces, or ameliorates the progression of the disease and/or the symptoms associated therewith.
  • the transgene encodes, in certain embodiments, a therapeutic antibody that binds and neutralizes and/or prevents infection by a viral pathogen.
  • the viral pathogen may be a human viral pathogen but may also infect one or more mammalian species.
  • compositions and methods provided herein systemically deliver anti-viral antibodies, as well, in certain embodiments, anti-viral peptides, from a depot of viral genomes, for example, in the subject’s liver (and/or muscle) at a serum level that is therapeutically or prophylactically effective to treat, ameliorate the symptoms or, prevent infection of or reduce the incidence of infection of a human pathogenic virus.
  • Useful antibodies bind the virus (or an antigen thereof, such as a spike protein of a coronavirus, for example) with a dissociation constant (KD) in the picomolar or nanomolar range as determined using a binding assay known in the art and a viral neutralization potency (IC50) at least in the nanomolar range and, in embodiments, in the picomolar range according to a viral neutralization assay accepted in the art.
  • KD dissociation constant
  • IC50 viral neutralization potency
  • viral vectors for delivery of transgenes encoding the therapeutic anti-viral antibodies to cells in the human subject including, in embodiments, liver cells, and regulatory elements operably linked to the nucleotide sequence encoding the heavy and light chains of the anti-viral antibody that promote the expression of the antibody in the cells, in embodiments, in the liver cells.
  • regulatory elements including liver specific regulatory elements, are provided in Table 1 herein.
  • such viral vectors may be delivered to the human subject at appropriate dosages, for example 10E11 to 10E14 vg/kg, such that at least 20, 30, 40, 50 or 60 days after administration, the anti-viral antibody is present in the serum of said human subject at a level of at least 1.5 pg/ml to 15 pg/ml.
  • the HuPTM mAb or HuPTM antigen-binding fragment encoded by the transgene can include, but is not limited to, a full-length or an antigen-binding fragment of a therapeutic antibody that binds and has neutralizing activity for a respiratory virus (e.g. influenza virus, rhinovirus, or coronavirus (e.g. Middle East Respiratory Syndome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-Co-V), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)), arbovirus, hepatitis virus (hepatitis C virus (HPC), hepatitis B virus (HBV)), herpesvirus (e.g.
  • a respiratory virus e.g. influenza virus, rhinovirus, or coronavirus (e.g. Middle East Respiratory Syndome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-Co-V), or severe acute respiratory syndrome coronavirus
  • herpes simplex type I HSV I
  • herpes simplex type II HSV II
  • CMV cytomegalus virus
  • EBV Epstein-Barr virus
  • papillomavirus e.g. human papillomaviruse (HPV)
  • HPV human papillomaviruse
  • EBOV Zaire ebolavirus
  • lyssaviruses e.g. rabies virus (RBV)
  • retroviral viruses e g. HIV
  • the HuPTM mAb or HuPTM antigen-binding fragment encoded by the transgene can include, but is not limited to, a full-length or an antigen-binding fragment of a therapeutic antibody that binds to and has neutralizing activity for one or more strains of SARS- CoV-2.
  • the anti-SARS-CoV-2 mAb binds specifically to the receptor binding domain (RBD) region of the SARS-CoV-2 spike protein.
  • the antibody binds specifically to RBD having a binding affinity KD in the range 1 x 10' 9 to 1 x 10' 12 .
  • the anti-SARS-CoV-2 mAb binds to the non-RBD region of the SARS-CoV-2 spike.
  • the HuPTM mAb or HuPTM antigen-binding fragment encoded by the transgene can include, but is not limited to, a full-length or an antigen-binding fragment of a therapeutic antibody that binds to SARS-CoV-2, including but not limited to, CC12.1 (FIG.
  • the anti-SARS-CoV-2 mAb or antigen-binding fragment encoded by the transgene is CC12.1, or CC12.23.
  • the amino acid sequences of the heavy and light chains of antigen binding fragments of the foregoing are provided in Table 7, infra.
  • the HuPTM mAb or HuPTM antigen-binding fragment encoded by the transgene can include, but is not limited to, a full-length or an antigen-binding fragment of a therapeutic antibody, such as a Fab fragment, or antigen-binding fragments engineered to contain additional glycosylation sites on the Fab domain (e.g., see Courtois et al., 2016, mAbs 8: 99-112 which is incorporated by reference herein in its entirety for its description of derivatives of antibodies that are hyperglycosylated on the Fab domain of the full-length antibody).
  • the recombinant vector used for delivering the transgene includes non-replicating recombinant adeno-associated virus vectors (“rAAV”).
  • rAAVs are particularly attractive vectors for a number of reasons -they can be modified to preferentially target a specific organ of choice; and there are hundreds of capsid serotypes to choose from to obtain the desired tissue specificity, and/or to avoid neutralization by pre-existing patient antibodies to some AAVs.
  • Such rAAVs include but are not limited to AAV based vectors comprising capsid components from one or more of AAV2, AAV3B, AAV-LK03, AAVS3, AAV5, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO, AAVhu37, AAVrh46, AAVrh73, AAVrh8, or AAV64R1.
  • AAV based vectors provided herein comprise capsids from one or more of AAV8, AAVrh46, AAVrh73, or AAVS3, or AAV-LK03 serotypes.
  • viral vectors including but not limited to lentiviral vectors; vaccinia viral vectors, or non-viral expression vectors referred to as “naked DNA” constructs. Expression of the transgene can be controlled by constitutive or tissue-specific expression control elements.
  • Gene therapy constructs are designed such that both the heavy and light chains are expressed.
  • the full length heavy and light chains of the antibody are expressed.
  • the coding sequences encode for a Fab or F(ab’)2 or an scFv.
  • the heavy and light chains should be expressed at about equal amounts, in other words, the heavy and light chains are expressed at approximately a 1 : 1 ratio of heavy chains to light chains.
  • the coding sequences for the heavy and light chains can be engineered in a single construct in which the heavy and light chains are separated by a cleavable linker or IRES so that separate heavy and light chain polypeptides are expressed.
  • the linker separating the heavy and light chains is a Furin-2A linker, for example a Furin-F2A linker RKRR(GSG)APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NOS: 105 or 106) or a Furin-T2 A linker RKRR(GSG)EGRGSLLTCGDVEENPGP (SEQ ID NOS: 103 or 104).
  • the construct expresses, from the N-terminus to C-terminus, NH2- VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • the construct expresses, from the N-terminus to C-terminus, NH2-signal or localization sequence- VL-linker-VH-COOH or NH2- signal or localization sequence- VH-linker-VL-COOH.
  • the constructs express an scFv in which the heavy and light chain variable domains are connected via a flexible, non- cleavable linker.
  • nucleic acids e.g., polynucleotides
  • nucleic acid sequences disclosed herein may be codon-optimized, for example, via any codon-optimization technique known to one of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59: 149-161) and may also be optimized to reduce CpG dimers.
  • Each heavy and light chain requires a signal sequence to ensure proper post-translation processing and secretion (unless expressed as an scFv, in which only the N-terminal chain requires a signal sequence sequence).
  • Useful signal sequences for the expression of the heavy and light chains of the therapeutic antibodies in human cells are disclosed herein. Exemplary recombinant expression constructs are shown in FIGS. 1 A and IB.
  • HuPTM mAb or HuPTM Fab should result in a “biobetter” molecule for the treatment of disease accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding a full-length HuPTM mAb or HuPTM Fab or other antigen binding fragment, such as an scFv, of a therapeutic mAb to a patient (human subject) diagnosed with a disease indication for that mAb, to create a permanent depot in the subject that continuously supplies the human-glycosylated, sulfated transgene product produced by the subject’s transduced cells.
  • a viral vector or other DNA expression construct encoding a full-length HuPTM mAb or HuPTM Fab or other antigen binding fragment, such as an scFv
  • the cDNA construct for the HuPTM mAb or HuPTM Fab or HuPTM scFv should include a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced human cells.
  • compositions suitable for administration to human subjects comprise a suspension of the recombinant vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant and optional excipients.
  • a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant and optional excipients.
  • Such formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
  • the full-length HuPTM mAb or HuPTM Fab or other antigen binding fragment thereof can be produced in human cell lines by recombinant DNA technology, and the glycoprotein can be administered to patients.
  • Human cell lines that can be used for such recombinant glycoprotein production include but are not limited to human embryonic kidney 293 cells (HEK293), fibrosarcoma HT-1080, HKB-11, CAP, HuH-7, and retinal cell lines, PER.C6, or RPE to name a few (e.g., see Dumont et al., 2015, Crit. Rev. Biotechnol.
  • HuPTM Fab glycoprotein e.g., HuPTM Fab glycoprotein
  • the cell line used for production can be enhanced by engineering the host cells to co-express a-2,6-sialyltransferase (or both a-2,3- and a-2,6-sialyltransferases) and/or TPST-1 and TPST-2 enzymes responsible for tyrosine-O-sulfation in human cells.
  • the goal of gene therapy treatment of the invention is to prevent or reduce the incidence of viral infection (by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%) or reduce the severity of symptoms or shorten the course of viral infection (by 1, 2, 3, 4, 5, 6 or 7 days or by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%). .
  • Combination therapies involving delivery of the full-length HuPTM mAb or HuPTM Fab or antigen binding fragment thereof to the patient accompanied by administration of other available treatments, including other anti-viral antibodies, are encompassed by the methods of the invention.
  • the additional treatments may be administered before, concurrently or subsequent to the gene therapy treatment.
  • Such additional treatments can include but are not limited to co-therapy with the therapeutic mAb.
  • kits for manufacturing the viral vectors particularly the AAV based viral vectors.
  • methods of producing recombinant AAVs comprising culturing a host cell containing an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding a therapeutic antibody operably linked to expression control elements that will control expression of the transgene in human cells; a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans; sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid proteins; and recovering recombinant AAV encapsidating the artificial genome from the cell culture.
  • Viral vectors or other DNA expression constructs encoding an anti-viral HuPTM mAb or antigen-binding fragment thereof, particularly a HuGlyFab, or a hyperglycosylated derivative of a HuPTM mAb antigen-binding fragment are provided herein.
  • the viral vectors and other DNA expression constructs provided herein include any suitable method for delivery of a transgene to a target cell.
  • the means of delivery of a transgene include viral vectors, liposomes, other lipid- containing complexes, other macromolecular complexes, synthetic modified mRNA, unmodified mRNA, small molecules, non-biologically active molecules (e.g., gold particles), polymerized molecules (e.g., dendrimers), naked DNA, plasmids, phages, transposons, cosmids, or episomes.
  • the vector is a targeted vector, e.g., a vector targeted liver cells or a vector that has a tropism for liver cells.
  • the disclosure provides for a nucleic acid for use, wherein the nucleic acid comprises a nucleotide sequence that encodes a HuPTM mAb or HuGlyFab or other antigenbinding fragment thereof, as a transgene described herein, operatively linked to an ubiquitous promoter, a liver-specific and/or muscle-specific promoter, or an inducible promoter, wherein the promoter is selected for expression in tissue targeted for expression of the transgene.
  • Promoters may, for example, be a CB7/CAG promoter (SEQ ID NO:36) and associated upstream regulatory sequences, cytomegalovirus (CMV) promoter, EF-1 alpha promoter (SEQ ID NO:39), mUla (SEQ ID NO:38), UB6 promoter, chicken beta-actin (CBA) promoter, and liver-specific promoters, such as TBG (Thyroxine-binding Globulin) promoter (SEQ ID NO:40), APOA2 promoter, SERPINA1 (hAAT) promoter, ApoE.hAAT (SEQ ID NO:21), CRE selected from Table 16, CRE (Table 16).
  • CMV cytomegalovirus
  • EF-1 alpha promoter SEQ ID NO:39
  • mUla SEQ ID NO:38
  • UB6 promoter EF-1 alpha promoter
  • CBA chicken beta-actin
  • TBG Thyroxine-binding Globulin
  • SERPINA1 h
  • hAAT or muscle-specific promoters, such as a human desmin promoter, CK8 promoter (SEQ ID NO:37) or Pitx3 promoter, inducible promoters, such as a hypoxia-inducible promoter or a rapamycin-inducible promoter, or a combination thereof.
  • the promoter is a liver-specific promoter or a liver- and muscle-specific (dual) promoter.
  • the promoter is the liver-specific ApoE.hAAT (SEQ ID NO:21) promoter.
  • the promoter is one, two, or three liver-specific cis- regulatory elements selected from the sequences in Table 16 or a dual promoter comprising one of the cis-regulatory elements selected from the sequences in Table 16 and the hAAT promoter.
  • transgene expression is controlled by engineered nucleic acid regulatory elements that have more than one regulatory element (promoter or enhancer), including regulatory elements that are arranged in tandem (two or three copies) that promote liver-specific expression, or both liver-specific expression and muscle-specific expression.
  • LSPX1 SEQ ID NO:9
  • LSPX2 SEQ ID NO: 10
  • LTP1 SEQ ID NO: 11
  • LTP2 SEQ ID NO: 12
  • LTP3 SEQ ID NO: 13
  • LMTP6 SEQ ID NO: 14
  • LMTP13 SEQ ID NO: 15
  • LMTP14 SEQ ID NO: 16
  • LMTP 15 SEQ ID NO : 17
  • LMTP 18 SEQ ID NO : 18
  • LMTP 19 SEQ ID NO: 19
  • LMTP20 SEQ ID NO:20
  • nucleic acids e.g., polynucleotides
  • the nucleic acids may comprise DNA, RNA, or a combination of DNA and RNA.
  • the DNA comprises one or more of the sequences selected from the group consisting of promoter sequences, the sequence of the gene of interest (the transgene, e.g., the nucleotide sequences encoding the heavy and light chains of the HuPTMmAb or HuGlyFab or other antigen-binding fragment), untranslated regions, and termination sequences.
  • viral vectors provided herein comprise a promoter operably linked to the gene of interest.
  • nucleic acids e.g., polynucleotides
  • nucleic acid sequences disclosed herein may be codon-optimized, for example, via any codon-optimization technique known to one of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59: 149- 161).
  • the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) one or more control elements, b) a optional intron and c) a rabbit 0-globin poly A signal; and (3) nucleic acid sequences coding for the heavy and light chains of a mAb or Fab, separated by a self-cleaving furin (F)/(F/T)2A linker (SEQ ID NOS: 103, 104, 105 or 105), ensuring expression of equal amounts of the heavy and the light chain polypeptides.
  • Exemplary constructs are shown in FIGS. lAto 1C.
  • the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) ApoE.hAAT promoter, b) a optional intron and c) a rabbit 0-globin poly A signal; and (3) nucleic acid sequences coding for a full-length antibody comprising the heavy and light chain sequences using sequences that encode the Fab portion of the heavy chain, including the hinge region sequence, plus the Fc polypeptide of the heavy chain for the appropriate isotype and the light chain, wherein heavy and light chain nucleotide sequences are separated by a self-cleaving furin (F)/(F/T)2A linker (SEQ ID NOS: 198, 199, 200 or 201), ensuring expression of equal amounts of the heavy and the light chain polypeptides.
  • An exemplary construct is shown in FIG. IB.
  • the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) a CRE selected from Table 16.hAAT promoter, b) a optional intron and c) a rabbit P-globin poly A signal; and (3) nucleic acid sequences coding for a full-length antibody comprising the heavy and light chain sequences using sequences that encode the Fab portion of the heavy chain, including the hinge region sequence, plus the Fc polypeptide of the heavy chain for the appropriate isotype and the light chain, wherein heavy and light chain nucleotide sequences are separated by a self-cleaving furin (F)/(F/T)2A linker (SEQ ID NOS: 103, 104, 105 or 105), ensuring expression of equal amounts of the heavy and the light chain polypeptides.
  • An exemplary construct is shown in FIG. 1C.
  • the vectors provided herein are modified mRNA encoding for the gene of interest (e.g., the transgene, for example, HuPTMmAb or HuGlyFab or other antigen binding fragment thereof).
  • the transgene for example, HuPTMmAb or HuGlyFab or other antigen binding fragment thereof.
  • the synthesis of modified and unmodified mRNA for delivery of a transgene to retinal pigment epithelial cells is taught, for example, in Hansson et al., J. Biol. Chem., 2015, 290(9):5661-5672, which is incorporated by reference herein in its entirety.
  • provided herein is a modified mRNA encoding for a HuPTMmAb, HuPTM Fab, or HuPTM scFv.
  • Viral vectors include adenovirus, adeno-associated virus (AAV, e.g., AAV8, AAV9, AAVrhlO, AAVS3), lentivirus, helper-dependent adenovirus, herpes simplex virus, poxvirus, hemagglutinin virus of Japan (HVJ), alphavirus, vaccinia virus, and retrovirus vectors.
  • Retroviral vectors include murine leukemia virus (MLV) and human immunodeficiency virus (HlV)-based vectors.
  • Alphavirus vectors include semliki forest virus (SFV) and Sindbis virus (SIN).
  • the viral vectors provided herein are recombinant viral vectors.
  • the viral vectors provided herein are altered such that they are replication-deficient in humans.
  • the viral vectors are hybrid vectors, e.g., an AAV vector placed into a “helpless” adenoviral vector.
  • viral vectors comprising a viral capsid from a first virus and viral envelope proteins from a second virus.
  • the second virus is vesicular stomatitus virus (VSV).
  • VSV vesicular stomatitus virus
  • the envelope protein is VSV- G protein.
  • the viral vectors provided herein are HIV based viral vectors.
  • HIV-based vectors provided herein comprise at least two polynucleotides, wherein the gag and pol genes are from an HIV genome and the env gene is from another virus.
  • the viral vectors provided herein are herpes simplex virusbased viral vectors.
  • herpes simplex virus-based vectors provided herein are modified such that they do not comprise one or more immediately early (IE) genes, rendering them non-cytotoxic.
  • IE immediately early
  • the viral vectors provided herein are MLV based viral vectors.
  • MLV-based vectors provided herein comprise up to 8 kb of heterologous DNAin place of the viral genes.
  • the viral vectors provided herein are lentivirus-based viral vectors.
  • lentiviral vectors provided herein are derived from human lentiviruses.
  • lentiviral vectors provided herein are derived from non-human lentiviruses.
  • lentiviral vectors provided herein are packaged into a lentiviral capsid.
  • lentiviral vectors provided herein comprise one or more of the following elements: long terminal repeats, a primer binding site, a polypurine tract, att sites, and an encapsidation site.
  • the viral vectors provided herein are alphavirus-based viral vectors.
  • alphavirus vectors provided herein are recombinant, replicationdefective alphaviruses.
  • alphavirus replicons in the alphavirus vectors provided herein are targeted to specific cell types by displaying a functional heterologous ligand on their virion surface.
  • the viral vectors provided herein are AAV based viral vectors.
  • the AAV-based vectors provided herein do not encode the AAV rep gene (required for replication) and/or the AAV cap gene (required for synthesis of the capsid proteins) (the rep and cap proteins may be provided by the packaging cells in trans). Multiple AAV serotypes have been identified.
  • AAV-based vectors provided herein comprise components from one or more serotypes of AAV.
  • AAV-based vectors provided herein comprise components from one or more serotypes of AAV with tropism to liver and/or muscle.
  • AAV based vectors provided herein comprise capsid components from one or more of AAV2 (SEQ ID NO:25), AAV7 (SEQ ID NO:1), AAV8 (SEQ ID NO:2), AAV9 (SEQ ID NO:3), AAVS3 (SEQ ID NO:7), AAVrh46 (SEQ ID NO:5), AAVrh73 (SEQ ID NO:6), AAV-LK03 (SEQ ID NO:8), or AAVrhlO (SEQ ID NO:4).
  • AAV based vectors provided herein are or comprise components from one or more of AAV8, AAVS3, AAV-LK03, AAVrh46, AAVrh73, or AAVrhlO serotypes.
  • the encoded AAV capsid has the sequence of SEQ ID NO:2
  • FIG. 4 provides a comparative alignment of the amino acid sequences of the capsid proteins of different AAV serotypes with potential amino acids that may be substituted at certain positions in the aligned sequences based upon the comparison in the row labeled SUBS.
  • the AAV vector comprises an AAV8, AAVS3, orAAV-LK03, capsid variant that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
  • amino acid sequence for AAV8, AAVS3, or AAV-LK03 capsids are provided in FIG. 4.
  • the amino acid sequence of hu37 capsid can be found in international application PCT
  • WO 2005/033321 SEQ ID NO: 88 thereof
  • amino acid sequence for the rh8 capsid can be found in international application PCT WO 03/042397 (SEQ ID NO:97).
  • the amino acid sequence for the rh64Rl sequence is found in W02006/110689 (a R697W substitution of the Rh.64 sequence, which is SEQ ID NO: 43 of WO 2006/110689).
  • the rh64Rl sequence is:
  • AAV-based vectors comprise components from one or more serotypes of AAV.
  • AAV based vectors provided herein comprise capsid components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.rh46, AAV.rh73, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV
  • AAV based vectors provided herein comprise components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.rh46, AAV.rh73, AAVRh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAVPHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAVLK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8,
  • rAAV particles comprise a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e.
  • AAV capsid serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.rh46, AAV.rh73, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAVPHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC
  • the recombinant AAV for us in compositions and methods herein is AAVS3 (including variants thereof) (see e.g., US Patent Application No. 20200079821, which is incorporated herein by reference in its entiretiy).
  • rAAV particles comprise the capsids of AAV-LK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety.
  • the AAV for use in compositions and methods herein is any AAV disclosed in US 10,301,648, such as AAV.rh46 or AAV.rh73.
  • the recombinant AAV for use in compositions and methods herein is Anc80 or Anc80L65 (see, e.g., Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety).
  • the AAV for use in compositions and methods herein is any AAV disclosed in US 9,585,971, such as AAV-PHP.B.
  • the AAV for use in compositions and methods herein is an AAV2/Rec2 or AAV2/Rec3 vector, which has hybrid capsid sequences derived from AAV8 and serotypes cy5, rh20 or rh39 (see, e.g., Issa et al., 2013, PLoS One 8(4): e60361, which is incorporated by reference herein for these vectors).
  • the AAV for use in compositions and methods herein is an AAV disclosed in any of the following, each of which is incorporated herein by reference in its entirety: US 7,282,199; US 7,906,111; US 8,524,446; US 8,999,678; US 8,628,966; US 8,927,514; US 8,734,809; US9,284,357; US 9,409,953; US 9,169,299; US 9,193,956; US 9,458,517; US 9,587,282; US 2015/0374803; US 2015/0126588; US 2017/0067908; US 2013/0224836; US 2016/0215024; US 2017/0051257; PCT/US2015/034799; and PCT/EP2015/053335.
  • rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos.
  • rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,840,719 and WO 2015/013313, such as AAV.Rh74 and RHM4-1, each of which is incorporated herein by reference in its entirety.
  • rAAV particles comprise any AAV capsid disclosed in WO 2014/172669, such as AAV rh.74, which is incorporated herein by reference in its entirety.
  • rAAV particles comprise the capsid of AAV2/5, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety.
  • rAAV particles comprise any AAV capsid disclosed in WO 2017/070491, such as AAV2tYF, which is incorporated herein by reference in its entirety.
  • rAAV particles comprise any AAV capsid disclosed in US Pat Nos. 8,628,966; US 8,927,514; US 9,923,120 and WO 2016/049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16, each of which is incorporated by reference in its entirety.
  • rAAV particles have a capsid protein disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g., SEQ ID NO: 2 of '051 publication), WO 2005/033321 (see, e.g., SEQ ID NOs: 123 and 88 of '321 publication), WO 03/042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of '397 publication), WO 2006/068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of '888 publication), WO 2006/110689, (see, e.g., SEQ ID NOs: 5-38 of '689 publication) W02009/104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of '964 publication), WO 2010/127097 (see, e.g., SEQ ID NOs:
  • rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in Inti. Appl. Publ. No.
  • WO 2003/052051 see, e.g., SEQ ID NO: 2 of '051 publication
  • WO 2005/033321 see, e.g., SEQ ID NOs: 123 and 88 of '321 publication
  • WO 03/042397 see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of '397 publication
  • WO 2006/068888 see, e.g., SEQ ID NOs: 1 and 3-6 of '888 publication
  • WO 2006/110689 see, e.g., SEQ ID NOs: 5-38 of '689 publication
  • W02009/104964 see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of 964 publication
  • W0 2010/127097 see, e.g., SEQ ID NOs: 5-38 of '097 publication
  • WO 2015/191508 see, e.g., SEQ ID NOs: 80-294 of
  • rAAV particles comprise a pseudotyped AAV capsid.
  • the pseudotyped AAV capsids are rAAV2/8 or rAAV2/9 pseudotyped AAV capsids.
  • Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74: 1524-1532 (2000); Zolotukhin et al., Methods 28: 158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001).
  • AAV3 -based, AAV8-based, AAV9-based, and AAVrh 10-based viral vectors are used in certain of the methods described herein.
  • Nucleotide sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in United States Patent No. 7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No. 8,318,480 B2, United States Patent No. 8,962,332 B2 and International Patent Application No. PCT/EP2014/076466, each of which is incorporated herein by reference in its entirety.
  • AAV e.g., AAV3, AAV8, AAV9 or AAVrh
  • a transgene e.g., an HuPTM Fab
  • AAV capsids including AAV8, AAV9 and AAVrhlO are provided in Figure 4.
  • ssAAV single-stranded AAV
  • a self-complementary vector e.g., scAAV
  • scAAV self-complementary vector
  • the viral vectors used in the methods described herein are adenovirus based viral vectors.
  • a recombinant adenovirus vector may be used to transfer in the transgene encoding the HuPTMmAb or HuGlyFab or antigen-binding fragment.
  • the recombinant adenovirus can be a first-generation vector, with an El deletion, with or without an E3 deletion, and with the expression cassette inserted into either deleted region.
  • the recombinant adenovirus can be a second-generation vector, which contains full or partial deletions of the E2 and E4 regions.
  • a helperdependent adenovirus retains only the adenovirus inverted terminal repeats and the packaging signal (phi).
  • the transgene is inserted between the packaging signal and the 3’ITR, with or without stuffer sequences to keep the genome close to wild-type size of approximately 36 kb.
  • An exemplary protocol for production of adenoviral vectors may be found in Alba et al., 2005, “Gutless adenovirus: last generation adenovirus for gene therapy,” Gene Therapy 12:S18-S27, which is incorporated by reference herein in its entirety.
  • the viral vectors used in the methods described herein are lentivirus based viral vectors.
  • a recombinant lenti virus vector may be used to transfer in the transgene encoding the HuPTM mAb antigen binding fragment.
  • Four plasmids are used to make the construct: Gag/pol sequence containing plasmid, Rev sequence containing plasmids, Envelope protein containing plasmid (e.g., VSV-G), and Cis plasmid with the packaging elements and the anti-VEGF antigen-binding fragment gene.
  • the four plasmids are co-transfected into cells (e.g., HEK293 based cells), whereby polyethylenimine or calcium phosphate can be used as transfection agents, among others.
  • the lentivirus is then harvested in the supernatant (lentiviruses need to bud from the cells to be active, so no cell harvest needs/should be done).
  • the supernatant is filtered (0.45 pm) and then magnesium chloride and benzonase added.
  • Further downstream processes can vary widely, with using TFF and column chromatography being the most GMP compatible ones. Others use ultracentrifugation with/without column chromatography.
  • Exemplary protocols for production of lentiviral vectors may be found in Lesch et al., 2011, “Production and purification of lentiviral vector generated in 293T suspension cells with baculoviral vectors,” Gene Therapy 18:531-538, andAusubel et al., 2012, “Production of CGMP-Grade Lentiviral Vectors,” Bioprocess Int. 10(2):32-43, both of which are incorporated by reference herein in their entireties.
  • a vector for use in the methods described herein is one that encodes an HuPTM mAb, such that, upon introduction of the vector into a relevant cell, a glycosylated and/or tyrosine sulfated variant of the HuPTM mAb is expressed by the cell.
  • the vectors provided herein comprise components that modulate gene delivery or gene expression (e.g., “expression control elements”). In certain embodiments, the vectors provided herein comprise components that modulate gene expression. In certain embodiments, the vectors provided herein comprise components that influence binding or targeting to cells. In certain embodiments, the vectors provided herein comprise components that influence the localization of the polynucleotide (e.g., the transgene) within the cell after uptake. In certain embodiments, the vectors provided herein comprise components that can be used as detectable or selectable markers, e.g., to detect or select for cells that have taken up the polynucleotide.
  • the viral vectors provided herein comprise one or more promoters that control expression of the transgene.
  • These promoters may be constitutive (promote ubiquitous expression) or may specifically or selectively express in the liver (including promoting expression in the liver only or expressing in the liver at least at 1 to 100 fold greater levels than in a non-liver tissue).
  • the promoter is a constitutive promoter.
  • the promoter is a CB7 (also referred to as a CAG promoter) (see Dinculescu et al., 2005, Hum Gene Ther 16: 649-663, incorporated by reference herein in its entirety).
  • the CAG or CB7 promoter (SEQ ID NO: 128) includes other expression control elements that enhance expression of the transgene driven by the vector.
  • the other expression control elements include chicken P-actin intron and/or rabbit P- globin polyA signal.
  • the promoter comprises a TATA box.
  • the promoter comprises one or more elements. In certain embodiments, the one or more promoter elements may be inverted or moved relative to one another.
  • the elements of the promoter are positioned to function cooperatively. In certain embodiments, the elements of the promoter are positioned to function independently.
  • the viral vectors provided herein comprise one or more promoters selected from the group consisting of the human CMV immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus (RS) long terminal repeat, and rat insulin promoter. In certain embodiments, the vectors provided herein comprise one or more long terminal repeat (LTR) promoters selected from the group consisting of AAV, MLV, MMTV, SV40, RSV, HIV-1, and HIV-2 LTRs.
  • LTR long terminal repeat
  • the vectors provided herein comprise one or more tissue specific promoters (e.g., a liver-specific promoter or a dual liver-muscle specific promoter).
  • the viral vectors provided herein comprises a liver cell specific promoter, such as, a TBG (Thyroxine-binding Globulin) promoter (SEQ ID NO: 132), liver specific cisregulating element selected from Table 16, an APOA2 promoter, a SERPINA1 (hAAT) promoter, an ApoE.hAAT promoter (SEQ ID NO:21), or a CRE (Table 16).
  • hAAT promoter such as, a TBG (Thyroxine-binding Globulin) promoter (SEQ ID NO: 132), liver specific cisregulating element selected from Table 16, an APOA2 promoter, a SERPINA1 (hAAT) promoter, an ApoE.hAAT promoter (SEQ ID NO:21), or a CRE (Table 16).
  • the viral vector provided herein comprises a muscle specific promoter, such as a human desmin promoter (Jonuschies et al., 2014, Curr. Gene Then 14:276-288), a CK8 promoter (SEQ ID NO: 129; Himeda et al., 2011 Muscle Gene Therapy: Methods and Protocols, Methods in Molecular Biology, Dongsheng Duan (ed.), 709:3-19), or aPitx3 promoter (Coulon et al., 2007, JBC 282:33192).
  • the viral vector comprises a VMD2 promoter.
  • nucleic acid regulatory elements that are chimeric with respect to arrangements of elements in tandem in the expression cassette. Regulatory elements, in general, have multiple functions as recognition sites for transcription initiation or regulation, coordination with cellspecific machinery to drive expression upon signaling, and to enhance expression of the downstream gene.
  • nucleic acid regulatory elements that promote transgene expression in liver tissue, or liver and muscle (skeletal and/or cardiac) tissue.
  • certain elements are arranged with two or more copies of the individual enhancer and promoter elements arranged in tandem and operably linked to a transgene to promote expression, particularly tissue specific expression.
  • Exemplary nucleotide sequences of the individual promoter and enhancer elements are provided in Table 1.
  • Table 1 Also provided in Table 1 are exemplary composite nucleic acid regulatory elements comprising the individual tandem promoter and enhancer elements.
  • the downstream promoter is an hAAT promoter (SEQ ID NO:30) (in certain embodiments the hAAT promoter is an hAAT(AATG) promoter (SEQ ID NO:31)) and the other promoter is another hAAT promoter or is a TBG promoter (SEQ ID NO:40).
  • transgene expression from tandem promoters i.e. two promoter sequences driving expression of the same transgene
  • This approach was employed to improve transgene expression from tandem tissue-specific promoter cassettes (such as those targeting the liver) as well as promoter cassettes to achieve dual expression in two separate tissue populations (such as liver and skeletal muscle, and in certain embodiments cardiac muscle, and liver and bone).
  • tissue-specific promoter cassettes such as those targeting the liver
  • promoter cassettes to achieve dual expression in two separate tissue populations (such as liver and skeletal muscle, and in certain embodiments cardiac muscle, and liver and bone).
  • these designs aim to improve the therapeutic efficacy of gene transfer by providing more robust levels of transgene expression, improved stability/persistence, and induction of immune tolerance to the transgene product.
  • the hAAT promoter with the start codon deleted (AATG) is used in an expression cassette provided herein.
  • nucleic acid regulatory elements that comprise or consist of promoters and/or other nucleic acid elements, such as enhancers, that promote liver expression, such as liver-specifc cis-regulating enhances of Table 16, ApoE enhancers, Mic/BiKE elements or hAAT promoters. These may be present as single copies or with two or more copies in tandem.
  • the nucleic acid regulatory element may also comprise, in addition to the one or more elements that promote liver specific expression, one or more elements that promote muscle specific expression (including skeletal and/or cardiac muscle), for example, one or more copies, for example two copies, of the MckE element, which may be arranged as two or more copies in tandem or an MckE and MhcE elements arranged in tandem.
  • a promoter element is deleted for the initiation codon to prevent translation initiation at that site, and preferably, the element with the modified start codon is the promoter that is the element at the 3’ end or the downstream end of the nucleic acid regulatory element, for example, closest within the nucleic acid sequence of the expression cassette to the transgene.
  • the composite nucleic acid regulatory element comprises an hAAT promoter, in embodiments an hAAT which is start-codon modified (AATG) as the downstream promoter, and a second promoter in tandem with the hAAT promoter, which is an hAAT promoter, a CK8 promoter, an Spc5.12 promoter or an minSpc5.12 promoter.
  • Nucleotide sequences are provided in Table 1.
  • the nucleotide sequence encoding the anti-viral antibody heavy and light chains is operably linked to a composite nucleic acid regulatory element comprising a) two copies of Mic/BiKE arranged in tandem or two copies of ApoE arranged in tandem or two copies of Mic/BiKE arranged in tandem with one copy of ApoE, b) one promoter or, in tandem promoter embodiments, two promoters arranged in tandem comprising at least one copy of hAAT which is start-codon modified (AATG) (where in certain embodiments the hAAT promoter is the downstream or 3’ promoter).
  • the composite nucleic acid regulatory element comprises LSPX1, LSPX2, LTP1, LTP2, or LTP3 of Table 1.
  • nucleotide sequence encoding the heavy and light chains of the anti-viral antibody is operably linked to a nucleic acid regulatory element comprising a) one copy of ApoE, two or three copies of MckE arranged in tandem, one copy of each MckE, MhcE, and ApoE arrange in tandem, or two or three copies of MckE arranged in tandem with one copy of ApoE, b) two copies of a promoter arranged in tandem comprising at least one copy of hAAT which is start-codon modified (AATG).
  • AATG start-codon modified
  • the second and upstream promoter is a CK8 promoter, an Spc5.12 promoter or a minSpc5.12 promoter.
  • the composite nucleic acid regulatory element comprises LMTP6, LMTP13, LMTP14, LMTP15, LMTP18, LMTP19, or LMTP20 of Table 1.
  • the nucleotide sequence encoding the anti-viral antibody heavy and light chains is operably linked to a composite nucleic acid regulatory element comprising a) two copies of a CRE selected from Table 16 arranged in tandem or two copies of Mic/BiKE arranged in tandem with one copy of a CRE selected from Table 16, b) one promoter or, in tandem promoter embodiments, two promoters arranged in tandem comprising at least one copy of hAAT which is startcodon modified (AATG) (where in certain embodiments the hAAT promoter is the downstream or 3’ promoter).
  • the composite nucleic acid regulatory element comprises LSPX1, LSPX2, LTP1, LTP2, or LTP3 of Table 1.
  • nucleic acid regulatory element comprising a) one copy of a CRE selected from Table 16, one copy of each MckE, MhcE, and a CRE selected from Table 16 arrange in tandem, or two or three copies of MckE arranged in tandem with one copy of a CRE selected from Table 16, b) two copies of a promoter arranged in tandem comprising at least one copy of hAAT which is start-codon modified (AATG).
  • the second and upstream promoter is a CK8 promoter, an Spc5.12 promoter or a minSpc5.12 promoter.
  • the composite nucleic acid regulatory element comprises LMTP6, LMTP13, LMTP14, LMTP15, LMTP18, LMTP19, or LMTP20 of Table 1.
  • the anti-viral therapeutic antibody coding sequence is operably linked to composite nucleic acid regulatory elements for enhancing gene expression in the liver LSPX1 (SEQ ID NO: 9), LSPX2 (SEQ ID NO: 10), LTP1 (SEQ ID NO: 11), LTP2 (SEQ ID NO: 12), or LTP3 (SEQ ID NO: 13), liver and muscle expression, LMTP6 (SEQ ID NO: 14), LMTP13 (SEQ ID NO: 15), LMTP14 (SEQ ID NO: 16), LMTP15 (SEQ ID NO: 17), LMTP18 (SEQ ID NO: 18), LMTP19 (SEQ ID NO: 19), or LMTP20 (SEQ ID NO:20), the sequences of which are provided in Table 1 below.
  • composite regulatory elements that enhance gene expression in the liver, and in certain embodiments, also muscle or bone, which have 99%, 95%, 90%, 85% or 80% sequence identity with one of nucleic acid sequences LSPX1 (SEQ ID NO:9), LSPX2 (SEQ ID NO: 10), LTP1 (SEQ ID NO: 11), LTP2 (SEQ ID NO: 12), or LTP3 (SEQ ID NO: 13), LMTP6 (SEQ ID NO: 14), LMTP13 (SEQ ID NO: 15), LMTP14 (SEQ ID NO: 16), LMTP15 (SEQ ID NO: 17), LMTP18 (SEQ ID NO: 18), LMTP19 (SEQ ID NO: 19), or LMTP20 (SEQ ID NO:20).
  • LSPX1 SEQ ID NO:9
  • LSPX2 SEQ ID NO: 10
  • LTP1 SEQ ID NO: 11
  • LTP2 SEQ ID NO: 12
  • LTP3 SEQ ID NO: 13
  • LMTP6
  • the constructs described herein result in preferred transcription start sites within the promoter region.
  • the constructs described herein have a tandem or composite nucleic acid regulatory sequence that comprises an hAAT promoter (particularly a modified start codon hAAT promoter) and has a transcription start site of TCTCC (corresponding to nt 1541-1545 of LMTP6 (SEQ ID NO: 14), which overlaps with the active TTS found in hAAT (nt 355-359 of SEQ ID NO:30) or GGTACAATGACTCCTTTCG (SEQ ID NO: 160), which corresponds to nucleotides 139-157 of SEQ ID NO:30, or GGTACAGTGACTCCTTTCG (SEQ ID NO: 161), which corresponds to nucleotides 139-157 of SEQ ID NO:31.
  • the constructs described herein have a tandem or composite regulatory sequence that comprises a CK8 promoter and has a transcription start site at TCATTCTACC (SEQ ID NO: 162), which corresopnds to nucleotides 377-386 of SEQ ID NO:37, particularly starting at the nucleotide corresponding to nucleotide 377 of SEQ ID NO: 14 or corresponding to nucleotide 1133 of SEQ ID NO: 14.
  • the promoter is an inducible promoter. In certain embodiments the promoter is a hypoxia-inducible promoter. In certain embodiments, the promoter comprises a hypoxia-inducible factor (HIF) binding site. In certain embodiments, the promoter comprises a HIF- la binding site. In certain embodiments, the promoter comprises a HIF -2a binding site. In certain embodiments, the HIF binding site comprises an RCGTG motif. For details regarding the location and sequence of HIF binding sites, see, e.g., Schodel, et al., Blood, 2011, 117(23):e207-e217, which is incorporated by reference herein in its entirety.
  • the promoter comprises a binding site for a hypoxia induced transcription factor other than a HIF transcription factor.
  • the viral vectors provided herein comprise one or more IRES sites that is preferentially translated in hypoxia.
  • the hypoxia-inducible promoter is the human N-WASP promoter, see, e.g., Salvi, 2017, Biochemistry and Biophysics Reports 9: 13-21 (incorporated by reference for the teaching of the N-WASP promoter) or is the hypoxia-induced promoter of human Epo, see, e.g., Tsuchiya et al., 1993, J. Biochem. 113:395-400 (incorporated by reference for the disclosure of the Epo hypoxia-inducible promoter).
  • the promoter is a drug inducible promoter, for example, a promoter that is induced by administration of rapamycin or analogs thereof.
  • rapamycin inducible promoters in PCT publications WO94/18317, WO 96/20951, WO 96/41865, WO 99/10508, WO 99/10510, WO 99/36553, and WO 99/41258, and US 7,067,526, which are hereby incorporated by reference in their entireties for the disclosure of drug inducible promoters.
  • constructs containing certain ubiquitous and tissue-specific promoters include synthetic and tandem promoters. Examples and nucleotide sequences of promoters are provided in Table 1 below. Table 1 also includes the nucleotide sequences of other regulatory elements useful for the expression cassettes provided herein
  • the viral vectors provided herein comprise one or more regulatory elements other than a promoter. In certain embodiments, the viral vectors provided herein comprise an enhancer. In certain embodiments, the viral vectors provided herein comprise a repressor. In certain embodiments, the viral vectors provided herein comprise an intron (e.g. VH4 intron (SEQ ID NO:42) SV40 Intron (SEQ ID NO:43) or a chimeric intron (P-globin/Ig Intron) (SEQ ID NO:41).
  • VH4 intron SEQ ID NO:42
  • SEQ ID NO:43 SV40 Intron
  • P-globin/Ig Intron SEQ ID NO:41
  • the viral vectors provided herein comprise a polyadenylation sequence downstream of the coding region of the transgene.
  • Any polyA site that signals termination of transcription and directs the synthesis of a polyA tail is suitable for use in AAV vectors of the present disclosure.
  • Exemplary polyA signals are derived from, but not limited to, the following: the SV40 late gene, the rabbit P-globin gene (SEQ ID NO:45), the bovine growth hormone (BPH) gene, the human growth hormone (hGH) gene, the synthetic polyA (SPA) site, and the bovine growth hormone (bGH) gene. See, e.g., Powell and Rivera-Soto, 2015, Discov. Med., 19(102):49-57.
  • the vectors provided herein comprise components that modulate protein delivery.
  • the viral vectors provided herein comprise one or more signal peptides.
  • Signal peptides also referred to as “signal sequences” may also be referred to herein as “leader sequences” or “leader peptides”.
  • the signal peptides allow for the transgene product to achieve the proper packaging (e.g., glycosylation) in the cell.
  • the signal peptides allow for the transgene product to achieve the proper localization in the cell.
  • the signal peptides allow for the transgene product to achieve secretion from the cell.
  • a signal sequence for protein production in a gene therapy context or in cell culture There are two general approaches to select a signal sequence for protein production in a gene therapy context or in cell culture.
  • One approach is to use a signal peptide from proteins homologous to the protein being expressed.
  • a human antibody signal peptide may be used to express IgGs in CHO or other cells.
  • Another approach is to identify signal peptides optimized for the particular host cells used for expression. Signal peptides may be interchanged between different proteins or even between proteins of different organisms, but usually the signal sequences of the most abundant secreted proteins of that cell type are used for protein expression.
  • the signal peptide of human albumin the most abundant protein in plasma, was found to substantially increase protein production yield in CHO cells.
  • the signal peptide may retain function and exert activity after being cleaved from the expressed protein as “post-targeting functions”.
  • the signal peptide is selected from signal peptides of the most abundant proteins secreted by the cells used for expression to avoid the post-targeting functions.
  • the signal sequence is fused to both the heavy and light chain sequences.
  • An exemplary sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO:50) which can be encoded by a nucleotide sequence of SEQ ID NO: 146 (see Table 2).
  • signal sequences that are appropriate for expression, and may cause selective expression or directed expression of the HuPTM mAb or Fab or scFv in muscle, or liver are provided in Tables 2 and 3, respectively, below.
  • a single construct can be engineered to encode both the heavy and light chains separated by a cleavable linker or IRES so that separate heavy and light chain polypeptides are expressed by the transduced cells.
  • the viral vectors provided herein provide polycistronic (e.g., bicistronic) messages.
  • the viral construct can encode the heavy and light chains separated by an internal ribosome entry site (IRES) elements (for examples of the use of IRES elements to create bicistronic vectors see, e.g., Gurtu et al., 1996, Biochem. Biophys. Res. Comm. 229(l):295-8, which is herein incorporated by reference in its entirety).
  • IRES internal ribosome entry site
  • the bicistronic message is contained within a viral vector with a restraint on the size of the polynucleotide(s) therein.
  • the bicistronic message is contained within an AAV virus-based vector (e.g., an AAV8- based, AAV9-based or A AVrh 10-based vector).
  • Furin-2A linkers encode the heavy and light chains separated by a cleavable linker such as the self-cleaving 2A and 2A-like peptides, with or without upstream furin cleavage sites, e.g. Furin/2A linkers, such as furin/F2A (F/F2A) or furin/T2A (F/T2A) linkers (Fang et al., 2005, Nature Biotechnology 23: 584-590, Fang, 2007, Mol Ther 15: 1153-9, and Chang, J. et al, MAbs 2015, 7(2):403-412, each of which is incorporated by reference herein in its entirety).
  • a furin/2A linker may be incorporated into an expression cassette to separate the heavy and light chain coding sequences, resulting in a construct with the structure:
  • a 2A site or 2A-like site such as an F2A site comprising the amino acid sequence RKRR(GSG)APVKQTLNFDLLKLAGDVESNPGP(SEQ ID NOS: 105 or 106) or a T2A site comprising the amino acid sequence RKRR(GSG)EGRGSLLTCGDVEENPGP (SEQ ID NOS: 103 or 104), is self-processing, resulting in “cleavage” between the final G and P amino acid residues.
  • Several linkers, with or without an upstream flexible Gly-Ser-Gly (GSG) linker sequence SEQ ID NO: 185
  • linkers with or without an upstream flexible Gly-Ser-Gly (GSG) linker sequence (SEQ ID NO: 185), that could be used include but are not limited to:
  • T2A (GSG)EGRGSLLTCGDVEENPGP (SEQ ID NOS:95 or 96);
  • P2A (GSG)ATNFSLLKQAGDVEENPGP (SEQ ID NOS:97 or 98);
  • E2A (GSG)QCTNYALLKLAGDVESNPGP (SEQ ID NOS:99 or 100);
  • F2A (GSG)APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NOS: 101 or 102)
  • an additional proteolytic cleavage site e.g. a furin cleavage site
  • the self-processing cleavage site e.g. 2A or 2A like sequence
  • a peptide bond is skipped when the ribosome encounters the 2A sequence in the open reading frame, resulting in the termination of translation, or continued translation of the downstream sequence (the light chain).
  • This self-processing sequence results in a string of additional amino acids at the end of the C-terminus of the heavy chain.
  • additional amino acids can then be cleaved by host cell Furin at the furin cleavage site(s), e.g. located immediately prior to the 2A site and after the heavy chain sequence, and further cleaved by carboxypeptidases.
  • the resultant heavy chain may have one, two, three, or more additional amino acids included at the C-terminus, or it may not have such additional amino acids, depending on the sequence of the Furin linker used and the carboxypeptidase that cleaves the linker in vivo (See, e.g. , Fang et al., 17 April 2005, Nature Biotechnol.
  • Furin linkers that may be used comprise a series of four basic amino acids, for example, RKRR (SEQ ID NO:91), RRRR (SEQ ID NO:92), RRKR (SEQ ID NO:93), or RKKR (SEQ ID NO:94).
  • linker Once this linker is cleaved by a carboxypeptidase, additional amino acids may remain, such that an additional zero, one, two, three or four amino acids may remain on the C- terminus of the heavy chain, for example, R, RR, RK, RKR, RRR, RRK, RKK, RKRR (SEQ ID NO:91), RRRR (SEQ ID NO:92), RRKR (SEQ ID NO:93), or RKKR (SEQ ID NO:94).
  • R, RR, RK, RKR, RRR, RRK, RKK, RKRR SEQ ID NO:91
  • RRRR SEQ ID NO:92
  • RRKR SEQ ID NO:93
  • RKKR SEQ ID NO:94
  • the furin linker has the sequence R-X-K/R-R, such that the additional amino acids on the C-terminus of the heavy chain are R, RX, RXK, RXR, RXKR, or RXRR, where X is any amino acid, for example, alanine (A).
  • X is any amino acid, for example, alanine (A).
  • no additional amino acids may remain on the C-terminus of the heavy chain.
  • a single construct can be engineered to encode both the heavy and light chains (e.g. the heavy and light chain variable domains) separated by a flexible peptide linker such as those encoding a scFv.
  • a flexible peptide linker can be composed of flexible residues like glycine and serine so that the adjacent heavy chain and light chain domains are free to move relative to one another.
  • the construct may be arranged such that the heavy chain variable domain is at the N-terminus of the scFv, followed by the linker and then the light chain variable domain.
  • the construct may be arranged such that the light chain variable domain is at the N-terminus of the scFv, followed by the linker and then the heavy chain variable domain. That is, the components may be arranged as NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • an expression cassette described herein is contained within a viral vector with a restraint on the size of the polynucleotide(s) therein.
  • the expression cassette is contained within an AAV virus-based vector. Due to the size restraints of certain vectors, the vector may or may not accommodate the coding sequences for the full heavy and light chains of the therapeutic antibody but may accommodate the coding sequences of the heavy and light chains of antigen binding fragments, such as the heavy and light chains of a Fab or F(ab’)2 fragment or an scFv.
  • the AAV vectors described herein may accommodate a transgene of approximately 4.7 kilobases. Substitution of smaller expression elements would permit the expression of larger protein products, such as full-length therapeutic antibodies.
  • the viral vectors provided herein comprise one or more untranslated regions (UTRs), e.g., 3’ and/or 5’ UTRs.
  • UTRs are optimized for the desired level of protein expression.
  • the UTRs are optimized for the mRNA half-life of the transgene.
  • the UTRs are optimized for the stability of the mRNA of the transgene.
  • the UTRs are optimized for the secondary structure of the mRNA of the transgene. 5.1.7 Inverted terminal repeats
  • the viral vectors provided herein comprise one or more inverted terminal repeat (ITR) sequences.
  • ITR sequences may be used for packaging the recombinant gene expression cassette into the virion of the viral vector.
  • the ITR is from an AAV, e.g., AAV8 or AAV2 (see, e.g., Yan et al., 2005, J. Virol., 79(l):364-379; United States Patent No. 7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No. 8,318,480 B2, United States PatentNo. 8,962,332 B2 and International Patent Application No.
  • nucleotide sequences encoding the ITRs may, for example, comprise the nucleotide sequences of SEQ ID NOS: 138 (5’-ITR) or 140 (3 ’-ITR).
  • the modified ITRs used to produce self- complementary vector e.g, sc AAV, may be used (see, e.g, Wu, 2007, Human Gene Therapy, 18(2): 171-82, McCarty et al, 2001, Gene Therapy, Vol 8, Number 16, Pages 1248-1254; and U.S. Patent Nos.
  • nucleotide sequences encoding the modified ITRs may, for example, comprise the nucleotide sequences of SEQ ID NOS: 139 (5’-ITR) or 141 (3’-ITR).
  • the transgenes encode a HuPTM mAb, either as a full-length antibody or an antigen binding fragment thereof, e.g. a Fab fragment (an HuGlyFab) or a F(ab’)2, nanobody, or an scFv based upon a therapeutic antibody disclosed herein.
  • the HuPTM mAb or antigen binding fragment, particularly the HuGlyFab are engineered to contain additional glycosylation sites on the Fab domain (e.g., see Courtois et al., 2016, mAbs 8: 99-112 which is incorporated by reference herein in its entirety for it description of sites of hyperglycosylation on a Fab domain).
  • 3A-3B provides alignments of the Fab heavy and light chains of the therapeutic antibodies disclosed herein and highlights in green residues that may be substituted with an asparagine or, in some instances, a serine, resulting in hyperglycosylation.
  • the Fc domain may be engineered to alter the glycosylation site at N297 to prevent glycosylation at that site (for example, a substitution at N297 for another amino acid and/or a substitution at T297 for a residue that is not a T or S to knock out the glycosylation site).
  • Such Fc domains are “aglycosylated”.
  • the transgenes encode a full length heavy chain (including the heavy chain variable domain, the heavy chain constant domain 1 (CHI), the hinge and Fc domain) and a full length light chain (light chain variable domain and light chain constant domain) that upon expression associate to form antigen-binding antibodies with Fc domains.
  • the recombinant AAV constructs express the intact (i.e., full length) or substantially intact HuPTM mAb in a cell, cell culture, or in a subject.
  • the nucleotide sequences encoding the heavy and light chains may be codon optimized for expression in human cells and have reduced incidence of CpG dimers in the sequence to promote expression in human cells.
  • the transgenes may encode any full-length antibody. In preferred embodiments, the transgenes encode a full-length form of any of the therapeutic antibodies disclosed herein, for example, the Fab fragment of which depicted in FIG. 2 herein and including, in certain embodiments, the associated Fc domain provided in Table 6.
  • the full length mAb encoded by the transgene described herein preferably have the Fc domain of the full-length therapeutic antibody or is an Fc domain of the same type of immunoglobulin as the therapeutic antibody to be expressed.
  • the Fc region is an IgG Fc region, but in other embodiments, the Fc region may be an IgA, IgD, IgE, or IgM.
  • the Fc domain is preferably of the same isotype as the therapeutic antibody to be expressed, for example, if the therapeutic antibody is an IgGl isotype, then the antibody expressed by the transgene comprises an IgGl Fc domain.
  • the antibody expressed from the transgene may have an IgGl, IgG2, IgG3 or IgG4 Fc domain.
  • the Fc region of the intact mAb has one or more effector functions that vary with the antibody isotype.
  • the effector functions can be the same as that of the wild-type or the therapeutic antibody or can be modified therefrom to add, enhance, modify, or inhibit one or more effector functions using the Fc modifications disclosed in Section 5.1.9, infra.
  • the HuPTM mAb transgene encodes a mAb comprising an Fc polypeptide comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the Fc domain polypeptides of the therapeutic antibodies described herein or an exemplary Fc domain of an IgGl, IgG2 or IgG4 isotype as set forth in Table 6.
  • the HuPTM mAb comprises a Fc polypeptide of a sequence that is a variant of the Fc polypeptide sequence in Table 6 in that the sequence has been modified with one or more of the techniques described in Section 5.1.9, infra, to alter the Fc polypeptide’s effector function.
  • recombinant AAV constructs such as the constructs shown in FIGS. 1 A and IB, for gene therapy administration to a human subject in order to express an intact or substantially intact HuPTM mAb in the subject.
  • Gene therapy constructs are designed such that both the heavy and light chains are expressed in tandem from the vector including the Fc domain polypeptide of the heavy chain.
  • the transgene encodes a transgene with heavy and light chain Fab fragment polypeptides as shown in Tables 7 or 8, yet have a heavy chain that further comprises an Fc domain polypeptide C terminal to the hinge region of the heavy chain (including an IgGl, IgG2 or IgG4 Fc domain as in Table 6).
  • the transgene is a nucleotide sequence that encodes the following: Signal sequence-heavy chain Fab portion (including hinge region)-heavy chain Fc polypeptide-Furin-2A linker-signal sequence-light chain Fab portion.
  • the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) Control elements, which include a) an inducible promoter, preferably a hypoxia-inducible promoter, b) a chicken P-actin intron and c) a rabbit P-globin poly A signal; and (3) nucleic acid sequences coding for the heavy chain Fab of an anti-viral mAb (e.g., an anti-SARS-CoV-2 Ab, see Tables 7 and 8); an Fc polypeptide associated with the therapeutic antibody or of the same isotype as the native form of the therapeutic antibody, such as an IgG isotype amino acid sequence from Table 6; and the light chain of an anti-viral mAb (e.g.
  • an anti- SARS-CoV-2 mAb wherein the heavy chain (Fab and Fc region) and the light chain are separated by a self-cleaving furin (F)/F2A or T2A or flexible linker, ensuring expression of equal amounts of the heavy and the light chain polypeptides.
  • Exemplary constructs are provided in FIGS. 1 A and IB.
  • AAV vectors comprising a viral capsid that is at least 95% identical to the amino acid sequence of an AAV8 capsid (SEQ ID NO: 104); and an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding an intact or substantially intact anti-viral mAb; operably linked to one or more regulatory sequences that control expression of the transgene in human liver or muscle cells.
  • ITRs AAV inverted terminal repeats
  • the rAAV vectors that encode and express the full-length therapeutic antibodies may be administered to treat or prevent or ameliorate symptoms of a disease or condition amenable to treatment, prevention or amelioration of symptoms with the therapeutic antibodies. Also provided are methods of expressing HuPTM mAbs in human cells using the rAAV vectors and constructs encoding them.
  • the transgenes express antigen binding fragments, e.g. a Fab fragment (an HuGlyFab) or a F(ab’)2, nanobody, an scFv, or a Nb based upon a therapeutic antibody disclosed herein.
  • FIG. 2 and Section 5.4 provide the amino acid sequences of the heavy and light chains of the Fab fragments of the therapeutic antibodies (see also Tables 7 and 8, which provide the amino acid sequences of the Fab heavy and light chains of the therapeutic antibodies).
  • the transgene may encode a Fab fragment using nucleotide sequences encoding the amino acid sequences provided in Tables 7 and 8, but not including the portion of the hinge region on the heavy chain that forms interchain di-sulfide bonds (e.g., the portion containing the sequence CPPCPA (SEQ ID NO: 113)).
  • Heavy chain Fab domain sequences that do not contain a CPPCP (SEQ ID NO: 112) sequence of the hinge region at the C-terminus will not form intrachain disulfide bonds and, thus, will form Fab fragments with the corresponding light chain Fab domain sequences, whereas those heavy chain Fab domain sequences with a portion of the hinge region at the C-terminus containing the sequence CPPCP (SEQ ID NO: 112) will form intrachain disulfide bonds and, thus, will form Fab2 fragments.
  • the transgene may encode a scFv comprising a light chain variable domain and a heavy chain variable domain connected by a flexible linker in between (where the heavy chain variable domain may be either at the N-terminal end or the C-terminal end of the scFv), and optionally, may further comprise a Fc polypeptide e.g., IgGl, IgG2, IgG3, or IgG4) on the C-terminal end of the heavy chain.
  • a Fc polypeptide e.g., IgGl, IgG2, IgG3, or IgG4
  • the transgene may encode F(ab’)2 fragments comprising a nucleotide sequence that encodes the light chain and the heavy chain sequence that includes at least the sequence CPPCA(SEQ ID NO: 209) of the hinge region, as depicted in FIG. 2 which depict various regions of the hinge region that may be included at the C-terminus of the heavy chain sequence.
  • Pre-existing anti-hinge antibodies may cause immunogenicity and reduce efficacy.
  • C-terminal ends with D221 or ends with a mutation T225L or with L242 can reduce binding to AHA.
  • the viral vectors provided herein comprise the following elements in the following order: a) a constitutive or inducible (e.g., hypoxia-inducible or rifamycin- inducible) promoter sequence or a tissue specific promoter/regulatory region, for example, one of the regulatory regions provided in Table 1, and b) a sequence encoding the transgene (e.g., a HuGlyFab).
  • the sequence encoding the transgene comprises multiple ORFs separated by IRES elements.
  • the ORFs encode the heavy and light chain domains of the HuGlyFab.
  • the sequence encoding the transgene comprises multiple subunits in one ORF separated by F/F2A sequences or F/T2A sequences. In certain embodiments, the sequence comprising the transgene encodes the heavy and light chain domains of the HuGlyFab separated by an F/F2A sequence or a F/T2A sequence. In certain embodiments, the sequence comprising the transgene encodes the heavy and light chain variable domains of the HuGlyFab separated by a flexible peptide linker (as an scFv).
  • the viral vectors provided herein comprise the following elements in the following order: a) a constitutive or an inducible promoter sequence or a tissue specific promoter, such as one of the promoters or regulatory regions in Table 1, and b) a sequence encoding the transgene (e.g., a HuGlyFab), wherein the transgene comprises a nucleotide sequence encoding a signal peptide, a light chain and a heavy chain Fab portion separated by an IRES element.
  • a constitutive or an inducible promoter sequence or a tissue specific promoter such as one of the promoters or regulatory regions in Table 1
  • a sequence encoding the transgene e.g., a HuGlyFab
  • the viral vectors provided herein comprise the following elements in the following order: a) a constitutive or a hypoxia-inducible promoter sequence or regulatory element listed in Table 1, and b) a sequence encoding the transgene comprising a signal peptide, a light chain and a heavy chain sequence separated by a cleavable F/F2A sequence (SEQ ID NOS: 105 or 106) or a F/T2A sequence (SEQ ID NOS: 103 or 104) or a flexible peptide linker.
  • the viral vectors provided herein comprise the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or an inducible promoter sequence or a tissue specific promoter or regulatory region, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a sequence encoding the transgene (e.g., a HuGlyFab), i) a second UTR sequence, j) a fourth linker sequence, k) a poly A sequence, 1) a fifth linker sequence, and m) a second ITR sequence.
  • a first ITR sequence e.g., a HuGlyFab
  • the viral vectors provided herein comprise the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or an inducible promoter sequence or a tissue specific regulatory region, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a sequence encoding the transgene (e.g., HuGlyFab), i) a second UTR sequence, j) a fourth linker sequence, k) a poly A sequence, 1) a fifth linker sequence, and m) a second ITR sequence, wherein the transgene comprises a signal, and wherein the transgene encodes a light chain and a heavy chain sequence separated by a cleavable F/2A sequence.
  • a first ITR sequence e.g., HuGlyFab
  • the transgenes encode full length or substantially full length heavy and light chains that associate to form a full length or intact antibody. (“Substantially intact” or “substantially full length” refers to a mAb having a heavy chain sequence that is at least 95% identical to the full-length heavy chain mAb amino acid sequence and a light chain sequence that is at least 95% identical to the full-length light chain mAb amino acid sequence). Accordingly, the transgenes comprise nucleotide sequences that encode, for example, the light and heavy chains of the Fab fragments including the hinge region of the heavy chain and C-terminal of the heavy chain of the Fab fragment, an Fc domain peptide. Table 6 provides the amino acid sequences of the Fc polypeptides for certain of the therapeutic antibodies described herein. Alternatively, an IgGl, IgG2, or IgG4 Fc domain, the sequences of which are provided in Table 6 may be utilized.
  • Fc region refers to a dimer of two "Fc polypeptides” (or “Fc domains”), each "Fc polypeptide” comprising the heavy chain constant region of an antibody excluding the first constant region immunoglobulin domain.
  • an "Fc region” includes two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers.
  • Fc polypeptide refers to at least the last two constant region immunoglobulin domains of IgA, IgD, and IgG, or the last three constant region immunoglobulin domains of IgE and IgM and may also include part or all of the flexible hinge N-terminal to these domains.
  • Fc polypeptide comprises immunoglobulin domains Cgamma2 (Cy2, often referred to as CH2 domain) and Cgamma3 (Cy3, also referred to as CH3 domain) and may include the lower part of the hinge domain between Cgammal (Cyl, also referred to as CHI domain) and CH2 domain.
  • the human IgG heavy chain Fc polypeptide is usually defined to comprise residues starting at T223 or C226 or P230, to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Services, Springfield, Va.).
  • Fc polypeptide comprises immunoglobulin domains Calpha2 (Ca2) and Calpha3 (Ca3) and may include the lower part of the hinge between Calphal (Cal) and Ca2.
  • the Fc polypeptide is that of the therapeutic antibody or is the Fc polypeptide corresponding to the isotype of the therapeutic antibody).
  • the Fc polypeptide is an IgG Fc polypeptide.
  • the Fc polypeptide may be from the IgGl, IgG2, or IgG4 isotype (see Table 6 and FIG. 6) or may be an IgG3 Fc domain, depending, for example, upon the desired effector activity of the therapeutic antibody.
  • the engineered heavy chain constant region (CH), which includes the Fc domain is chimeric. As such, a chimeric CH region combines CH domains derived from more than one immunoglobulin isotype and/or subtype.
  • the chimeric (or hybrid) CH region comprises part or all of an Fc region from IgG, IgA and/or IgM.
  • the chimeric CH region comprises part or all a CH2 domain derived from a human IgGl, human IgG2, or human IgG4 molecule, combined with part or all of a CH3 domain derived from a human IgGl, human IgG2, or human IgG4 molecule.
  • the chimeric CH region contains a chimeric hinge region.
  • the recombinant vectors encode therapeutic antibodies comprising an engineered (mutant) Fc regions, e.g. engineered Fc regions of an IgG constant region.
  • Modifications to an antibody constant region, Fc region or Fc fragment of an IgG antibody may alter one or more effector functions such as Fc receptor binding or neonatal Fc receptor (FcRn) binding and thus half-life, CDC activity, ADCC activity, and/or ADPC activity, compared to a corresponding antibody having a wild-type IgG constant region, or an IgG heavy chain constant region without the recited modification(s).
  • the antibody may be engineered to provide an antibody constant region, Fc region or Fc fragment of an IgG antibody that exhibits altered binding (as compared to a reference or wild-type constant region without the recited modification(s)) to one or more Fc receptors (e g., FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, FcyRIIIB, FcyRI V, or FcRn receptor).
  • Fc receptors e g., FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, FcyRIIIB, FcyRI V, or FcRn receptor.
  • the antibody an antibody constant region, Fc region or Fc fragment of an IgG antibody that exhibits a one or more altered effector functions such as CDC, ADCC, or ADCP activity, compared to a corresponding antibody having a wild-type IgG constant region, or an IgG constant without the recited modification(s).
  • Effective function refers to a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include FcyR-mediated effector functions such as ADCC and ADCP and complement-mediated effector functions such as CDC.
  • effector cell refers to a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include but are not limited to monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, and T cells, and may be from any organism including but not limited to humans, mice, rats, rabbits, and monkeys.
  • ADCC antibody dependent cell-mediated cytotoxicity
  • FcyRs cytotoxic effector cells that express FcyRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • ADCP antibody dependent cell-mediated phagocytosis
  • CDC complement-dependent cytotoxicity
  • the modifications of the Fc domain include, but are not limited to, the following modifications and combinations thereof, with reference to EU numbering of an IgG constant region (see FIG. 6): 233, 234, 235, 236, 237, 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296,
  • the Fc region comprises an amino acid addition, deletion, or substitution of one or more of amino acid residues 251-256, 285-290, 308-314, 385-389, and 428-436 of the IgG.
  • 251-256, 285-290, 308-314, 385-389, and 428-436 (EU numbering of Kabat; see FIG. 6) is substituted with histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine.
  • a non-histidine residue is substituted with a histidine residue.
  • a histidine residue is substituted with a non-histidine residue.
  • Enhancement of FcRn binding by an antibody having an engineered Fc leads to preferential binding of the affinity-enhanced antibody to FcRn as compared to antibody having wildtype Fc, and thus leads to a net enhanced recycling of the FcRn-affinity-enhanced antibody, which results in further increased antibody half-life.
  • An enhanced recycling approach allows highly effective targeting and clearance of antigens, including e.g. "high titer" circulating antigens, such as C5, cytokines, or bacterial or viral antigens.
  • modified constant region, Fc region or Fc fragment of an IgG antibody with enhanced binding to FcRn in serum as compared to a wild-type Fc region are engineered modifications.
  • antibodies e.g. IgG antibodies
  • antibodies, e.g. IgG antibodies are engineered to exhibit enhanced binding (e.g. increased affinity or KD) to FcRn in endosomes (e.g.
  • an acidic pH e.g. , at or below pH 6.0
  • a wildtype IgG and/or reference antibody binding to FcRn at an acidic pH as well as in comparison to binding to FcRn in serum (e.g., at a neutral pH, e.g., at or above pH 7.4).
  • serum e.g., at a neutral pH, e.g., at or above pH 7.4
  • an engineered antibody constant region, Fc region or Fc fragment of an IgG antibody that exhibits an improved serum or resident tissue half-life, compared to a corresponding antibody having a wild-type IgG constant region, or an IgG constant without the recited modification(s);
  • Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., LN/Y/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434.
  • a modification at position 250 e.g., E or Q
  • 250 and 428 e.g., L or F
  • 252 e.g., LN/Y/W or T
  • 254 e.g., S or T
  • the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V2591), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or 308P) (EU numbering; see FIG. 6).
  • a 428L e.g., M428L
  • 434S e.g., N434S
  • a 428L, 2591 e.g., V2591
  • 308F e.g.
  • the Fc region can be a mutant form such as hlgGl Fc including M252 mutations, e.g. M252Y and S254T and T256E (“YTE mutation”) exhibit enhanced affinity for human FcRn (Dall’Acqua, et al., 2002, J Immunol 169:5171-5180) and subsequent crystal structure of this mutant antibody bound to hFcRn resulting in the creation of two salt bridges (Oganesyan, et al. 2014, JBC 289(11): 7812-7824).
  • Antibodies having the YTE mutation have been administered to monkeys and humans, and have significantly improved pharmacokinetic properties (Haraya, et al., 2019, Drug Metabolism and Pharmacokinetics, 34(1):25-41).
  • modifications to one or more amino acid residues in the Fc region may reduce half-life in systemic circulation (serum), however result in improved retainment in tissues (e.g. in the eye) by disabling FcRn binding (e.g. H435A, EU numbering of Kabat) (Ding et al., 2017, MAbs 9:269-284; and Kim, 1999, Eur J Immunol 29:2819).
  • FcRn binding e.g. H435A, EU numbering of Kabat
  • the Fc domain may be engineered to activate all, some, or none of the normal Fc effector functions, without affecting the Fc polypeptide’s (e.g. antibody's) desired pharmacokinetic properties.
  • Fc polypeptides having altered effector function may be desirable as they may reduce unwanted side effects, such as activation of effector cells, by the therapeutic protein.
  • Methods to alter or even ablate effector function may include mutation(s) or modification(s) to the hinge region amino acid residues of an antibody.
  • IgG Fc domain mutants comprising 234A, 237A, and 238S substitutions, according to the EU numbering system, exhibit decreased complement dependent lysis and/or cell mediated destruction.
  • Deletions and/or substitutions in the lower hinge e.g. where positions 233-236 within a hinge domain (EU numbering) are deleted or modified to glycine, have been shown in the art to significantly reduce ADCC and CDC activity.
  • the Fc domain is an aglycosylated Fc domain that has a substitution at residue 297 or 299 to alter the glycosylation site at 297 such that the Fc domain is not glycosylated.
  • Such aglycosylated Fc domains may have reduced ADCC or other effector activity.
  • Non-limiting examples of proteins comprising mutant and/or chimeric CH regions having altered effector functions, and methods of engineering and testing mutant antibodies, are described in the art, e.g. K.L. Amour, et al., Eur. J. Immunol. 1999, 29:2613-2624; Lazar et al., Proc. Natl. Acad. Sci. USA 2006, 103:4005; US Patent Application Publication No. 20070135620A1 published June 14, 2007; US Patent Application Publication No. 20080154025 Al, published June 26, 2008; US Patent Application Publication No. 20100234572 Al, published September 16, 2010; US Patent Application Publication No. 20120225058 Al, published September 6, 2012; US Patent Application Publication No.
  • the C-terminal lysines (-K) conserved in the heavy chain genes of all human IgG subclasses are generally absent from antibodies circulating in serum - the C-terminal lysines are cleaved off in circulation, resulting in a heterogeneous population of circulating IgGs.
  • van den Bremer et al., 2015, mAbs 7:672-680 the DNA encoding the C-terminal lysine (-K) or glycine-lysine (-GK) of the Fc terminus can be deleted to produce a more homogeneous antibody product in situ.
  • the viral vectors provided herein may be manufactured using host cells.
  • the viral vectors provided herein may be manufactured using mammalian host cells, for example, A549, WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, and myoblast cells.
  • the viral vectors provided herein may be manufactured using host cells from human, monkey, mouse, rat, rabbit, or hamster.
  • the host cells are stably transformed with the sequences encoding the transgene and associated elements (e.g., the vector genome), and the means of producing viruses in the host cells, for example, the replication and capsid genes (e.g., the rep and cap genes of AAV).
  • the replication and capsid genes e.g., the rep and cap genes of AAV.
  • Genome copy titers of said vectors may be determined, for example, by TAQMAN® analysis.
  • Virions may be recovered, for example, by CsCh sedimentation.
  • baculovirus expression systems in insect cells may be used to produce AAV vectors.
  • AAV vectors See Aponte-Ubillus et al., 2018, Appl. Microbiol. Biotechnol. 102: 1045- 1054 which is incorporated by reference herein in its entirety for manufacturing techniques.
  • in vitro assays e.g., cell culture assays
  • transgene expression from a vector described herein thus indicating, e.g., potency of the vector.
  • in vitro neutralization assays can be used to measure the activity of the transgene expressed from a vector described herein.
  • Vero-E6 cells a cell line derived from the kidney of an African green monkey, or HeLa cells engineered to stably express the ACE2 receptor (HeLa-ACE2), can be used to assess neutralization activity of transgenes expressed from a vector described herein.
  • glycosylation and tyrosine sulfation patterns associated with the HuGlyFab can be determined, for example determination of the glycosylation and tyrosine sulfation patterns associated with the HuGlyFab. Glycosylation patterns and methods of determining the same are discussed in Section 5.3, while tyrosine sulfation patterns and methods of determining the same are discussed in Section 5.3.
  • benefits resulting from glycosylation/sulfation of the cell-expressed HuGlyFab can be determined using assays known in the art, e.g., the methods described in Section 5.3.
  • Vector genome concentration (GC) or vector genome copies can be evaluated using digital PCR (dPCR) or ddPCRTM (BioRad Technologies, Hercules, CA, USA).
  • dPCR digital PCR
  • ddPCRTM BioRad Technologies, Hercules, CA, USA
  • liver biopsies are obtained at several timepoints.
  • mice are sacrificed at various timepoints post injection.
  • Liver tissue samples are subjected to total DNA extraction and dPCR assay for vector copy numbers.
  • Copies of vector genome (transgene) per gram of tissue may be measured in a single biopsy sample, or measured in various tissue sections at sequential timepoints will reveal spread of AAV througout the liver.
  • Total DNA from collected liver tissue is extracted with the DNeasy Blood & Tissue Kit and the DNA concentration measured using a Nanodrop spectrophotometer.
  • the copy number of delivered vector in a specific tissue section per diploid cell is calculated as: (vector copy number)/(endogenous control)*2.
  • Vector copy in specific cell types, such as liver cells, over time may indicate sustained expression of the transgene by the tissue.
  • compositions suitable for administration to human subjects comprise a suspension of the recombinant vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant and optional excipients.
  • a formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
  • the pharmaceutical composition comprises rAAV combined with a pharmaceutically acceptable carrier for administration to a subject.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant (e.g., Freund's complete and incomplete adjuvant), excipient, or vehicle with which the agent is administered.
  • adjuvant e.g., Freund's complete and incomplete adjuvant
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, e.g., peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a common carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • compositions include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin and gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM as known in the art.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • low molecular weight polypeptides proteins, such as serum albumin and gelatin
  • hydrophilic polymers such as
  • the pharmaceutical composition of the present invention can also include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative, in addition to the above ingredients.
  • a lubricant e.g., talc, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol
  • methods for treating infectious diseases or disorders in a subject in need thereof comprising the administration of recombinant AAV particles comprising an expression cassette encoding anti-viral antibodies and antibody-binding fragments and variants thereof, or peptides, are provided.
  • a subject in need thereof includes a subject suffering from an infectious disease or disorder, or a subj ect pre-disposed thereto, e.g. , a subj ect at risk of developing or having a recurrence of the infectious disease or disorder.
  • Subjects to whom such gene therapy is administered can be those responsive to anti-infectious therapy.
  • the methods encompass treating patients who have been diagnosed with an infectious disease (including having detectable viral antigen or nucleic acid, for example, by PCR assay, in a biological sample such as blood, sputum or nasal swab; or detectable antibodies agains the virus), or have one or more symptoms associated therewith, such as fever, cough, rhinitis, body aches, respiratory distress, etc., and, in certain embodiments, identified as responsive to treatment with an anti-viral antibody or considered a good candidate for therapy with an anti-viral antibody.
  • the patients have previously been treated with an anti-iviral antibody.
  • the anti-viral antibody or antigen-binding fragment transgene product may be administered directly to the subject.
  • the transgene may encode a therapeutic antibody or peptide that binds to a respiratory virus (e.g. influenza virus, rhinovirus, and coronavirus (e.g. Middle East Respiratory Syndome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-Co-V), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)), arbovirus, hepatitis virus (hepatitis C virus (HPC), hepatitis B virus (HBV)), herpesvirus (e.g.
  • a respiratory virus e.g. influenza virus, rhinovirus, and coronavirus (e.g. Middle East Respiratory Syndome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-Co-V), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
  • MERS-CoV Middle East Respiratory Syndome coronavirus
  • SARS-Co-V severe acute respiratory syndrome coron
  • herpes simplex type I HSV I
  • herpes simplex type II HSV II
  • CMV cytomegalus virus
  • EBV Epstein-Barr virus
  • papillomavirus e.g. human papillomaviruse (HPV)
  • HPV human papillomaviruse
  • EBOV ebola virus
  • lyssaviruses e.g. rabies virus (RBV)
  • retroviral viruses e.g. HIV
  • kits for treating a SARS-CoV-2 infection in a human subject in need thereof comprising: administering to the liver or muscle of said subject a therapeutically effective amount of a recombinant nucleotide expression vector comprising a transgene encoding a substantially full-length or full-length anti-SARS-CoV-2 mAb having an Fc region, or an antigen-binding fragment thereof, or a peptide, operably linked to one or more regulatory sequences that control expression of the transgene in human liver and/or muscle cells, so that a depot is formed that releases a HuPTM form of mAb or antigen-binding fragment thereof.
  • Recombinant vectors and pharmaceutical compositions for treating infectious diseases or disorders in a subject in need thereof are described in Section 5.1.
  • Such vectors should have a tropism for human liver and/or muscle cells and can include non-replicating rAAV, particularly those bearing an AAV3B, AAVrh8, AAVhu37, AAV64R.1, AAV5, AAV7, AAV8, AAAV9, AAVS3, AAV-LK03, AAVrh46, AAVrhlO, or AAVrh73 capsid.
  • the recombinant vectors can be administered in any manner such that the recombinant vector enters liver and or muscle cells, e.g., by introducing the recombinant vector into circulation.
  • Such vectors should further comprise one or more regulatory sequences that control expression of the transgene in human liver cells and/or human liver and muscle cells include, but are not limited to, an ApoE.hAAT (SEQ ID NO:21) regulatory sequence, a LSPX1 promoter (SEQ ID NO:9), a LSPX2 promoter (SEQ ID NO: 10), a LTP1 promoter (SEQ ID NO: 11), a LTP2 (SEQ ID NO: 12) promoter, a CRE selected from Table 16, a CRE (Table 16).hAAT, or a LTP3 (SEQ ID NO: 13) promoter (see also Table 1).
  • an ApoE.hAAT SEQ ID NO:21
  • LSPX1 promoter SEQ ID NO:9
  • LSPX2 promoter SEQ ID NO: 10
  • LTP1 promoter SEQ ID NO: 11
  • LTP2 SEQ ID NO: 12
  • Anti-viral antibodies that are useful in the methods of treatment described herein may meet certain threshold binding and viral neutralization parameters.
  • the functional properties of the compositions of the invention can be assessed using standard assays available in the art (e.g., flow cytometry, binding assays) and/or described herein, such as those set forth in Examples 15 to 20.
  • antibody binding assays and an in vitro pseudo-coronavirus such as SARS-CoV-2 pseudovirion or wild-type virus neutralization assay can be used to measure the quality and function of compositions of the invention elicited by different SARS-CoV-2 spike antigens.
  • binding activity is used as a surrogate method to analyze the anti-viral antibodies. Binding to a viral antigen can be tested using binding assays described in the art or described in the Example 20. Surface plasmon resonance (SPR)-based technology (e.g. BIAcore analysis), enzyme-linked immunosorbent assay (ELISA), or flow cytometry may be used to demonstrate antigen binding of the antibodies of the current invention. Binding can be assayed against the virus itself or viral proteins, particularly those of the viral coat or capsid that elicit antibodies in an immune response. Antibodies binding to a virus, particular viral protein, or epitope thereof are likely to be neutralizing.
  • SPR Surface plasmon resonance
  • ELISA enzyme-linked immunosorbent assay
  • flow cytometry may be used to demonstrate antigen binding of the antibodies of the current invention. Binding can be assayed against the virus itself or viral proteins, particularly those of the viral coat or capsid that elicit antibodies in an immune response. Antibodies binding to a
  • ELISA and SPR-based technology are widely used in the art for ligand-binding, including antibody-antigen binding analysis.
  • four different forms of ELISA assay can be used to evaluate the binding ability of mAbs.
  • the test antibody With an appropriate antigen coated on 96-well plates, the test antibody can bind to the surface specifically, following by a wash. The test antibody that binds the antigen can then be captured by a labeled secondary antibody, and subsequently assessed through the detection of the optical density (OD). With serial dilution, the binding curve reflecting the binding ability of mAb products is obtained, and the EC50 value (the effective concentration needed for 50% of maximal binding) can be derived by four- parameter logistic fit.
  • the ratio of EC50 values (or relative EC50, rEC50) can be obtained by the EC50 value of the standard over the EC50 value of a test mAb. This value indicates the binding activity of the test mAb compared to that of the standard. The greater the rEC50 value, the higher the binding activity.
  • KD is the equilibrium dissociation constant, a ratio of koff/kon, between the antibody and its antigen. KD and affinity are inversely related. The KD value relates to the concentration of antibody (the amount of antibody needed for a particular experiment) and so the lower the KD value (lower concentration) and thus the higher the affinity of the antibody.
  • compositions of the invention display KD values in the low micromolar (10‘ 6 ) to nanomolar (10‘ 7 to 10' 9 ) range (that is 1-9 X 10' 7 to 10' 9 ).
  • Preferred compositions of the invention display KD values in the low nanomolar (1-9 x 10' 9 ) range.
  • more preferred compositions display very high affinity to an anti-viral antigen with the KD value being in the picomolar (1-9 x 10" 10 to 10' 12 ) range.
  • the binding affinity of anti-SARS-CoV-2 antibodies can be determined using binding assays as disclosed in Rogers et al, 2020; Hansen et al, 2020; Wrapp et al, 2020; Yuan et al, 2020; Ju et al; 2020; Lv et al, 2020; Wu et al, 2020; Zhou et al, 2020; Shi et al, 2020; Barnes et al, 2020; Robianni et al, 2020; Hurlburt et al, 2020; Brouwer et al, 2020; Pinto et al, 2020; Pak et al, 2009; Chen et al, 2020; Zost et al, 2020; Kreer et al, 2020; Liu et al, 2020; Cao et al, 2020; Andreano et al, 2020; Wan et al, 2020; Wang et al, 2020; and Seydoux et al, 2020, all of
  • Neutralizing antibodies not only bind to a virus, they bind in a manner that blocks or reduces infection of a cell by the virus. Only a small subset of the many antibodies that bind a virus are capable of neutralization.
  • a classic neutralization assay comprises four stages. First, virus and antibody are incubated together. Second, the virus is allowed to adsorb to target cells. Third, viral replication proceeds to produce viral product or induce the expression of a reporter molecule. Fourth, the product is measured in an assay and converted to a signal and compared with no-antibody and no-virus (background, noise) controls. The outcome can differ greatly in accordance with variations.
  • Neutralization assays can be performed and measured in different ways, including the use of techniques such as plaque reduction (comparing counts of virus plaques in control wells with those in inoculated cultures), microneutralization (performed in microtiter plates filled with small amounts of sera), and colorimetric assays (depend on biomarkers indicating metabolic inhibition of the virus).
  • plaque reduction comparing counts of virus plaques in control wells with those in inoculated cultures
  • microneutralization performed in microtiter plates filled with small amounts of sera
  • colorimetric assays depending on biomarkers indicating metabolic inhibition of the virus.
  • compositions of the invention display neutralization activity (as determined by the inhibitory concentrations at which 50% (or 90%) neutralization is attained) in the nanomolar (1-9 x 10' 7 to 10' 9 ) range.
  • Preferred compositions of the invention display neutralization activity in the low nanomolar (1-9 x 10' 9 ) range.
  • compositions display anti-viral neutralization activity in the picomolar (1-9 x 10' 12 ) range.
  • an anti-SARS-CoV-2 antibody of the invention with a very high potency exhibits a 50% virus-inhibitory concentration (IC50) of less than 10' 12 .
  • the amino acid sequence (primary sequence) of HuGlyFabs or HuPTM Fabs, HuPTMmAbs, and HuPTM scFvs disclosed herein each comprises at least one site at which N- glycosylation or tyrosine sulfation takes place (see exemplary FIG. 2) for glycosylation and/or sulfation positions within the amino acid sequences of the Fab fragments of the therapeutic antibodies).
  • Post-translational modification also occurs in the Fc domain of full length antibodies, particularly at residue N297 (by EU numbering, see Table 6).
  • mutations may be introduced into the Fc domain to alter the glycosylation site at residue N297 (EU numbering, see Table 6), in particular substituting another amino acid for the asparagine at 297 or the threonine at 299 to remove the glycosylation site resulting in an aglycosylated Fc domain.
  • the canonical N-glycosylation sequence is known in the art to be Asn-X-Ser(or Thr), wherein X can be any amino acid except Pro.
  • Asn asparagine residues of human antibodies can be glycosylated in the context of a reverse consensus motif, Ser (or Thr)-X-Asn, wherein X can be any amino acid except Pro.
  • Ser (or Thr)-X-Asn Asparagine (Asn) residues of human antibodies can be glycosylated in the context of a reverse consensus motif, Ser (or Thr)-X-Asn, wherein X can be any amino acid except Pro.
  • certain HuGlyFabs and HuPTM scFvs disclosed herein comprise such reverse consensus sequences.
  • glutamine (Gin) residues of human antibodies can be glycosylated in the context of a non-consensus motif, Gln-Gly-Thr. See Valliere-Douglass et al., 2010, J. Biol. Chem. 285: 16012-16022.
  • certain of the HuGlyFab fragments disclosed herein comprise such non-consensus sequences.
  • O-glycosylation comprises the addition of N-acetyl -galactosamine to serine or threonine residues by the enzyme. It has been demonstrated that amino acid residues present in the hinge region of antibodies can be O-glycosylated.
  • O-glycosylation confers another advantage to the therapeutic antibodies provided herein, as compared to, e.g., antigen-binding fragments produced in E. coli, again because the E. coli naturally does not contain machinery equivalent to that used in human O-glycosylation. (Instead, O-glycosylation in E. coli has been demonstrated only when the bacteria is modified to contain specific O-glycosylation machinery. See, e.g., Farid-Moayer et al., 2007, J. Bacteriol. 189:8088-8098.)
  • a nucleic acid encoding a HuPTM mAb, HuGlyFab or HuTPM scFv is modified to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites (including the canonical N-glycosylation consensus sequence, reverse N-glycosylation site, and non-consensus N- glycosylation sites) than would normally be associated with the HuPTM mAb, HuGlyFab or HuPTM scFv (e.g., relative to the number of N-glycosylation sites associated with the HuPTM mAb, HuGlyFab or HuPTM scFv in its unmodified state).
  • introduction of glycosylation sites is accomplished by insertion of N-glycosylation sites (including the canonical N- glycosylation consensus sequence, reverse N-glycosylation site, and non-consensus N-glycosylation sites) anywhere in the primary structure of the antigen-binding fragment, so long as said introduction does not impact binding of the antibody or antigen-binding fragment to its antigen.
  • N-glycosylation sites including the canonical N- glycosylation consensus sequence, reverse N-glycosylation site, and non-consensus N-glycosylation sites
  • glycosylation sites can be accomplished by, e.g., adding new amino acids to the primary structure of the antigen-binding fragment, or the antibody from which the antigen-binding fragment is derived (e.g., the glycosylation sites are added, in full or in part), or by mutating existing amino acids in the antigen-binding fragment, or the antibody from which the antigen-binding fragment is derived, in order to generate the N-glycosylation sites (e.g., amino acids are not added to the antigen-binding fragment/antibody, but selected amino acids of the antigen-binding fragment/antibody are mutated so as to form N-glycosylation sites).
  • amino acid sequence of a protein can be readily modified using approaches known in the art, e.g. , recombinant approaches that include modification of the nucleic acid sequence encoding the protein.
  • a HuGlyMab or antigen-binding fragment is modified such that, when expressed in mammalian cells, such as retina, CNS, liver or muscle cells, it can be hyperglycosylated. See Courtois et al., 2016, mAbs 8:99-112 which is incorporated by reference herein in its entirety.
  • biologies Unlike small molecule drugs, biologies usually comprise a mixture of many variants with different modifications or forms that could have a different potency, pharmacokinetics, and/or safety profile. It is not essential that every molecule produced either in the gene therapy or protein therapy approach be fully glycosylated and sulfated. Rather, the population of glycoproteins produced should have sufficient glycosylation (including 2,6-sialylation) and sulfation to demonstrate efficacy.
  • the goal of gene therapy treatment provided herein can be, for example, to slow or arrest the progression of a disease or abnormal condition or to reduce the severity of one or more symptoms associated with the disease or abnormal condition.
  • the N- glycosylation sites of the antigen-binding fragment can be glycosylated with various different glycans.
  • N-glycans of antigen-binding fragments and the Fc domain have been characterized in the art. For example, Bondt et al., 2014, Mol. & Cell. Proteomics 13.11 :3029-3039 (incorporated by reference herein in its entirety for its disclosure of Fab-associated N-glycans; see also, FIG.
  • Glycosylation of the Fc domain has been characterized and is a single N-linked glycan at asparagine 297 (EU numbering; see Table 6 and FIG. 6).
  • the glycan plays an integral structural and functional role, impacting antibody effector function, such as binding to Fc receptor (see, for example, Jennewein and Alter, 2017, Trends In Immunology 38:358 for a discussion of the role of Fc glycosylation in antibody function). Removal of the Fc region glycan almost completely ablates effector function (Jennewien and Alter at 362).
  • the composition of the Fc glycan has been shown to impact effector function, for example hypergalactosylation and reduction in fucosylation have been shown to increase ADCC activity while sialylation correlates with anti-inflammatory effects (Id. at 364).
  • Disease states, genetics and even diet can impact the composition of the Fc glycan in vivo.
  • the glycan composition can differ significantly by the type of host cell used for recombinant expression and strategies are available to control and modify the composition of the glycan in therapeutic antibodies recombinantly expressed in cell culture, such as CHO to alter effector function (see, for example, US 2014/0193404 by Hansen et al.).
  • the HuPTM mAbs provided herein may advantageously have a glycan at N297 that is more like the native, human glycan composition than antibodies expressed in non-human host cells.
  • the need for in vitro production in prokaryotic host cells e.g., E. colt
  • eukaryotic host cells e.g., CHO cells or NSO cells
  • N-glycosylation sites of the HuPTM mAb, HuGlyFab or HuPTM scFv are advantageously decorated with glycans relevant to and beneficial to treatment of humans. Such an advantage is unattainable when CHO cells, NSO cells, or E.
  • coli are utilized in antibody/anti gen -binding fragment production, because e.g., CHO cells (1) do not express 2,6 sialyltransferase and thus cannot add 2,6 sialic acid during N-glycosylation; (2) can add Neu5Gc as sialic acid instead of Neu5Ac; and (3) can also produce an immunogenic glycan, the a-Gal antigen, which reacts with anti-a-Gal antibodies present in most individuals, which at high concentrations can trigger anaphylaxis; and because (4) E. coli does not naturally contain components needed for N-glycosylation.
  • Assays for determining the glycosylation pattern of antibodies, including antigenbinding fragments are known in the art.
  • hydrazinolysis can be used to analyze glycans.
  • polysaccharides are released from their associated protein by incubation with hydrazine (the Ludger Liberate Hydrazinolysis Glycan Release Kit, Oxfordshire, UK can be used).
  • the nucleophile hydrazine attacks the glycosidic bond between the polysaccharide and the carrier protein and allows release of the attached glycans.
  • N-acetyl groups are lost during this treatment and have to be reconstituted by re-N-acetylation.
  • Glycans may also be released using enzymes such as glycosidases or endoglycosidases, such as PNGase F and Endo H, which cleave cleanly and with fewer side reactions than hydrazines.
  • the free glycans can be purified on carbon columns and subsequently labeled at the reducing end with the fluorophor 2-amino benzamide.
  • the labeled polysaccharides can be separated on a GlycoSep-N column (GL Sciences) according to the HPLC protocol of Royle et al, Anal Biochem 2002, 304(l):70-90. The resulting fluorescence chromatogram indicates the polysaccharide length and number of repeating units.
  • Structural information can be gathered by collecting individual peaks and subsequently performing MS/MS analysis. Thereby the monosaccharide composition and sequence of the repeating unit can be confirmed and additionally in homogeneity of the polysaccharide composition can be identified. Specific peaks of low or high molecular weight can be analyzed by MALDI-MS/MS and the result used to confirm the glycan sequence. Each peak in the chromatogram corresponds to a polymer, e.g., glycan, consisting of a certain number of repeat units and fragments, e.g., sugar residues, thereof. The chromatogram thus allows measurement of the polymer, e.g., glycan, length distribution.
  • the elution time is an indication for polymer length, while fluorescence intensity correlates with molar abundance for the respective polymer, e.g., glycan.
  • fluorescence intensity correlates with molar abundance for the respective polymer, e.g., glycan.
  • Other methods for assessing glycans associated with antigen-binding fragments include those described by Bondt et al., 2014, Mol. & Cell. Proteomics 13.11 :3029-3039, Huang et al., 2006, Anal. Biochem. 349: 197-207, and/or Song et al., 2014, Anal. Chem. 86:5661-5666.
  • Homogeneity or heterogeneity of the glycan patterns associated with antibodies can be assessed using methods known in the art, e.g., methods that measure glycan length or size and hydrodynamic radius.
  • HPLC such as size exclusion, normal phase, reversed phase, and anion exchange HPLC, as well as capillary electrophoresis, allows the measurement of the hydrodynamic radius. Higher numbers of glycosylation sites in a protein lead to higher variation in hydrodynamic radius compared to a carrier with less glycosylation sites.
  • Glycan length can be measured by hydrazinolysis, SDS PAGE, and capillary gel electrophoresis.
  • homogeneity can also mean that certain glycosylation site usage patterns change to a broader/narrower range. These factors can be measured by Glycopeptide LC-MS/MS.
  • the HuPTM mAbs, or antigen binding fragments thereof also do not contain detectable NeuGc and/or a-Gal.
  • detectable NeuGc or “detectable a-Gal” or “does not contain or does not have NeuGc or a-Gal” means herein that the HuPTM mAb or antigen-binding fragment, does not contain NeuGc or a-Gal moieties detectable by standard assay methods known in the art.
  • NeuGc may be detected by HPLC according to Hara et al., 1989, “Highly Sensitive Determination of - Acetyl -and N-Glycolylneuraminic Acids in Human Serum and Urine and Rat Serum by Reversed-Phase Liquid Chromatography with Fluorescence Detection.” J. Chromatogr., B: Biomed. 377, 111-119, which is hereby incorporated by reference for the method of detecting NeuGc.
  • NeuGc may be detected by mass spectrometry.
  • the a-Gal may be detected using an ELISA, see, for example, Galili et al., 1998, “A sensitive assay for measuring a-Gal epitope expression on cells by a monoclonal anti-Gal antibody.” Transplantation. 65(8): 1129-32, or by mass spectrometry, see, for example, Ayoub et al., 2013, “Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques.” Austin Bioscience. 5(5):699-710.
  • N-glycosylation confers numerous benefits on the HuPTM mAb, HuGlyFab or HuPTM scFv described herein. Such benefits are unattainable by production of antigen-binding fragments in E. coli, because E. coli does not naturally possess components needed for N-glycosylation.
  • CHO cells or murine cells such as NS0 cells
  • CHO cells lack components needed for addition of certain glycans (e.g, 2,6 sialic acid and bisecting GlcNAc) and because either CHO or murine cell lines add N-N- Glycolylneuraminic acid (“Neu5Gc” or “NeuGc”) which is not natural to humans (and potentially immunogenic), instead of N-Acetylneuraminic acid (“Neu5Ac”) the predominant human sialic acid.
  • Neu5Gc N-N- Glycolylneuraminic acid
  • Ne5Ac N-Acetylneuraminic acid
  • CHO cells can also produce an immunogenic glycan, the a-Gal antigen, which reacts with anti-a-Gal antibodies present in most individuals, which at high concentrations can trigger anaphylaxis. See, e.g., Bosques, 2010, Nat. Biotech. 28: 1153-1156.
  • the human glycosylation pattern of the HuGlyFab of HuPTM scFv described herein should reduce immunogenicity of the transgene product and improve efficacy.
  • Fab glycosylation may affect the stability, half-life, and binding characteristics of an antibody.
  • any technique known to one of skill in the art may be used, for example, enzyme linked immunosorbent assay (ELISA), or surface plasmon resonance (SPR).
  • any technique known to one of skill in the art may be used, for example, by measurement of the levels of radioactivity in the blood or organs in a subject to whom a radiolabelled antibody has been administered.
  • any technique known to one of skill in the art may be used, for example, differential scanning calorimetry (DSC), high performance liquid chromatography (HPLC), e.g., size exclusion high performance liquid chromatography (SEC-HPLC), capillary electrophoresis, mass spectrometry, or turbidity measurement.
  • DSC differential scanning calorimetry
  • HPLC high performance liquid chromatography
  • SEC-HPLC size exclusion high performance liquid chromatography
  • capillary electrophoresis capillary electrophoresis
  • mass spectrometry or turbidity measurement.
  • sialic acid on HuPTM mAb, HuGlyFab or HuPTM scFv used in the methods described herein can impact clearance rate of the HuPTM mAb, HuGlyFab or HuPTM scFv. Accordingly, sialic acid patterns of a HuPTM mAb, HuGlyFab or HuPTM scFv can be used to generate a therapeutic having an optimized clearance rate. Methods of assessing antigen-binding fragment clearance rate are known in the art. See, e.g., Huang et al., 2006, Anal. Biochem. 349: 197-207.
  • a benefit conferred by N-glycosylation is reduced aggregation.
  • Occupied N-glycosylation sites can mask aggregation prone amino acid residues, resulting in decreased aggregation.
  • Such N-glycosylation sites can be native to an antigen-binding fragment used herein or engineered into an antigen-binding fragment used herein, resulting in HuGlyFab or HuPTM scFv that is less prone to aggregation when expressed, e.g., expressed in human cells.
  • Methods of assessing aggregation of antibodies are known in the art. See, e.g., Courtois et al., 2016, mAbs 8:99-112 which is incorporated by reference herein in its entirety.
  • a benefit conferred by N-glycosylation is reduced immunogenicity.
  • Such N-glycosylation sites can be native to an antigen-binding fragment used herein or engineered into an antigen-binding fragment used herein, resulting in HuPTM mAb, HuGlyFab or HuPTM scFv that is less prone to immunogenicity when expressed, e.g., expressed in human retinal cells, human CNS cells, human liver cells or human muscle cells.
  • N-glycosylation is protein stability.
  • N-glycosylation of proteins is well-known to confer stability on them, and methods of assessing protein stability resulting from N-glycosylation are known in the art. See, e.g., Sola and Griebenow, 2009, J Pharm Sci., 98(4): 1223-1245.
  • a benefit conferred by N-glycosylation is altered binding affinity. It is known in the art that the presence of N-glycosylation sites in the variable domains of an antibody can increase the affinity of the antibody for its antigen. See, e.g., Bovenkamp et al., 2016, J. Immunol. 196: 1435-1441. Assays for measuring antibody binding affinity are known in the art. See, e.g., Wright et al., 1991, EMBO J. 10:2717-2723; and Leibiger et al., 1999, Biochem. J. 338:529-538.
  • Tyrosine sulfation occurs at tyrosine (Y) residues with glutamate (E) or aspartate (D) within +5 to -5 position of Y, and where position -1 of Y is a neutral or acidic charged amino acid, but not a basic amino acid, e.g., arginine (R), lysine (K), or histidine (H) that abolishes sulfation.
  • the HuGlyFabs and HuPTM scFvs described herein comprise tyrosine sulfation sites (see exemplary FIG. 2).
  • tyrosine-sulfated anti gen -binding fragments cannot be produced in E. coli, which naturally does not possess the enzymes required for tyrosine-sulfation.
  • CHO cells are deficient for tyrosine sulfation-they are not secretory cells and have a limited capacity for post- translational tyrosine-sulfation. See, e.g., Mikkelsen & Ezban, 1991, Biochemistry 30: 1533-1537.
  • the methods provided herein call for expression of HuPTM Fab in human cells that are secretory and have capacity for tyrosine sulfation.
  • Tyrosine sulfation is advantageous for several reasons.
  • tyrosine-sulfation of the antigen-binding fragment of therapeutic antibodies against targets has been shown to dramatically increase avidity for antigen and activity. See, e.g., Loos et al., 2015, PNAS 112: 12675- 12680, and Choe et al., 2003, Cell 114: 161-170.
  • Assays for detection tyrosine sulfation are known in the art. See, e.g., Yang et al., 2015, Molecules 20:2138-2164.
  • O-glycosylation comprises the addition of N-acetyl-galactosamine to serine or threonine residues by the enzyme. It has been demonstrated that amino acid residues present in the hinge region of antibodies can be O-glycosylated.
  • the HuGlyFab comprise all or a portion of their hinge region, and thus are capable of being O-glycosylated when expressed in human cells. The possibility of O-glycosylation confers another advantage to the HuGlyFab provided herein, as compared to, e.g., antigen-binding fragments produced in E. coli, again because the E. coli naturally does not contain machinery equivalent to that used in human O-glycosylation.
  • O- glycosylation in E. coli has been demonstrated only when the bacteria is modified to contain specific O-glycosylation machinery. See, e.g., Farid-Moayer et al., 2007, J. Bacteriol. 189:8088-8098.
  • O- glycosylated HuGlyFab by virtue of possessing glycans, shares advantageous characteristics with N- glycosylated HuGlyFab (as discussed above).
  • Severe acute respiratory syndrome coronavirus 2 belongs to the Betacoronavirus genus in the Coronaviridae family.
  • the SARS-CoV-2 virus has a positive-sense RNA genome that encodes four structural proteins (spike (S), envelope, membrane, and nucleocapsid (N)) and 16 non- structural proteins.
  • the structural proteins are largely responsible for receptor recognition on the host cell, membrane fusion, and subsequent viral entry.
  • the S protein forms homotrimers on the viral membrane in which each monomer is composed of two subunits - the N-terminal SI, that is largely responsible for receptor recognition, and the C-terminal S2, that is implicated in membrane fusion and viral entry.
  • the SI subunit contains the receptor-binding domain (RBD), the region of the protein that makes direct contact with the host cell receptor, angiotensin-converting-enzyme 2 (ACE2).
  • RBD receptor-binding domain
  • ACE2 angiotensin-converting-enzyme 2
  • the S2 subunit contains the fusion peptide, the heptad repeat 1 (HR1), and heptad repeat 2 (HR2).
  • HR1 heptad repeat 1
  • HR2 heptad repeat 2
  • the HuPTM mAb has the amino acid sequence of CC12.1 (heavy and light chain amino acid sequences of SEQ ID NOS:305 and 306), CC12.23 (heavy and light chain amino acid sequences SEQ ID NOs:307 and 308), JS016 or LY-C0VOI6 (etesevimab) (having heavy and light chain amino acid sequences SEQ ID Nos:348 and 349), LY- CoV555 (also bamlanivimab) (having heavy and light chain amino acid sequences SEQ ID Nos: 340 and 342), VIR-7831 (sotrovimab) (having heavy and light chain amino acid sequences SEQ ID Nos: 344 and 345), TY027
  • the HuPTM mAb has the amino acid sequence of an anti-SARS- CoV-2 antibody disclosed in any of the following publications, each of which is incorporated herein by reference in its entirety: Rogers et al, 2020; Hansen et al, 2020; Wrapp et al, 2020; Yuan et al, 2020; Ju et al; 2020; Lv et al, 2020; Wu et al, 2020; Zhou et al, 2020; Shi et al, 2020; Barnes et al, 2020; Robianni et al, 2020; Hurlburt et al, 2020; Brouwer et al, 2020; Pinto et al, 2020; Pak et al, 2009; Chen et al, 2020; Zost et al, 2020; Kreer et al, 2020; Liu et al, 2020; Cao et al, 2020; Andreano et al, 2020; Wan et al, 2020; Wang et al,
  • Delivery may be accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding an SARS -Co V-2 -binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to patients (human subjects) diagnosed with, or having one or more symptoms of COVID-19 infection or to prevent COVID-19 infection in a human subject to create a permanent depot that continuously supplies the human PTM, e.g., human-glycosylated, transgene product.
  • Transgenes e.g., a viral vector or other DNA expression construct encoding an SARS -Co V-2 -binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof)
  • the transgene is a nucleic acid comprising the nucleotide sequences encoding an antigen binding fragment of an antibody that binds to SARS-CoV-2, such as CC12.1, CC12.23, JS016, LY-Cov555, VIR-7831, TY027, BRII-96, BRII-98, CT-P59, SCTA01, STI1499, AZD8895, AZS1061, SAB-185, S309, VHH72, VHH55, CR3022, H014, P2B-2F6, B38, H4, EY6A, CAI, CB6, C105, CV30, COVA2-39, COVA2-04, F26G19, ADI-55689, ADI-56046, AZD1061, AZD7442, 311mab31B5, or 311mab32D4 or variants thereof as detailed herein.
  • SARS-CoV-2 such as CC12.1, CC12.23, JS016,
  • the transgene may also encode an SARS-CoV-2 antigen binding fragment that contains additional glycosylation sites (e.g., see Courtois et al.).
  • Table 7 provides the VH and VL amino acid sequences of the antibodies, in some cases also including CH or CL domains of Fab fragments (or the entire constant domain of the heavy chain) underlined if present in the sequence and CDR sequences may be bolded.
  • the anti-SARS-CoV-2 antigen-binding fragment transgene comprises the nucleotide sequences encoding the heavy and light chains of the Fab portion of an anti- SARS-CoV-2 antibody or may be a single domain (e.g., a VHH).
  • the nucleotide sequences may be codon optimized for expression in human cells.
  • Nucleotide sequences may, for example, comprise the nucleotide sequences as disclosed in any of the following publications, each of which is incorporated herein by reference in its entirety: Rogers et al, 2020; Hansen et al, 2020; Wrapp et al, 2020; Yuan et al, 2020; Ju et al; 2020; Lv et al, 2020; Wu et al, 2020; Zhou et al, 2020; Shi et al, 2020; Barnes et al, 2020; Robianni et al, 2020; Hurlburt et al, 2020; Brouwer et al, 2020; Pinto et al, 2020; Pak et al, 2009; Chen et al, 2020; Zost et al, 2020; Kreer et al, 2020; Liu et al, 2020; Cao et al, 2020; Andreano et al, 2020; Wan et al, 2020, Wang et al, 2020; Babb
  • the heavy and light chain sequences both have a signal or leader sequence at the N-terminus appropriate for expression and secretion in human cells, in particular, human liver cells (e.g., hepatocytes) or muscle cells.
  • the signal sequence may have the amino acid sequence of MYRMQLLLLIALSLALVTNS (SEQ ID NO:50).
  • the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Tables 2 or 3 that correspond to the proteins secreted by myocytes or hepatocytes, respectively.
  • the transgenes may comprise, at the C-terminus of the heavy chain CHI domain sequence, all or a portion of the hinge region.
  • the anti-SARS-CoV-2-antigen binding domain has a heavy chain Fab domain with additional hinge region sequence starting after the C-terminal valine (V), contains all or a portion of the amino acid sequence EPKSCDKTHTCPPCPAPEAAGG (SEQ ID NO: 123), and specifically, EPKSCDKTHL (SEQ ID NO: 117), EPKSCDKTHT (SEQ ID NO: 118), EPKSCDKTHTCPPCPA (SEQ ID NO: 119), EPKSCDKTHLCPPCPA (SEQ ID NO: 120), EPKSCDKTHTCPPCPAPEAAGGPSVFL (SEQ ID NO: 124) or EPKSCDKTHLCPPCPAPEAAGGPSVFL (SEQ ID NO: 125).
  • the transgenes comprise the amino acid sequences encoding the full length (or substantially full length) heavy and light chains of the antibody, comprising the Fc domain at the C terminus of the heavy chain, e.g., having an amino acid sequence of encoding an IgGl Fc domain of SEQ ID NO: 141 (Table 6), or a mutant or variant thereof.
  • the Fc domain may be engineered for altered binding to one or more Fc receptors and/or effector function as disclosed in Section 5.1.9, infra.
  • the anti-SARS-CoV-2 -binding fragment transgene encodes an SARS-CoV-2 antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS: 306, 308, 310, 314, 316, 318, 320,322, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, or 349.
  • the anti-SARS- CoV-2 antigen-binding fragment transgene encodes an SARS-CoV-2 antigen-binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS: 305, 307, 309, 313, 315, 317, 319, 321, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, or 348.
  • the anti-SARS-CoV-2 antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS: 306, 308, 310, 314, 316, 318, 320,322, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, or 349 and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS: 305, 307, 309, 313, 315, 317, 319, 321, 324
  • the SARS-CoV-2 antigen binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NOS: 305, 307, 309, 313, 315, 317, 319, 321, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, or 348 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, for example, in the framework regions (e.g., those regions outside of the CDRs) or are substitutions with an amino acid present at that position in the heavy chain of one or more of the other therapeutic antibodies, for example, as identified by the alignment in FIG.
  • the SARS-CoV-2 antigen binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NOS: 306, 308, 310, 314, 316, 318, 320,322, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, or 349 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs) or are substitutions with an amino acid present at that position in the light chain of one or more of the other therapeutic antibodies, for example, as identified by the alignment in FIG. 3B.
  • the anti-SARS-CoV-2 antigen-binding fragment transgene encodes a hyperglycosylated Fab, comprising a heavy chain and a light chain of SEQ ID NOS: 305, 307, 309, 313, 315, 317, 319, 321, 324, 326, 328, 330, 332, 334, 317, 319, 321, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, or 348 and 306, 308, 310, 314, 316, 318, 320,322, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, or 349, respectively, with one or more mutations(see FIGS. 3 A (heavy chain) and 3B (light chain)).
  • the anti-SARS-CoV-2 antigen-binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequences encoding the six CDRs which are, for example, underlined in the heavy and light chain variable domain sequences of Table 7 and FIG2, which are spaced between framework regions, generally human framework regions, and associated with constant domains depending upon the form of the antigen-binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-SARS-CoV-2 antibody or antigen-binding fragment thereof.
  • AAV vectors comprising a viral capsid that is at least 95% identical to the amino acid sequence of an AAV8 capsid (SEQ ID NO:2), AAVS3 capsid (SEQ ID NO:8) or AAV-LK03 capsid (SEQ ID NO:7); and an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding an anti-SARS-CoV-2 mAb, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in human liver or muscle cells.
  • ITRs AAV inverted terminal repeats
  • the regulatory sequence is the ApoE.hAAT regulatory sequence (SEQ ID NO:21) or one of LSPX1, LSPX2, LTP1, LTP2, or LTP3, SEQ ID NOS:9, 10, 11, 12, or 13, respectively).
  • the regulatory sequence is one of the liverspecific CRE sequence selected from Table 16 or a CRE sequence of Table 16 operable linked with a hAAT sequence.
  • the promoter is an LMTP6 promoter (SEQ ID NO: 14).
  • Useful antibodies bind the SARS-CoV-2 virus (or an antigen thereof, such as the SARS-CoV-2 spike protein, for example) with a dissociation constant (KD) in the picomolar or nanomolar range as determined using a binding assay known in the art and a viral neutralization potency (IC50) at least nanomolar and, in embodiments, picomolar range according to a SARS-CoV- 2 neutralization assay accepted in the art.
  • the binding assay is an ELISA- or SPR based binding assay and the neutralization assay and the neutralization assay is a pseudotyped or replicating virus neutralization assay.
  • Particular rAAV gene therapy vectors described herein when administered to a human subject at a dosage of 10E11 to 10E14 result in expression of an anti-SARS-CoV-2 antibody or binding fragment thereof which exhibits a KD value in the picomolar (IO' 10 to 1 O' 12 ) or low nanomolar range (10‘ 9 ) in an appropriate assay, for example ELISA- or SPR-based binding assay, and exhibits viral neutralization activity in an appropriate assay, for example, in a pseudotyped or replicating virus neutralization assay of an IC50 in at least the nanomolar range (10‘ 7 to 10' 9 ), and, in particular embodiments, picomolar range (IO' 10 to 1 O' 12 ).
  • the rAAV gene therapy vector results in expression of an anti-SARS-CoV-2 antibody or binding fragment thereof which exhibits a KD value of less than 10' 9 , IO' 10 , 10’ 11 , or 10' 12 using an ELISA- or SPR-based binding assay.
  • the KD value is less than 10' 11 or less than 10' 12 .
  • the rAAV gene therapy vector results in expression of an anti-SARS-CoV-2 antibody or binding fragment thereof which exhibits neutralization activity (IC50) of less than 10' 7 , 10' 8 , 10' 9 , IO' 10 , 10’ 11 , or 10' 12 using an ELISA- or SPR-based binding assay.
  • the neutralization activity is less than IO' 10 , 10’ 11 , or 10' 12 and in even more preferred embodiments, the neutralization activity is less than 10' 12 .
  • the binding affinity and neutralization activity of the expressed anti-SARS-CoV-2 antibody or antigen-binding fragment thereof is determined by using binding assays and neutralization assays such as the ones outlined in Examples 17 to 21 or disclosed in Rogers et al, 2020; Hansen et al, 2020; Wrapp et al, 2020; Yuan et al, 2020; Ju et al; 2020; Lv et al, 2020; Wu et al, 2020; Zhou et al, 2020; Shi et al, 2020; Barnes et al, 2020; Robianni et al, 2020; Hurlburt et al, 2020; Brouwer et al, 2020; Pinto et al, 2020; Pak et al, 2009; Chen et al, 2020; Zost et al, 2020; Kreer et al, 2020; Liu et al, 2020; Cao et al, 2020; Andreano et al, 2020; Wa
  • the binding assay determines the binding affinity (KD value) of the expressed anti-SARS-CoV-2 antibody or antigen-binding fragment thereof to the SARS-CoV-2 virus, a SARS-CoV-2 viral protein, or epitope thereof.
  • the neutralization assay determines the neutralization activity (IC50) of the expressed anti-SARS-CoV-2 antibody or antigen-binding fragment thereof against SARS-CoV- 2 virus, a SARS-CoV-2 viral protein, or epitope thereof.
  • the binding affinity of the expressed anti-SARS-CoV-2 antibody or antigen-binding fragment exhibits at least equivalent or better binding affinity to the SARS-CoV-2 virus, a SARS-CoV-2 viral protein, or epitope thereof compared to the binding affinity of CC12.1 when using the binding assay, or a variation thereof, described in Example 20 or a binding assay disclosed in Rogers et al, 2020 (CC12.1) or Hansen et al, 2020.
  • the neutralization activity (IC50) of the expressed anti-SARS-CoV-2 antibody or antigen-binding fragment is equivalent or better compared to the neutralization activity of CC12.1 using the pseudotyped and/or replicating virus neutralization assay described in Rogers et al, 2020.
  • the expressed anti-SARS-CoV-2 antibody or antigen-binding fragment thereof exhibits a lower than at least 0.1 pg/mL, 0.05 pg/mL, or 0.02 pg/mL in vitro neutralization IC50 value in a pseudovirus neutralization assay or a lower than at least 0.1 pg/mL, 0.05 pg/mL, or 0.02 pg/mL in vitro neutralization IC50 value in a live replicating virus neutralization assay using a neutralization described herein or disclosed by Rogers et al., 2020.
  • the neutralization activity of the expressed anti-SARS-CoV-2 antibody or antigen-binding fragment thereof is measured at 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks after said administration.
  • rAAV gene therapy vectors which when administered to a human subject result in expression of an anti-SARS-CoV-2 antibody to achieve a maximum or steady state serum concentration (for example, 20, 30, 40, 50, 60 or 90 days after administration) of 1.5 pg/ml to 15 pg/ml (or 1.5 pg/ml to 12 pg/ml or 1.5 pg/ml to 10 pg/ml, or 3 pg/ml to 15 pg/ml or 3 pg/ml to 10 pg/ml or 3 pg/ml to 5 pg/ml or 5 pg/ml to 15 pg/ml or 5 pg/ml to 10 pg/ml or 7 pg/ml to 15 pg/ml or 7 pg/ml to 10 pg/ml or 10 pg/ml to 15 pg/ml) anti-SARS-CoV-2 antibody
  • Gene Therapy Methods Provided are methods of preventing or reducing the incidence of in human subj ects and treating human subjects for COVID-19 by administration of a viral vector containing a transgene encoding an anti-SARS-CoV-2 antibody, or antigen binding fragment thereof.
  • the antibody may be CC12.1, CC12.23, LY-C0VOI6, LY-CoV555, VIR-7831, TY027, BRII-96, BRII-98, CT-P59, SCTA01, STI1499, AZD8895, AZS1061, SAB-185, S309, VHH72, VHH55, CR3022, H014, P2B- 2F6, B38, H4, EY6A, CAI, CB6, C105, CV30, COVA2-39, COVA2-04, F26G19, ADI-55689, ADI- 56046, AZD7442, AZD1061, 311mab31B5, 311mab32D4, or BGB-DXP593 and is, for example, a full length antibody or Fab fragment thereof, or other antigen-binding fragment thereof.
  • the patient has been diagnosed with and/or has symptoms associated with COVID-19.
  • the subject is at risk of infection with COVID-19.
  • Recombinant vector used for delivering the transgene are described in Section 5.1.
  • such vectors should have a tropism for human liver cells and can include non-replicating rAAV, particularly those bearing an AAV8, AAVS3, or AAV-LK03 capsid.
  • the recombinant vectors, such as those shown in FIG. 1, can be administered in any manner such that the recombinant vector enters the liver or muscle tissue, e.g. by introducing the recombinant vector into the bloodstream. See Section 5.2 for details regarding the methods of treatment.
  • Subjects to whom such gene therapy is administered can be those responsive to anti- SARS-CoV-2 therapy.
  • the methods encompass treating patients who have been diagnosed with COVID-19, or have one or more symptoms associated therewith, and identified as responsive to treatment with an anti-SARS-CoV-2 antibody or considered a good candidate for therapy with an anti-SARS-CoV-2 antibody.
  • the patients have previously been treated with CC12.1, CC12.23, LY-C0VOI6, LY-CoV555, VIR-7831, TY027, BRII-96, BRII-98, CT-P59, SCTA01, STI1499, AZD8895, AZS1061, SAB-185, S309, VHH72, VHH55, CR3022, H014, P2B-2F6, B38, H4, EY6A, CAI, CB6, C105, CV30, COVA2-39, COVA2-04, F26G19, ADI-55689, ADI-56046, 311mab31B5, 311mab32D4, or BGB-DXP593, and have been found to be responsive to CC12.1, CC12.23, LY-C0VOI6, VIR-7831, TY027, BRII-96, BRII-98, CT-P59, SCTA01, STI1499, AZD8895
  • the production of the anti-SARS-CoV-2 HuPTM mAb or HuPTM Fab should result in a “biobetter” molecule for the treatment of COVID-19 accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding the anti-SARS-CoV-2 HuPTM Fab, subcutaneously, intramuscularly, or intravenously to human subjects (patients) diagnosed with or having one or more symptoms of MS, to create a permanent depot in the liver or muscle tissue that continuously supplies the fully-human post-translationally modified, such as human-glycosylated, sulfated transgene product produced by transduced liver or muscle cells.
  • the cDNA construct for the anti-SARS-CoV-2 HuPTMmAb or anti-SARS-CoV-2 HuPTM Fab should include a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced liver or muscle cells.
  • the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO:50).
  • the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Tables 2 or 3 that correspond to the proteins secreted by myocytes or hepatocytes, respectively.
  • the anti-SARS-CoV-2 HuPTM mAb or HuPTM Fab can be produced in human cell lines by recombinant DNA technology, and administered to patients diagnosed with COVID-19, or for whom therapy or prohylaxis for COVID-19 is considered appropriate.
  • the anti-SARS-CoV-2 HuPTM mAb or antigen-binding fragment thereof does not contain detectable NeuGc moieties and/or does not contain detectable alphaGai moieties.
  • the HuPTM mAb is a full length or substantially full length mAb with an Fc region.
  • the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1% or 2% glycosylated and/or sulfated and may be at least 5%, 10% or even 50% or 100% glycosylated and/or sulfated.
  • the goal of gene therapy treatment provided herein is to prevent symptomatic COVID-19 disease or prevent severe COVID-19 disease and SARS-CoV-2 infection. Efficacy may be monitored by scoring the symptoms (e.g. fever, chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, new loss of taste or smell, sore throat, congestion or runny nose, nausea or vomiting, diarrhea), laboratory features (e.g. elevated inflammatory markers, liver enzymes), percentage organ involvement (e.g. kidney, lung), all-cause mortality, respiratory failure, need for invasive mechanical ventilation and sustained clinical recovery.
  • symptoms e.g. fever, chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, new loss
  • Section 5.2 describes recombinant vectors that contain a transgene encoding a HuPTM mAb or HuPTM Fab (or other antigen binding fragment of the HuPTM mAb) that binds to SARS- CoV-2.
  • Therapeutically effective doses of any such recombinant vector should be administered in any manner such that the recombinant vector enters the liver or muscle (e.g., skeletal muscle), e.g. by introducing the recombinant vector into the bloodstream.
  • the vector may be administered directly to the liver through hepatic blood flow, e.g., via the suprahepatic veins or via the hepatic artery.
  • the vector is administered subcutaneously, intramuscularly or intravenously.
  • Intramuscular, subcutaneous, intravenous or hepatic administration should result in expression of the soluble transgene product in cells of the liver or muscle.
  • the vector may be administered directly to the liver through hepatic blood flow, e.g., via the suprahepatic veins or via the hepatic artery.
  • the expression of the transgene encoding an anti-SARS-CoV-2 antibody creates a permanent depot in liver and/or muscle of the patient that constinuously supplies the anti- SARS-CoV-2 HuPTM mAb, or antigen binding fragment of the anti-SARS-CoV-2 mAb to the circulation of the subject.
  • the range of a therapeutically or prophylactically effective amount of an AAV gene therapy vector or pharmaceutical composition is 1E11 to 1E14 genome copies (gc)/kg, preferably between 1E11 to 1E13, and even more preferably 1E12.
  • the dose ranges described herein are exemplary only and do not limit the dose ranges that can be selected.
  • intraveneous administration of an AAV gene therapy vector encoding an anti-SARS-CoV-2 antibody results in at least 1.5 g/mL, 2 pg/mL, 5 pg/mL, 7 pg/ml, 10 pg/mL, or at least 15 pg/mL and up to 15 pg/ml, 20 pg/ml or 30 pg/ml transgene product expression in human serum at least 20, 30, 40, 50 or 60 days after administration.
  • the administration results in a human serum concentration (Cmin) of the transgene product of at least about 1.0 pg/mL, 1.1 pg/mL, 1.2 pg/mL, 1.3 pg/mL, 1.4 pg/mL, 1.5 pg/mL, 1.6 pg/mL, 1.7 pg/mL, 1.8 pg/mL, 1.9 pg/mL, 2.0 pg/mL to about 15 pg/mL mAb and associated with a neutralization titer of 500 to 5000.
  • Cmin human serum concentration
  • doses that maintain a serum concentration of the anti-SARS- CoV-2 antibody transgene product at a Cmin of at least 1.5 pg/mL or at least 15 pg/mL e.g., Cmin of 1.5 to 5 pg/ml, 5 to 10 pg/ml or 10 to 15 pg/mL) at least 30, 40, 50 or 69 days after administration are provided.
  • a dose of 1E11 maintains a serum concentration of the anti-SARS- CoV-2 antibody transgene product of at least 1.5 pg/mL.
  • a dose of 1E12 maintains a serum concentration of the anti-SARS-CoV-2 antibody transgene product of at least 1.5 pg/mL.
  • the administratin results in a serum level of transgene product that is effective to treat or prevent viral infection, or ameliorate the symptoms of or to reduce the incidence or risk of infection.
  • incidence of infection is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%.
  • Infection by the virus can be detected by a method known in the art, such as PCR detection of viral nucleic acid or viral antigen in a biological sample, such as sputum or a nasal swab, of the subject.
  • the method of the invention results in sufficient transgene product in the patient serum to ameliorate, treat or prevent symptoms of viral infection such as, but not limited to, fever (over 101 °F), cough, pneumonia, body aches, headache, and any other symptoms of SARS-CoV2 virus.
  • viral infection such as, but not limited to, fever (over 101 °F), cough, pneumonia, body aches, headache, and any other symptoms of SARS-CoV2 virus.
  • doses that maintain a serum concentration of the anti-SARS- CoV-2 antibody transgene product reach a neutralization titer of at least 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000 or 5000.
  • compositions suitable for intravenous, intramuscular, subcutaneous or hepatic administration comprise a suspension of the recombinant vector comprising the transgene encoding the anti-SARS-CoV-2 antibody, or antigen-binding fragment thereof, in a formulation buffer comprising a physiologically compatible aqueous buffer.
  • the formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
  • Combinations of delivery of the anti-SARS-CoV-2 HuPTM mAb or antigen-binding fragment thereof, to the liver or muscles accompanied by delivery of other available treatments are encompassed by the methods provided herein.
  • the additional treatments may be administered before, concurrently, or subsequent to the gene therapy treatment.
  • Interferon alpha 2b interferon beta la
  • corticosteroids immunomodulators (e.g, azathioprine, 6-mercaptopurine, and/or methotrexate), mitoxantrone, tyrosine kinase inhibitors (e.g. ruxolitinib, imatinib), anti-viral medications (e.g. lopinavir, ritonavir, remdesivir, favipiravir, oseltamivir, sofosbuvir), anti-inflammatory monoclonal antibodies (e.g.
  • peptides that exhibit potent antiviral activity include CoV-Pep (SEQ ID NO:342), a 40 amino acid peptide corresponding to the HR2 domain of the spike protein of COVID-19, fragments and/or analogs of CoV-Pep, and peptides which are homologous to CoV-Pep.
  • Such peptides of the invention are used to inhibit/interfere with the viral entry of the SARS-CoV-2 virus into cells.
  • the peptide CoV-PEP (SEQ ID NO:342) of the invention corresponds to amino acid residues I 163 to 1202 of the HR2 domain from die SARS-CoV-2 virus (SARS-CoV_Tor2: GenBank MN908947), and has the 40 amino acid sequence (NH2 to COOH terminus): NH2 ⁇ DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQE-COOH (SEQ ID NO:342).
  • the peptides of the invention may include truncations of the CoV-PEP peptide which exhibit antiviral activity.
  • Such truncated CoV-PEP peptides may comprise peptides of between 3 and 40 amino acid residues (i.e., peptides ranging in size from a tripeptide to a 40-mer polypeptide).
  • the antiviral peptides of the invention also include analogs of CoV-PEP and/or CoV- PEP truncations which may include, but are not limited to, peptides comprising the CoV-PEP (SEQ ID N::342) sequence, or CoV-PEP truncated sequence, containing one or more amino acid substitutions, insertions and/or deletions. Analogs of CoV-PEP homologs are also within the scope of the invention.
  • the CoV-PEP analogs of the invention exhibit antiviral activity, and may, further, possess additional advantageous features, such as, for example, increased bioavailability, and/or stability, or reduced host immune recognition.
  • the anti-viral peptide can be expressed from a DNA construct comprising a nucleic acid sequence encoding for the anti-viral peptide, operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells or human muscle cells.
  • the peptides may be combined with the gene therapy provided herein.
  • the peptides may be administered before, concurrently, or subsequent to the gene therapy treatment.
  • the anti-viral peptide and a HuPTM mAb or HuPTM antigen-binding fragment provided herein are expressed from a single construct.
  • the peptides of the invention may also be synthesized or prepared by techniques well known in the art.
  • the peptides of the invention, fragments, analogs, and homologs may be used as inhibitors of human coronavirus transmission to uninfected cells.
  • the human coronavirus whose transmission may be inhibited by the peptides include, but are not limited to all strains of SARS-CoV- 1 and SARS-CoV-2.
  • the peptides of the invention may be used as a therapeutic in the treatment of COVID-19.
  • the peptides may be used as a prophylactic measure in previously uninfected individuals after acute exposure to a coronavirus.
  • the peptide is fused to a signal sequence to promote secretion from the liver cell, for example the mutant IL-2 signal sequence of SEQ ID NO: 145, or any signal sequence listed in Tables 2 or 3.
  • the peptide is included in the expression cassette with the anti-viral pathogen antibody such that both the antibody and the peptide are expressed from the integrated genome of the gene therapy vector.
  • the structure of the construct is Signal Sequence - Anti-viral antibody heavy chain - Furin site - 2A site - signal sequence - Anti-viral antibody light chain - PolyA or Furin/2A site - signal sequence-anti-viral peptide - PolyA or, alternatively, signal sequence - anti-viral antibody heavy chain - Furin site - 2A site - signal sequence - anti-viral antibody light chain - PolyA or Furin/2A site - signal sequence - anti-viral peptide- PolyA.
  • Effective dosages of the peptides of the invention to be administered may be determined through procedures well known to those in the art which address such parameters as biological half-life, bioavailability, and toxicity.
  • compositions and methods are described for the delivery of HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fabs, that bind to Influenza A and indicated for treating, ameliorating the symptoms of, preventing, or reducing the incidence and/or severity of Influenza infection (“the flu”).
  • the HuPTM mAb has the amino acid sequence of VIS410 (having heavy and light chain amino acid sequences SEQ ID NOS:338 and 3395), MHAA4549A (having heavy and light chain amino acid sequences SEQ ID NOS: 36 and 37), CR6261 (having heavy and light chain amino acid sequences SEQ ID NOS: 38 and 39), CR8020 (having heavy and light chain amino acid sequences SEQ ID NOS: 40 and 41), TCN-032 (having heavy and light chain amino acid sequences SEQ ID NOS: 42 and 43), or VIR-2482, or an antigen binding fragment of the foregoing.
  • VIS410 having heavy and light chain amino acid sequences SEQ ID NOS:338 and 3395
  • MHAA4549A having heavy and light chain amino acid sequences SEQ ID NOS: 36 and 37
  • CR6261 having heavy and light chain amino acid sequences SEQ ID NOS: 38 and 39
  • CR8020 having heavy and light chain amino acid sequences SEQ ID NO
  • Delivery may be accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding an Influenza A binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to patients (human subjects) diagnosed with, or having one or more symptoms of Influenza or to prevent, reduce the incidence of (by 10%, 20%, 30%, 40%, 50%, 60% 70%, 80%, 90%, 95%, 99%) or the severity of or course of Influenza infection in a human subject to create a permanent depot that continuously supplies the human PTM, e.g., human-glycosylated, transgene product.
  • a viral vector or other DNA expression construct encoding an Influenza A binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to patients (human subjects) diagnosed with, or having one or more symptoms of Influenza or to prevent, reduce the incidence of (by 10%, 20%, 30%, 40%, 50%,
  • transgene encoding a HuPTM mAb or HuPTM Fab (or other antigen binding fragment of the HuPTM mAb) that binds to Influenza A that can be administered to deliver the HuPTM mAb or antigen binding fragment in a patient.
  • the transgene is a nucleic acid comprising the nucleotide sequences encoding an antigen binding fragment of an antibody that binds to Influenza, such as VIS410, MHAA4549A, CR6261, CR8020, TCN-032, or VIR-2482, or variants thereof as detailed herein.
  • the transgene may also encode an Influenza A antigen binding fragment that contains additional glycosylation sites (e.g., see Courtois et al.).
  • Table 8 Provided below in Table 8 are the amino acid sequences for the heavy and light chain variable domains of anti-influenza antibodies (and antibodies against other viruses detailed elsewhere herein) with the CH or the CL sequences underlined if they are present and CDR sequences may be bolded.
  • the anti-influenza A antigen-binding fragment transgene comprises the nucleotide sequences encoding the heavy and light chains of the Fab portion of an antiInfluenza A antibody.
  • the nucleotide sequences may be codon optimized for expression in human cells.
  • the heavy and light chain sequences both have a signal or leader sequence at the N-terminus appropriate for expression and secretion in human cells, in particular, human liver cells (e.g., hepatocytes) or muscle cells.
  • the signal sequence may have the amino acid sequence of MYRMQLLLLIALSLALVTNS (SEQ ID NO:50).
  • the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Tables 2 or 3 that correspond to the proteins secreted by myocytes or hepatocytes, respectively.
  • the transgenes may comprise, at the C-terminus of the heavy chain CHI domain sequence, all or a portion of the hinge region.
  • the anti-influenza A antigen binding domain has a heavy chain Fab domain with additional hinge region sequence starting after the C-terminal valine (V), contains all or a portion of the amino acid sequence EPKSCDKTHTCPPCPAPEAAGG (SEQ ID NO: 123), and specifically, EPKSCDKTHL (SEQ ID NO: 117), EPKSCDKTHT (SEQ ID NO: 118), EPKSCDKTHTCPPCPA (SEQ ID NO: 119), EPKSCDKTHLCPPCPA (SEQ ID NO: 120), EPKSCDKTHTCPPCPAPEAAGGPSVFL (SEQ ID NO: 124) or EPKSCDKTHLCPPCPAPEAAGGPSVFL (SEQ ID NO: 125).
  • the transgenes comprise the amino acid sequences encoding the full length (or substantially full length) heavy and light chains of the antibody, comprising the Fc domain at the C terminus of the heavy chain, e.g., having an amino acid sequence of encoding an IgGl Fc domain of SEQ ID NO: 141 (Table 6), or a mutant or variant thereof.
  • the Fc domain may be engineered for altered binding to one or more Fc receptors and/or effector function as disclosed in Section 5.1.9, infra.
  • the anti -Influenza A-binding fragment transgene encodes an Influenza antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS: 341, 344, 346, 348, or 350.
  • the anti-influenza antigen-binding fragment transgene encodes an Influenza A antigen-binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS: 340, 343, 345, 347, or 348.
  • the anti -Influenza A antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS: 341, 344, 346, 348, or 350 and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS: 340, 343, 345, 347, or 349.
  • the Influenza A antigen binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NOS: 340, 343, 345, 347, or 349 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, for example, in the framework regions (e.g., those regions outside of the CDRs) or are substitutions with an amino acid present at that position in the heavy chain of one or more of the other therapeutic antibodies, for example, as identified by the alignment in FIG. 3 A.
  • the Influenza A antigen binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NOS: 341, 344, 346, 348, or 350 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs) or are substitutions with an amino acid present at that position in the light chain of one or more of the other therapeutic antibodies, for example, as identified by the alignment in FIG. 3B.
  • the anti-influenza A antigen-binding fragment transgene encodes a hyperglycosylated Fab, comprising a heavy chain and a light chain of SEQ ID NOS: 340, 343, 345, 347, or 349 and 341, 344, 346, 348, or 350, respectively, with one or more mutations (see FIGS. 3 A (heavy chain) and 3B (light chain)).
  • the anti-influenza A antigen-binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequences encoding the six CDRs which are, for example, underlined in the heavy and light chain variable domain sequences of Table 8, which are spaced between framework regions, generally human framework regions, and associated with constant domains depending upon the form of the antigen-binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti -Influenza A antibody or antigenbinding fragment thereof.
  • AAV vectors comprising a viral capsid that is at least 95% identical to the amino acid sequence of an AAV8 capsid (SEQ ID NO:2), AAVS3 capsid (SEQ ID NO:8) or AAV-LK03 capsid (SEQ ID NO:7); and an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding an anti-influenza A mAb, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in human liver or muscle cells.
  • ITRs AAV inverted terminal repeats
  • the regulatory sequence is the ApoE.HAAT regulatory sequence (SEQ ID NO:217) or one of LSPX1, LSPX2, LTP1, LTP2, or LTP3, SEQ ID NOS:9, 10, 11, 12, or 13, respectively).
  • the regulatory sequence is one of the liverspecific CRE sequence selected from Table 16 or a CRE sequence of Table 16 operable linked with a hAAT sequence.
  • Gene Therapy Methods Provided are methods of preventing human subjects of and treating human subjects for Influenza by administration of a viral vector containing a transgene encoding an anti -Influenza A antibody, or antigen binding fragment thereof.
  • the antibody may be VIS410, MHAA4549A, CR6261, CR8020, TCN-032, or VIR-2482 and is, for example, a full length antibody or Fab fragment thereof, or other antigen-binding fragment thereof.
  • the patient has been diagnosed with and/or has symptoms associated with Influenza.
  • Recombinant vector used for delivering the transgene are described in Section 5.1.
  • such vectors should have a tropism for human liver cells and can include non-replicating rAAV, particularly those bearing an AAV8, AAVS3, or AAV- LK03 capsid.
  • the recombinant vectors can be administered in any manner such that the recombinant vector enters the liver or muscle tissue, e.g. by introducing the recombinant vector into the bloodstream. See Section 5.2 for details regarding the methods of treatment.
  • Subjects to whom such gene therapy is administered can be those responsive to antiInfluenza therapy.
  • the methods encompass treating patients who have been diagnosed with Influenza, or have one or more symptoms associated therewith, and identified as responsive to treatment with an anti -Influenza A antibody or considered a good candidate for therapy with an anti-influenza A antibody.
  • the patients have previously been treated with VIS410, MHAA4549A, CR6261, CR8020, TCN-032, or VIR-2482, and have been found to be responsive to VIS410, MHAA4549A, CR6261, CR8020, TCN-032, or VIR-2482.
  • the anti -Influenza A antibody or antigen-binding fragment transgene product may be administered directly to the subject.
  • the production of the anti-influenza A HuPTM mAb or HuPTM Fab should result in a “biobetter” molecule for the treatment of Influenza accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding the anti-influenza A HuPTM Fab, subcutaneously, intramuscularly, or intravenously to human subjects (patients) diagnosed with or having one or more symptoms of Influenza, to create a permanent depot in the liver or muscle tissue that continuously supplies the fully-human post-translationally modified, such as human-glycosylated, sulfated transgene product produced by transduced liver or muscle cells.
  • the cDNA construct for the anti-influenza HuPTMmAb or anti -Influenza HuPTM Fab should include a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced liver or muscle cells.
  • the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 50).
  • the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Tables 2 or 3 that correspond to the proteins secreted by myocytes or hepatocytes, respectively.
  • the anti -Influenza HuPTM mAb or HuPTM Fab can be produced in human cell lines by recombinant DNA technology, and administered to patients diagnosed with Influenza, or for whom therapy or prohylaxis for Influenza is considered appropriate.
  • the anti-influenza HuPTM mAb or antigen-binding fragment thereof does not contain detectable NeuGc moieties and/or does not contain detectable alpha-Gal moieties.
  • the HuPTM mAb is a full length or substantially full length mAb with an Fc region.
  • the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1% or 2% glycosylated and/or sulfated and may be at least 5%, 10% or even 50% or 100% glycosylated and/or sulfated.
  • the goal of gene therapy treatment provided herein is to prevent symptomatic Influenza disease or prevent severe Influenza disease and Influenza A infection. Efficacy may be monitored by scoring the symptoms (e.g. fever, muscle aches, headache, lack of energy, dry cough, sore throat, nasal congestion, and runny nose), laboratory features (e.g. elevated inflammatory markers, liver enzymes), percentage organ involvement (e.g. kidney, lung), all-cause mortality, respiratory failure, need for invasive mechanical ventilation and sustained clinical recovery.
  • symptoms e.g. fever, muscle aches, headache, lack of energy, dry cough, sore throat, nasal congestion, and runny nose
  • laboratory features e.g. elevated inflammatory markers, liver enzymes
  • percentage organ involvement e.g. kidney
  • Section 5.1 describes recombinant vectors that contain a transgene encoding a HuPTM mAb or HuPTM Fab (or other antigen binding fragment of the HuPTM mAb) that binds to Influenza A.
  • Therapeutically effective doses of any such recombinant vector should be administered in any manner such that the recombinant vector enters the liver or muscle (e.g., skeletal muscle), e.g. by introducing the recombinant vector into the bloodstream.
  • the vector may be administered directly to the liver through hepatic blood flow, e.g., via the suprahepatic veins or via the hepatic artery.
  • the vector is administered subcutaneously, intramuscularly or intravenously.
  • Intramuscular, subcutaneous, intravenous or hepatic administration should result in expression of the soluble transgene product in cells of the liver or muscle.
  • the vector may be administered directly to the liver through hepatic blood flow, e.g., via the suprahepatic veins or via the hepatic artery.
  • the expression of the transgene encoding an anti -Influenza A antibody creates a permanent depot in liver and/or muscle of the patient that constinuously supplies the antiInfluenza A HuPTM mAb, or antigen binding fragment of the anti-influenza A mAb to the circulation of the subject.
  • the range of a therapeutically or prophylactically effective amount of an AAV gene therapy vector or pharmaceutical composition is 1E11 to 1E14 genome copies (gc)/kg, preferably between 1E11 to 1E13, and even more preferably 1E12.
  • the dose ranges described herein are exemplary only and do not limit the dose ranges that can be selected.
  • intraveneous administration of an AAV gene therapy vector encoding an anti -Influenza A antibody results in at least 1.5 pg/mL, 2 g/mL, 5 pg/mL, 10 pg/mL, or at least 15 pg/mL transgene product expression in human serum at least 30, 40, 50 or 60 days after administration.
  • the target human serum concentration (Cmin) of the transgene product is about 1.5 pg/mL to about 15 pg/mL mAb and associated with a neutralization titer of 500 to 5000.
  • doses that maintain a serum concentration of the anti-influenza A antibody transgene product at a Cmin of at least 1.5 pg/mL or at least 15 pg/mL e.g., Cmin of 1.5 to 5 pg/ml, 5 to 10 pg/ml or 10 to 15 pg/mL) at least 30, 40, 50 or 69 days after administration are provided.
  • a dose of 1E11 maintains a serum concentration of the antiInfluenza A antibody transgene product of at least 1.5 pg/mL.
  • a dose of 1E12 maintains a serum concentration of the anti-influenza A antibody transgene product of at least 1.5 pg/mL.
  • doses that maintain a serum concentration of the anti-influenza are administered to a patient.
  • a antibody transgene product reach a neutralization titer of at least 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000 or 5000.
  • the transgene product is continuously produced, maintenance of lower concentrations can be effective. Notwithstanding, because the transgene product is continuously produced, maintenance of lower concentrations can be effective.
  • the concentration of the transgene product can be measured in patient blood serum samples.
  • compositions suitable for intravenous, intramuscular, subcutaneous or hepatic administration comprise a suspension of the recombinant vector comprising the transgene encoding the anti-influenza A antibody, or antigen-binding fragment thereof, in a formulation buffer comprising a physiologically compatible aqueous buffer.
  • the formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
  • Combinations of delivery of the anti-influenza A HuPTM mAb or antigen-binding fragment thereof, to the liver or muscles accompanied by delivery of other available treatments are encompassed by the methods provided herein.
  • the additional treatments may be administered before, concurrently, or subsequent to the gene therapy treatment.
  • oseltamivir Tamiflu
  • zanamivir Relenza
  • peramivir Rapivab
  • baloxavir Xofluza
  • antiInfluenza A agents including but not limited to VIS410, MHAA4549A, CR6261, CR8020, TCN-032, or VIR-2482 and have been found to be responsive to VIS410, MHAA4549A, CR6261, CR8020, or TCN-032, VIR-2482.
  • compositions and methods are described for the delivery of HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fabs, that bind to human immunodeficiency virus (HIV) and indicated for prevention and/or treatment of acquired immunodeficiency syndrome (AIDS).
  • HuPTM mAbs and antigen-binding fragments thereof such as HuPTM Fabs
  • HAV human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • the HuPTM mAb has the amino acid sequence of 10-1074 (having heavy and light chain amino acid sequences SEQ ID NOS:351 and 352), 4E10, 2F5, 2G12 (described in Kunert et al ,1998, AIDS RESEARCH AND HUMAN RETROVIRUSES, Vol 14, No 13, p.1115-1128; Kunert et al, 2004, AIDS RESEARCH AND HUMAN RETROVIRUSES, Vol 20, No 7, p.
  • leronlimab having heavy and light chain amino acid sequences SEQ IDNOS:353 and 354
  • 3BNC117 having heavy and light chain amino acid sequences SEQ ID NOS:355 and 356
  • VRC01 having heavy and light chain amino acid sequences SEQ ID NOS:357 and 358
  • ibalizumab having heavy and light chain amino acid sequences SEQ ID NOS:359 and 360
  • Delivery may be accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding an HIV-binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to patients (human subjects) diagnosed with, or having one or more symptoms of AIDS or to prevent AIDS in a human subject to create a permanent depot that continuously supplies the human PTM, e.g., human- glycosylated, transgene product.
  • a viral vector or other DNA expression construct encoding an HIV-binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to patients (human subjects) diagnosed with, or having one or more symptoms of AIDS or to prevent AIDS in a human subject to create a permanent depot that continuously supplies the human PTM, e.g., human- glycosylated, transgene product.
  • transgene encoding a HuPTM mAb or HuPTM Fab (or other antigen binding fragment of the HuPTM mAb) that binds to HIV that can be administered to deliver the HuPTM mAb or antigen binding fragment in a patient.
  • the transgene is a nucleic acid comprising the nucleotide sequences encoding an antigen binding fragment of an antibody that binds to HIV, such as 10-1074, 4E10, 2F5, 2G12, leronlimab, 3BNC117, VRC01, or ibalizumab, or variants thereof as detailed herein.
  • the transgene may also encode an HIV binding fragment that contains additional glycosylation sites (e.g., see Courtois et al.).
  • the anti-HIV antigen-binding fragment transgene comprises the nucleotide sequences encoding the heavy and light chains of the Fab portion of an anti-HIV antibody.
  • the nucleotide sequences may be codon optimized for expression in human cells.
  • the heavy and light chain sequences both have a signal or leader sequence at the N-terminus appropriate for expression and secretion in human cells, in particular, human liver cells (e.g., hepatocytes) or muscle cells.
  • the signal sequence may have the amino acid sequence of MYRMQLLLLIALSLALVTNS (SEQ ID NO:50).
  • the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Tables 2 or 3 that correspond to the proteins secreted by myocytes or hepatocytes, respectively.
  • the transgenes may comprise, at the C-terminus of the heavy chain CHI domain sequence, all or a portion of the hinge region.
  • the anti-HIV antigen binding domain has a heavy chain Fab domain with additional hinge region sequence starting after the C-terminal valine (V), contains all or a portion of the amino acid sequence EPKSCDKTHTCPPCPAPEAAGG (SEQ ID NO: 123), and specifically, EPKSCDKTHL (SEQ ID NO: 117), EPKSCDKTHT (SEQ ID NO: 118 212), EPKSCDKTHTCPPCPA (SEQ ID NO: 119), EPKSCDKTHLCPPCPA (SEQ ID NO: 120 214), EPKSCDKTHTCPPCPAPEAAGGPSVFL (SEQ ID NO: 124 218) or
  • EPKSCDKTHLCPPCPAPEAAGGPSVFL (SEQ ID NO: 125 219). These hinge regions may be encoded by nucleotide sequences at the 3’ end of the heavy chain sequence.
  • the transgenes comprise the amino acid sequences encoding the full length (or substantially full length) heavy and light chains of the antibody, comprising the Fc domain at the C terminus of the heavy chain, e.g., having an amino acid sequence of encoding an IgGl Fc domain of SEQ ID NO: 142 (Table 6), or a mutant or variant thereof.
  • the Fc domain may be engineered for altered binding to one or more Fc receptors and/or effector function as disclosed in Section 5.1.9, infra.
  • the anti-HIV-binding fragment transgene encodes an HIV antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS:352, 354, 356, 358, or 360.
  • the HIV antigen-binding fragment transgene encodes an anti-HIV antigen-binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS:351, 353, 355, 357, or 359.
  • the HIV antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS:352, 354, 356, 358, or 360 and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS:351, 353, 355, 357, or 359.
  • the anti -HIV antigen binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NOS:351, 353, 355, 357, or 359 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, for example, in the framework regions (e.g., those regions outside of the CDRs) or are substitutions with an amino acid present at that position in the heavy chain of one or more of the other therapeutic antibodies, for example, as identified by the alignment in FIG. 3 A.
  • the HIV antigen binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NOS: 352, 354, 356, 358, or 360with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs) or are substitutions with an amino acid present at that position in the light chain of one or more of the other therapeutic antibodies, for example, as identified by the alignment in FIG. 3B.
  • the anti -HIV antigen-binding fragment transgene encodes a hyperglycosylated Fab, comprising a heavy chain and a light chain of SEQ ID NOS: 351, 353, 355, 357, or 359 and 352, 354, 356, 358, or 360, respectively, with one or more mutations (see FIGS. 3A (heavy chain) and 3B (light chain)).
  • the anti-HIV antigen-binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequences encoding the six CDRs which are, for example, underlined in the heavy and light chain variable domain sequences of Table 8, which are spaced between framework regions, generally human framework regions, and associated with constant domains depending upon the form of the antigen-binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-HIV antibody or antigen-binding fragment thereof.
  • AAV vectors comprising a viral capsid that is at least 95% identical to the amino acid sequence of an AAV8 capsid (SEQ ID NO:2), AAVS3 capsid (SEQ ID NO:8) or AAV-LK03 capsid (SEQ ID NO:7); and an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding an anti-HIV mAb, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in human liver or muscle cells.
  • ITRs AAV inverted terminal repeats
  • the regulatory sequence is the ApoE.HAAT regulatory sequence (SEQ ID NO:21) or one of LSPX1, LSPX2, LTP1, LTP2, or LTP3, SEQ ID NOS:9, 10, 11, 12, or 13, respectively).
  • the regulatory sequence is one of the liverspecific CRE sequence selected from Table 16 or a CRE sequence of Table 16 operable linked with a hAAT sequence.
  • the promoter is an LMTP6 promoter (SEQ ID NO: 14).
  • a viral vector containing a transgene encoding an anti- HIV antibody, or antigen binding fragment thereof may be 10-1074, 4E10, 2F5, 2G12, leronlimab, 3BNC117, VRC01, or ibalizumab and is, for example, a full length antibody or Fab fragment thereof, or other antigen-binding fragment thereof.
  • the patient has been diagnosed with and/or has symptoms associated with AIDS.
  • Recombinant vector used for delivering the transgene are described in Section 5.1.
  • such vectors should have a tropism for human liver cells and can include non-replicating rAAV, particularly those bearing an AAV8, AAVS3, or AAV-LK03 capsid.
  • the recombinant vectors can be administered in any manner such that the recombinant vector enters the liver or muscle tissue, e.g. by introducing the recombinant vector into the bloodstream. See Section 5.2 for details regarding the methods of treatment.
  • Subjects to whom such gene therapy is administered can be those responsive to anti- HIV therapy.
  • the methods encompass treating patients who have been diagnosed with HIV or AIDS, or have one or more symptoms associated therewith, and identified as responsive to treatment with an anti-HIV antibody or considered a good candidate for therapy with an anti-HIV antibody.
  • the patients have previously been treated with 10-1074, 4E10, 2F5, 2G12, leronlimab, 3BNC117, VRC01, or ibalizumab, and have been found to be responsive to 10-1074, 4E10, 2F5, 2G12, leronlimab, 3BNC117, VRC01, or ibalizumab.
  • the anti-HIV antibody or antigen-binding fragment transgene product may be administered directly to the subject.
  • the production of the anti -HIV HuPTM mAh or HuPTM Fab should result in a “biobetter” molecule for the treatment of AIDS accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding the anti -HIV HuPTM Fab, subcutaneously, intramuscularly, or intravenously to human subjects (patients) diagnosed with or having one or more symptoms of AIDS, to create a permanent depot in the liver or muscle tissue that continuously supplies the fully-human post-translationally modified, such as human-glycosylated, sulfated transgene product produced by transduced liver or muscle cells.
  • the cDNA construct for the anti-HIV HuPTMmAb or anti-HIV HuPTM Fab should include a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced liver or muscle cells.
  • the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO:50).
  • the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Tables 2 or 3 that correspond to the proteins secreted by myocytes or hepatocytes, respectively.
  • the anti-HIV HuPTM m Ab or HuPTM Fab can be produced in human cell lines by recombinant DNA technology, and administered to patients diagnosed with an HIV infection or AIDS or for whom therapy or prohylaxis for an HIV infection and/or AIDS is considered appropriate.
  • the anti-HIV HuPTM mAb or antigen-binding fragment thereof does not contain detectable NeuGc moieties and/or does not contain detectable alpha-Gal moieties.
  • the HuPTM mAb is a full length or substantially full length mAb with an Fc region.
  • the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1% or 2% glycosylated and/or sulfated and may be at least 5%, 10% or even 50% or 100% glycosylated and/or sulfated.
  • the goal of gene therapy treatment provided herein is to prevent a symptomatic HIV infection or AIDS or progression of HIV to AIDS. Efficacy may be monitored by scoring the HIV symptoms (e.g.
  • AIDS symptoms weight loss, recurring fever or profused night sweats, extreme and unexplained tiredness, prolonged swelling of the lymph glands in the armpits, groin, or neck, diarrhea that lasts for more than a week, sores of the mouth, anus, or genitals, pneumonia
  • laboratory features e.g. CD4 levels, elevated inflammatory markers, liver enzymes
  • percentage organ involvement e.g. kidney, lung
  • all-cause mortality kidney failure, respiratory failure, need for invasive mechanical ventilation, transfusion, occurrence of opportunistic infections, and sustained clinical recovery.
  • Section 5.1 describes recombinant vectors that contain a transgene encoding a HuPTM mAb or HuPTM Fab (or other antigen binding fragment of the HuPTM mAb) that binds to HIV.
  • Therapeutically effective doses of any such recombinant vector should be administered in any manner such that the recombinant vector enters the liver or muscle (e.g., skeletal muscle), e.g. by introducing the recombinant vector into the bloodstream.
  • the vector may be administered directly to the liver through hepatic blood flow, e.g., via the suprahepatic veins or via the hepatic artery.
  • the vector is administered subcutaneously, intramuscularly or intravenously.
  • Intramuscular, subcutaneous, intravenous or hepatic administration should result in expression of the soluble transgene product in cells of the liver or muscle.
  • the vector may be administered directly to the liver through hepatic blood flow, e.g., via the suprahepatic veins or via the hepatic artery.
  • the expression of the transgene encoding an anti-HIV antibody creates a permanent depot in liver and/or muscle of the patient that constinuously supplies the anti-HIV HuPTM mAb, or antigen binding fragment of the anti-HIV mAb to the circulation of the subject.
  • the range of a therapeutically or prophylactically effective amount of an AAV gene therapy vector or pharmaceutical composition is 1E11 to 1E14 genome copies (gc)/kg, preferably between 1E11 to 1E13, and even more preferably 1E12.
  • the dose ranges described herein are exemplary only and do not limit the dose ranges that can be selected.
  • intraveneous administration of an AAV gene therapy vector encoding an anti-HIV antibody results in at least 1.5 g/mL, 2 pg/mL, 5 pg/mL, 10 pg/mL, or at least 15 pg/mL transgene product expression in human serum at least 30, 40, 50 or 60 days after administration.
  • the target human serum concentration (Cmin) of the transgene product is about 1.5 pg/mL to about 15 pg/mL mAb and associated with a neutralization titer of 500 to 5000.
  • doses that maintain a serum concentration of the anti-HIV antibody transgene product at a Cmin of at least 1.5 pg/mL or at least 15 pg/mL e.g., Cmin of 1.5 to 5 pg/ml, 5 to 10 pg/ml or 10 to 15 pg/mL) at least 30, 40, 50 or 69 days after administration are provided.
  • a dose of 1E11 maintains a serum concentration of the anti-HIV antibody transgene product of at least 1.5 pg/mL.
  • a dose of 1E12 maintains a serum concentration of the anti-HIV antibody transgene product of at least 1.5 pg/mL.
  • doses that maintain a serum concentration of the anti-HIV antibody transgene product reach a neutralization titer of at least 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000 or 5000.
  • the transgene product is continuously produced, maintenance of lower concentrations can be effective. Notwithstanding, because the transgene product is continuously produced, maintenance of lower concentrations can be effective.
  • the concentration of the transgene product can be measured in patient blood serum samples.
  • compositions suitable for intravenous, intramuscular, subcutaneous or hepatic administration comprise a suspension of the recombinant vector comprising the transgene encoding the anti-HIV antibody, or antigen-binding fragment thereof, in a formulation buffer comprising a physiologically compatible aqueous buffer.
  • the formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
  • Combinations of delivery of the anti-HIV HuPTM mAb or antigen-binding fragment thereof, to the liver or muscles accompanied by delivery of other available treatments are encompassed by the methods provided herein.
  • the additional treatments may be administered before, concurrently, or subsequent to the gene therapy treatment.
  • ART antiretroviral therapy
  • NRTIs nucleoside and nucleotide reverse transcriptase inhibitors
  • NRTIs non-nucleoside reverse transcriptase inhibitors
  • PI protease inhibitors
  • compositions and methods are described for the delivery of HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fabs, that bind to human cytomegalovirus (HCMV) and indicated for prevention and/or treatment of patients with newly diagnosed and relapsed HCMV retinitis or prevention of HCMV infections in transplant patients.
  • HuPTM mAbs and antigen-binding fragments thereof such as HuPTM Fabs
  • the HuPTM mAb has the amino acid sequence of MCMV5322A, MCMV3068A, LIP538 (disclosed in WO 2016/055950A1, which is incorporated herein by reference in its entirety), LIP539 (disclosed in WO 2016/055950A1, which is incorporated herein by reference in its entirety), TCN-202 (SEQ ID NOS:361 and 362), or serivumab (SEQ ID NOS:363 and 364), or an antigen binding fragment of the foregoing.
  • Delivery may be accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding an HCMV-binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to patients (human subjects) diagnosed with, or having one or more symptoms of an HCMV infection in a human subject to create a permanent depot that continuously supplies the human PTM, e.g., human-glycosylated, transgene product.
  • a viral vector or other DNA expression construct encoding an HCMV-binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to patients (human subjects) diagnosed with, or having one or more symptoms of an HCMV infection in a human subject to create a permanent depot that continuously supplies the human PTM, e.g., human-glycosylated, transgene product.
  • transgene encoding a HuPTM mAb or HuPTM Fab (or other antigen binding fragment of the HuPTM mAb) that binds to HCMV that can be administered to deliver the HuPTM mAb or antigen binding fragment in a patient.
  • the transgene is a nucleic acid comprising the nucleotide sequences encoding an antigen binding fragment of an antibody that binds to HCMV, such as MCMV5322A, MCMV3068A, LIP538, LIP539, TNC-202, or serivumab, or variants thereof as detailed herein.
  • the transgene may also encode an HCMV binding fragment that contains additional glycosylation sites (e.g., see Courtois et al.).
  • the anti -HCMV antigen-binding fragment transgene comprises the nucleotide sequences encoding the heavy and light chains of the Fab portion of an anti- HCMV antibody.
  • the nucleotide sequences may be codon optimized for expression in human cells.
  • the heavy and light chain sequences both have a signal or leader sequence at the N-terminus appropriate for expression and secretion in human cells, in particular, human liver cells (e.g., hepatocytes) or muscle cells.
  • the signal sequence may have the amino acid sequence of MYRMQLLLLIALSLALVTNS (SEQ ID NO:50).
  • the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Tables 2 or 3 that correspond to the proteins secreted by myocytes or hepatocytes, respectively.
  • the transgenes may comprise, at the C-terminus of the heavy chain CHI domain sequence, all or a portion of the hinge region.
  • the anti-HCMV antigen binding domain has a heavy chain Fab domain with additional hinge region sequence starting after the C-terminal valine (V), contains all or a portion of the amino acid sequence EPKSCDKTHTCPPCPAPEAAGG (SEQ ID NO:123), and specifically, EPKSCDKTHL (SEQ ID NO:117), EPKSCDKTHT (SEQ ID NO:118), EPKSCDKTHTCPPCPA (SEQ ID NO: 119), EPKSCDKTHLCPPCPA (SEQ ID NO: 120), EPKSCDKTHTCPPCPAPEAAGGPSVFL (SEQ ID NO: 124) or EPKSCDKTHLCPPCPAPEAAGGPSVFL (SEQ ID NO: 125).
  • the transgenes comprise the amino acid sequences encoding the full length (or substantially full length) heavy and light chains of the antibody, comprising the Fc domain at the C terminus of the heavy chain, e.g., having an amino acid sequence of encoding an IgGl Fc domain of SEQ ID NO: 141 (Table 6), or a mutant or variant thereof.
  • the Fc domain may be engineered for altered binding to one or more Fc receptors and/or effector function as disclosed in Section 5.1.9, infra.
  • the anti-HCMV-binding fragment transgene encodes an HCMV antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS:362 or 364.
  • the HCMV antigen-binding fragment transgene encodes an anti-HCMV antigen-binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS:361 or 363.
  • the HCMV antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS:362 or 364 and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS:361 or 363
  • the anti-HCMV antigen binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NOS:361 or 363 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions
  • the HCMV antigen binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NOS:362 or 364 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs) or are substitutions with an amino acid present at that position in the light chain of one or more of the other therapeutic antibodies, for example, as identified by the alignment in FIG. 3B.
  • the anti-HCMV antigen-binding fragment transgene encodes a hyperglycosylated Fab, comprising a heavy chain and a light chain of SEQ ID NOS:361 or 363 and 362 or 364, respectively, with one or more mutations (see FIGS. 3A (heavy chain) and 3B (light chain)).
  • the anti-HCMV antigen-binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequences encoding the six CDRs which are, for example, underlined in the heavy and light chain variable domain sequences of Table 8, which are spaced between framework regions, generally human framework regions, and associated with constant domains depending upon the form of the antigen-binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-HCMV antibody or antigen-binding fragment thereof.
  • AAV vectors comprising a viral capsid that is at least 95% identical to the amino acid sequence of an AAV8 capsid (SEQ ID NO:2), AAVS3 capsid (SEQ ID NO:8) or AAV-LK03 capsid (SEQ ID NO:7); and an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding an anti-HCMV mAb, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in human liver or muscle cells.
  • ITRs AAV inverted terminal repeats
  • the regulatory sequence is the ApoE.HAAT regulatory sequence (SEQ ID NO:21) or one of LSPX1, LSPX2, LTP1, LTP2, or LTP3, SEQ ID NOS:9, 10, 11, 12, or 13, respectively).
  • the regulatory sequence is one of the liverspecific CRE sequence selected from Table 16 or a CRE sequence of Table 16 operable linked with a hAAT sequence.
  • the promoter is an LMTP6 promoter (SEQ ID NO: 14).
  • a viral vector containing a transgene encoding an anti-HCMV antibody, or antigen binding fragment thereof may be MCMV5322A, MCMV3068A, LIP538, LIP539, TNC-202, or serivumab, and is, for example, a full length antibody or Fab fragment thereof, or other antigen-binding fragment thereof.
  • the patient has been diagnosed with and/or has symptoms associated with an HCMV infection.
  • Recombinant vector used for delivering the transgene are described in Section 5.1.
  • such vectors should have a tropism for human liver cells and can include non-replicating rAAV, particularly those bearing an AAV8, AAVS3, or AAV-LK03 capsid.
  • the recombinant vectors can be administered in any manner such that the recombinant vector enters the liver or muscle tissue, e.g. by introducing the recombinant vector into the bloodstream. See Section 5.2 for details regarding the methods of treatment.
  • Subjects to whom such gene therapy is administered can be those responsive to anti- HCMV therapy.
  • the methods encompass treating patients who have been diagnosed with an HCMV infection, or have one or more symptoms associated therewith, and identified as responsive to treatment with an anti-HCMV antibody or considered a good candidate for therapy with an anti-HCMV antibody.
  • the patients have previously been treated with MCMV5322A, MCMV3068A, LIP538, LIP539, TNC-202, or serivumab, and have been found to be responsive to MCMV5322A, MCMV3068A, LIP538, LIP539, TNC-202, or serivumab.
  • the anti-HCMV antibody or antigen-binding fragment transgene product may be administered directly to the subject.
  • the production of the anti-HCMV HuPTM mAb or HuPTM Fab should result in a “biobetter” molecule for the treatment of HCMV infections accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding the anti-HCMV HuPTM Fab, subcutaneously, intramuscularly, or intravenously to human subjects (patients) diagnosed with or having one or more symptoms of an HCMV infection, to create a permanent depot in the liver or muscle tissue that continuously supplies the fully-human post-translationally modified, such as human-glycosylated, sulfated transgene product produced by transduced liver or muscle cells.
  • the cDNA construct for the anti-HCMV HuPTMmAb or anti-HCMV HuPTM Fab should include a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced liver or muscle cells.
  • the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 50).
  • the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Tables 2 or 3 that correspond to the proteins secreted by myocytes or hepatocytes, respectively.
  • the anti-HCMV HuPTM mAb or HuPTM Fab can be produced in human cell lines by recombinant DNA technology, and administered to patients diagnosed with an HCMV infection or for whom therapy or prohylaxis for an HCMV infection, e.g. patients undergoing an organ transplant or a weakened immune system.
  • the anti-HCMV HuPTM mAb or antigen-binding fragment thereof does not contain detectable NeuGc moieties and/or does not contain detectable alpha-Gal moieties.
  • the HuPTM mAb is a full length or substantially full length mAb with an Fc region.
  • the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1% or 2% glycosylated and/or sulfated and may be at least 5%, 10% or even 50% or 100% glycosylated and/or sulfated.
  • the goal of gene therapy treatment provided herein is to prevent or treate a symptomatic HCMV infection. Efficacy may be monitored by scoring the HCMV symptoms in healthy adults (e.g. fever, sore throat, fatigue, and muscle aches), in infants (e.g.
  • Section 5.1 describes recombinant vectors that contain a transgene encoding a HuPTM mAb or HuPTM Fab (or other antigen binding fragment of the HuPTM mAb) that binds to HCMV.
  • Therapeutically effective doses of any such recombinant vector should be administered in any manner such that the recombinant vector enters the liver or muscle (e.g., skeletal muscle), e.g. by introducing the recombinant vector into the bloodstream.
  • the vector may be administered directly to the liver through hepatic blood flow, e.g., via the suprahepatic veins or via the hepatic artery.
  • the vector is administered subcutaneously, intramuscularly or intravenously.
  • Intramuscular, subcutaneous, intravenous or hepatic administration should result in expression of the soluble transgene product in cells of the liver or muscle.
  • the vector may be administered directly to the liver through hepatic blood flow, e.g., via the suprahepatic veins or via the hepatic artery.
  • the expression of the transgene encoding an anti-HCMV antibody creates a permanent depot in liver and/or muscle of the patient that constinuously supplies the anti-HCMV HuPTM mAb, or antigen binding fragment of the anti-HCMV mAb to the circulation of the subject.
  • the range of a therapeutically or prophylactically effective amount of an AAV gene therapy vector or pharmaceutical composition is 1E11 to 1E14 genome copies (gc)/kg, preferably between 1E11 to 1E13, and even more preferably 1E12.
  • the dose ranges described herein are exemplary only and do not limit the dose ranges that can be selected.
  • intraveneous administration of an AAV gene therapy vector encoding an anti-HCMV antibody results in at least 1.5 g/mL, 2 pg/mL, 5 pg/mL, 10 pg/mL, or at least 15 pg/mL transgene product expression in human serum at least 30, 40, 50 or 60 days after administration.
  • the target human serum concentration (Cmin) of the transgene product is about 1.5 pg/mL to about 15 pg/mL mAb and associated with a neutralization titer of 500 to 5000.
  • doses that maintain a serum concentration of the anti-HCMV antibody transgene product at a Cmin of at least 1.5 pg/mL or at least 15 pg/mL e.g., Cmin of 1.5 to 5 pg/ml, 5 to 10 pg/ml or 10 to 15 pg/mL) at least 30, 40, 50 or 69 days after administration are provided.
  • a dose of 1E11 maintains a serum concentration of the anti-HCMV antibody transgene product of at least 1.5 pg/mL.
  • a dose of 1E12 maintains a serum concentration of the anti-HCMV antibody transgene product of at least 1.5 pg/mL.
  • doses that maintain a serum concentration of the anti-HCMV antibody transgene product reach a neutralization titer of at least 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000 or 5000.
  • the transgene product is continuously produced, maintenance of lower concentrations can be effective. Notwithstanding, because the transgene product is continuously produced, maintenance of lower concentrations can be effective.
  • the concentration of the transgene product can be measured in patient blood serum samples.
  • compositions suitable for intravenous, intramuscular, subcutaneous or hepatic administration comprise a suspension of the recombinant vector comprising the transgene encoding the anti-HCMV antibody, or antigen-binding fragment thereof, in a formulation buffer comprising a physiologically compatible aqueous buffer.
  • the formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil. 5.4.4.2. Combination Therapies with anti-HCMV Antibodies
  • Combinations of delivery of the anti-HCMV HuPTM mAb or antigen-binding fragment thereof, to the liver or muscles accompanied by delivery of other available treatments are encompassed by the methods provided herein.
  • the additional treatments may be administered before, concurrently, or subsequent to the gene therapy treatment.
  • HCMV infections that could be combined with the gene therapy provided herein include but are not limited to anti-viral inhibitors (e.g. ganciclovir), and administration with anti-HCMV agents, including but not limited to MCMV5322A, MCMV3068A, LIP538, LIP539, TNC-202, or serivumab and have been found to be responsive to MCMV5322A, MCMV3068A, LIP538, LIP539, TNC-202, or serivumab.
  • anti-viral inhibitors e.g. ganciclovir
  • administration with anti-HCMV agents including but not limited to MCMV5322A, MCMV3068A, LIP538, LIP539, TNC-202, or serivumab and have been found to be responsive to MCMV5322A, MCMV3068A, LIP538, LIP539, TNC-202, or serivumab.
  • compositions and methods are described for the delivery of HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fabs, that bind to respiratory syncytial virus (RSV) and indicated for prevention and/or treatment of patients diagnosed with an RSV infection.
  • HuPTM mAbs and antigen-binding fragments thereof such as HuPTM Fabs
  • RSV respiratory syncytial virus
  • the HuPTM mAb has the amino acid sequence of palivizumab (SEQ ID NOS: 365 and 366), motavizumab (SEQ ID NOS:367 and 368), MEDI-8897 (SEQ ID NOS:369 and 370)), suptavumab (SEQ ID NOS:371 and 372), or gontivimab (SEQ ID NOS:373), or an antigen binding fragment of the foregoing.
  • Delivery may be accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding an RSV-binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to patients (human subjects) diagnosed with, or having one or more symptoms of an RSV infection in a human subject to create a permanent depot that continuously supplies the human PTM, e.g., human-glycosylated, transgene product.
  • a viral vector or other DNA expression construct encoding an RSV-binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to patients (human subjects) diagnosed with, or having one or more symptoms of an RSV infection in a human subject to create a permanent depot that continuously supplies the human PTM, e.g., human-glycosylated, transgene product.
  • transgene encoding a HuPTM mAb or HuPTM Fab (or other antigen binding fragment of the HuPTM mAb) that binds to RSV that can be administered to deliver the HuPTM mAb or antigen binding fragment in a patient.
  • the transgene is a nucleic acid comprising the nucleotide sequences encoding an antigen binding fragment of an antibody that binds to RSV, such as palivizumab, motavizumab, MEDI-8897, suptavumab, or gontivimab, or variants thereof as detailed herein.
  • the transgene may also encode a RVS binding fragment that contains additional glycosylation sites (e.g., see Courtois et al.).
  • the anti-RSV antigen-binding fragment transgene comprises the nucleotide sequences encoding the heavy and light chains of the Fab portion of an anti-RSV antibody.
  • the nucleotide sequences may be codon optimized for expression in human cells.
  • the heavy and light chain sequences both have a signal or leader sequence at the N-terminus appropriate for expression and secretion in human cells, in particular, human liver cells (e.g., hepatocytes) or muscle cells.
  • the signal sequence may have the amino acid sequence of MYRMQLLLLIALSLALVTNS (SEQ ID NO:50).
  • the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Tables 2 or 3 that correspond to the proteins secreted by myocytes or hepatocytes, respectively.
  • the transgenes may comprise, at the C-terminus of the heavy chain CHI domain sequence, all or a portion of the hinge region.
  • the anti-RSV antigen binding domain has a heavy chain Fab domain with additional hinge region sequence starting after the C-terminal valine (V), contains all or a portion of the amino acid sequence EPKSCDKTHTCPPCPAPEAAGG (SEQ ID NO:123), and specifically, EPKSCDKTHL (SEQ ID NO:117), EPKSCDKTHT (SEQ ID NO:118), EPKSCDKTHTCPPCPA (SEQ ID NO: 119), EPKSCDKTHLCPPCPA (SEQ ID NO: 120), EPKSCDKTHTCPPCPAPEAAGGPSVFL (SEQ ID NO: 124) or
  • EPKSCDKTHLCPPCPAPEAAGGPSVFL (SEQ ID NO: 125). These hinge regions may be encoded by nucleotide sequences at the 3’ end of the heavy chain sequence.
  • the transgenes comprise the amino acid sequences encoding the full length (or substantially full length) heavy and light chains of the antibody, comprising the Fc domain at the C terminus of the heavy chain, e.g., having an amino acid sequence of encoding an IgGl Fc domain of SEQ ID NO: 141 (Table 6), or a mutant or variant thereof.
  • the Fc domain may be engineered for altered binding to one or more Fc receptors and/or effector function as disclosed in Section 5.1.9, infra.
  • the anti-RSV-binding fragment transgene encodes an RSV antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS:366, 368, 370, or 372.
  • the RSV antigen-binding fragment transgene encodes an anti-RSV antigen-binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS:365, 367, 369, 371, or 373.
  • the RSV antigen -binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS: 366, 368, 370, or 372 and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS: 365, 367, 369, 371, or 373.
  • the anti-RSV antigen binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NOS: 365, 367, 369, 371, or 373with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, for example, in the framework regions (e.g., those regions outside of the CDRs) or are substitutions with an amino acid present at that position in the heavy chain of one or more of the other therapeutic antibodies, for example, as identified by the alignment in FIG. 3 A.
  • the RSV antigen binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NOS: 366, 368, 370, or 372 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs) or are substitutions with an amino acid present at that position in the light chain of one or more of the other therapeutic antibodies, for example, as identified by the alignment in FIG. 3B.
  • the anti-RSV antigen-binding fragment transgene encodes a hyperglycosylated Fab, comprising a heavy chain and a light chain of SEQ ID NOS: 365, 367, 369, 371, or 373 and 366, 368, 370, or 372, respectively, with one or more mutations (see FIGS. 3 A (heavy chain) and 3B (light chain)).
  • the anti-RSV antigen-binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequences encoding the six CDRs which are, for example, underlined in the heavy and light chain variable domain sequences of Table 8, which are spaced between framework regions, generally human framework regions, and associated with constant domains depending upon the form of the antigen-binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-RSV antibody or antigen-binding fragment thereof.
  • AAV vectors comprising a viral capsid that is at least 95% identical to the amino acid sequence of an AAV8 capsid (SEQ ID NO:2), AAVS3 capsid (SEQ ID NO:8) or AAV-LK03 capsid (SEQ ID NO:7); and an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding an anti-RSV mAb, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in human liver or muscle cells.
  • ITRs AAV inverted terminal repeats
  • the regulatory sequence is the ApoE.HAAT regulatory sequence (SEQ ID NO:21) or one of LSPX1, LSPX2, LTP1, LTP2, or LTP3, SEQ ID NOS:9, 10, 11, 12, or 13, respectively).
  • the regulatory sequence is one of the liverspecific CRE sequence selected from Table 16 or a CRE sequence of Table 16 operable linked with a hAAT sequence.
  • the promoter is an LMTP6 promoter (SEQ ID NO: 14).
  • a viral vector containing a transgene encoding an anti-RSV antibody, or antigen binding fragment thereof may be palivizumab, motavizumab, MEDI-8897, suptavumab, or gontivimab, and is, for example, a full length antibody or Fab fragment thereof, or other antigen-binding fragment thereof.
  • the patient has been diagnosed with and/or has symptoms associated with an RSV infection.
  • Recombinant vector used for delivering the transgene are described in Section 5.1.
  • such vectors should have a tropism for human liver cells and can include non-replicating rAAV, particularly those bearing an AAV8, AAVS3, or AAV-LK03 capsid.
  • the recombinant vectors can be administered in any manner such that the recombinant vector enters the liver or muscle tissue, e.g. by introducing the recombinant vector into the bloodstream. See Section 5.2 for details regarding the methods of treatment.
  • Subjects to whom such gene therapy is administered can be those responsive to anti- RSV therapy.
  • the methods encompass treating patients who have been diagnosed with an RSV infection, or have one or more symptoms associated therewith, and identified as responsive to treatment with an anti-RSV antibody or considered a good candidate for therapy with an anti-RSV antibody.
  • the patients have previously been treated with palivizumab, motavizumab, MEDI-8897, suptavumab, or gontivimab, and have been found to be responsive to palivizumab, motavizumab, MEDI-8897, suptavumab, or gontivimab.
  • the anti-RSV antibody or antigen-binding fragment transgene product may be administered directly to the subject.
  • the production of the anti-RSV HuPTM mAb or HuPTM Fab should result in a “biobetter” molecule for the treatment of RSV infections accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding the anti-RSV HuPTM Fab, subcutaneously, intramuscularly, or intravenously to human subjects (patients) diagnosed with or having one or more symptoms of an RSV infection, to create a permanent depot in the liver or muscle tissue that continuously supplies the fully-human post-translationally modified, such as human- glycosylated, sulfated transgene product produced by transduced liver or muscle cells.
  • the cDNA construct for the anti-RSV HuPTMmAb or anti-RSV HuPTM Fab should include a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced liver or muscle cells.
  • the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO:50).
  • the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Tables 2 or 3 that correspond to the proteins secreted by myocytes or hepatocytes, respectively.
  • the anti-RSV HuPTM mAb or HuPTM Fab can be produced in human cell lines by recombinant DNA technology, and administered to patients diagnosed with an RSV infection or for whom therapy or prohylaxis for an RSV infection, e.g. patients undergoing an organ transplant or a weakened immune system.
  • the anti-RSV HuPTM mAh or antigen-binding fragment thereof does not contain detectable NeuGc moieties and/or does not contain detectable alpha-Gal moieties.
  • the HuPTM mAb is a full length or substantially full length mAb with an Fc region.
  • the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1% or 2% glycosylated and/or sulfated and may be at least 5%, 10% or even 50% or 100% glycosylated and/or sulfated.
  • the goal of gene therapy treatment provided herein is to prevent or treat a symptomatic RSV infection and prevent progression to a severe RSV infection in premature babies, older adults, infants and adults with heart and lung disease, or anyone with a very weak immune system (immunocompromised). Efficacy may be monitored by scoring the RSV symptoms (e.g. flow-grade ever, dry cough, congested or runny nose, sore throat, or mild headaches).
  • RSV symptoms include fever, severe cough, wheezing, rapid breathing or difficulty breathing, or bluish color of the skin due to lack of oxygen.
  • RSV symptoms include short, shallow or rapid breathing, cough, poor feeding, unusual tiredness, or irritability.
  • Section 5.1 describes recombinant vectors that contain a transgene encoding a HuPTM mAb or HuPTM Fab (or other antigen binding fragment of the HuPTM mAb) that binds to RSV.
  • Therapeutically effective doses of any such recombinant vector should be administered in any manner such that the recombinant vector enters the liver or muscle (e.g., skeletal muscle), e.g. by introducing the recombinant vector into the bloodstream.
  • the vector may be administered directly to the liver through hepatic blood flow, e.g., via the suprahepatic veins or via the hepatic artery.
  • the vector is administered subcutaneously, intramuscularly or intravenously.
  • Intramuscular, subcutaneous, intravenous or hepatic administration should result in expression of the soluble transgene product in cells of the liver or muscle.
  • the vector may be administered directly to the liver through hepatic blood flow, e.g., via the suprahepatic veins or via the hepatic artery.
  • the expression of the transgene encoding an anti-RSV antibody creates a permanent depot in liver and/or muscle of the patient that constinuously supplies the anti-RSVHuPTM mAh, or antigen binding fragment of the anti -RS V mAh to the circulation of the subject.
  • the range of a therapeutically or prophylactically effective amount of an AAV gene therapy vector or pharmaceutical composition is 1E11 to 1E14 genome copies (gc)/kg, preferably between 1E11 to 1E13, and even more preferably 1E12.
  • the dose ranges described herein are exemplary only and do not limit the dose ranges that can be selected.
  • intraveneous administration of an AAV gene therapy vector encoding an anti -RS V antibody results in at least 1.5 pg/mL, 2 pg/mL, 5 pg/mL, 10 pg/mL, or at least 15 pg/mL transgene product expression in human serum at least 30, 40, 50 or 60 days after administration.
  • the target human serum concentration (Cmin) of the transgene product is about 1.5 pg/mL to about 15 pg/mL mAb and associated with a neutralization titer of 500 to 5000.
  • doses that maintain a serum concentration of the anti -RS V antibody transgene product at a Cmin of at least 1.5 pg/mL or at least 15 pg/mL e.g., Cmin of 1.5 to 5 pg/ml, 5 to 10 pg/ml or 10 to 15 pg/mL) at least 30, 40, 50 or 69 days after administration are provided.
  • a dose of 1E11 maintains a serum concentration of the anti-RSV antibody transgene product of at least 1.5 pg/mL.
  • a dose of 1E12 maintains a serum concentration of the anti-RSV antibody transgene product of at least 1.5 pg/mL.
  • doses that maintain a serum concentration of the anti-RSV antibody transgene product reach a neutralization titer of at least 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000 or 5000.
  • the transgene product is continuously produced, maintenance of lower concentrations can be effective. Notwithstanding, because the transgene product is continuously produced, maintenance of lower concentrations can be effective.
  • the concentration of the transgene product can be measured in patient blood serum samples.
  • compositions suitable for intravenous, intramuscular, subcutaneous or hepatic administration comprise a suspension of the recombinant vector comprising the transgene encoding the anti-RSV antibody, or antigen-binding fragment thereof, in a formulation buffer comprising a physiologically compatible aqueous buffer.
  • the formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
  • Combinations of delivery of the anti-RSV HuPTM mAb or antigen-binding fragment thereof, to the liver or muscles accompanied by delivery of other available treatments are encompassed by the methods provided herein.
  • the additional treatments may be administered before, concurrently, or subsequent to the gene therapy treatment.
  • RSV infections include but are not limited to anti-viral medications (e.g. ribavirin), corticosteroids, antibacterial medications, oral or IV fluids, chest therapy, oxygen therapy, inhaled bronchodilators, and administration with anti-RSV agents, including but not limited to palivizumab, motavizumab, MEDI-8897, suptavumab, or gontivimab and have been found to be responsive to palivizumab, motavizumab, MEDI-8897, suptavumab, or gontivimab.
  • HuPTM mAbs and antigen-binding fragments thereof such as HuPTM Fabs, that bind to ebola virus (EBOV), Epstein- barr virus (EBV), hepatitis B virus (HBV), or C-C chemokine receptor type 4 (CCR4) and indicated for prevention and/or treatment of patients diagnosed with Ebola, mononucleosis, hepatitis B, or relapsed or refractory mycosis fungoides and Sezary disease, respectively.
  • HuPTM mAbs and antigen-binding fragments thereof such as HuPTM Fabs, that bind to ebola virus (EBOV), Epstein- barr virus (EBV), hepatitis B virus (HBV), or C-C chemokine receptor type 4 (CCR4) and indicated for prevention and/or treatment of patients diagnosed with Ebola, mononucleosis, hepatitis B, or relapsed or refractory mycosis fungoides
  • the HuPTM mAb has the amino acid sequence of AM001 (SEQ ID NOS:374 and 375), mogamulizumab (SEQ ID NOS:376 and 377), VIR-3434 (SEQ ID NOS:378 and 379), REGN-EB3 (which includes atoltivimab (REGN3470), maftivimab (REGN3479), and odesivimab (REGN3471), SEQ ID NOS:380-385), mAbll4 (SEQ ID NOS:386 and 387), or ZMapp (disclosed in US Patent No. 2019/0263894A1 and incorporated herein in its entirety), or an antigen binding fragment of the foregoing.
  • AM001 SEQ ID NOS:374 and 375
  • mogamulizumab SEQ ID NOS:376 and 377
  • VIR-3434 SEQ ID NOS:378 and 379
  • REGN-EB3 which includes atoltivimab (RE
  • Delivery may be accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding an EBOV-binding, EBV-binding, HBV-binding, or CCR4- binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to patients (human subjects) diagnosed with, or having one or more symptoms of Ebola, mononucleosis, hepatitis B, or relapsed or refractory mycosis fungoides and Sezary disease in a human subject to create a permanent depot that continuously supplies the human PTM, e.g., human- glycosylated, transgene product.
  • a viral vector or other DNA expression construct encoding an EBOV-binding, EBV-binding, HBV-binding, or CCR4- binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to
  • recombinant vectors containing a transgene encoding a HuPTM mAb or HuPTM Fab (or other antigen binding fragment of the HuPTM mAb) that binds to EBOV, EBV, HBV, or CCR4 that can be administered to deliver the HuPTM mAb or antigen binding fragment in a patient are provided.
  • the transgene is a nucleic acid comprising the nucleotide sequences encoding an antigen binding fragment of an antibody that binds to EBOV, EBV, HBV, or CCR4, such as AM001, mogamulizumab, VIR-3434, REGN-EB3 (atoltivimab (REGN3470), maftivimab (REGN3479), and odesivimab (REGN3471)), mAbll4, or ZMapp, or variants thereof as detailed herein.
  • the transgene may also encode an EBOV-binding, EBV-binding, HBV-binding, or CCR4-binding fragment that contains additional glycosylation sites e.g., see Courtois et al.).
  • the anti-EBOV, anti-EBV, anti-HBV-, or anti-CCR4 antigenbinding fragment transgene comprises the nucleotide sequences encoding the heavy and light chains of the Fab portion of an anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 antibody, respectively.
  • the nucleotide sequences may be codon optimized for expression in human cells.
  • the heavy and light chain sequences both have a signal or leader sequence at the N-terminus appropriate for expression and secretion in human cells, in particular, human liver cells (e.g., hepatocytes) or muscle cells.
  • the signal sequence may have the amino acid sequence of MYRMQLLLLIALSLALVTNS (SEQ ID NO:50).
  • the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Tables 2 or 3 that correspond to the proteins secreted by myocytes or hepatocytes, respectively.
  • the transgenes may comprise, at the C-terminus of the heavy chain CHI domain sequence, all or a portion of the hinge region.
  • the anti-EBOV, anti-EBV, anti-HBV, or anti- CCR4 antigen binding domain has a heavy chain Fab domain with additional hinge region sequence starting after the C-terminal valine (V), contains all or a portion of the amino acid sequence EPKSCDKTHTCPPCPAPEAAGG (SEQ ID NO: 123), and specifically, EPKSCDKTHL (SEQ ID NO: 117), EPKSCDKTHT (SEQ ID NO: 118), EPKSCDKTHTCPPCPA (SEQ ID NO: 119), EPKSCDKTHLCPPCPA (SEQ ID NO: 120), EPKSCDKTHTCPPCPAPEAAGGPSVFL (SEQ ID NO: 124) or EPKSCDKTHLCPPCPAPEAAGGPSVFL (SEQ ID NO: 125).
  • the transgenes comprise the amino acid sequences encoding the full length (or substantially full length) heavy and light chains of the antibody, comprising the Fc domain at the C terminus of the heavy chain, e.g., having an amino acid sequence of encoding an IgGl Fc domain of SEQ ID NO: 141 (Table 6), or a mutant or variant thereof.
  • the Fc domain may be engineered for altered binding to one or more Fc receptors and/or effector function as disclosed in Section 5.1.9, infra.
  • the anti-EBOV-, anti-EBV, anti-HBV, or anti-CCR4-binding, fragment transgene encodes an EBOV, EBV, HBV, or CCR4 antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS: 375, 377, 379, 381, 383, 385, or 387.
  • the EBOV, EBV, HBV, or CCR4 antigen-binding fragment transgene encodes an anti- EBOV, anti-EBV, anti-HBV, or anti-CCR4 antigen-binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS: 374, 376, 378, 380, 382, 384, or 386.
  • the EBOV, EBV, HBV, or CCR4 antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS: 375, 377, 379, 381, 383, 385, or 387and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS: 374, 376, 378, 380, 382, 384, or 386.
  • the anti- EBOV, anti-EBV, anti-HBV, or anti-CCR4 antigen binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NOS: 374, 376, 378, 380, 382, 384, or 386with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, for example, in the framework regions (e.g., those regions outside of the CDRs) or are substitutions with an amino acid present at that position in the heavy chain of one or more of the other therapeutic antibodies, for example, as identified by the alignment in FIG. 3 A.
  • the EBOV antigen binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NOS: 375, 377, 379, 381, 383, 385, or 387with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs) or are substitutions with an amino acid present at that position in the light chain of one or more of the other therapeutic antibodies, for example, as identified by the alignment in FIG. 3B.
  • the anti- EBOV, anti-EBV, anti-HBV, or anti-CCR4 antigenbinding fragment transgene encodes a hyperglycosylated Fab, comprising a heavy chain and a light chain of SEQ ID NOS: 374, 376, 378, 380, 382, 384, or 386and 375, 377, 379, 381, 383, 385, or 387, respectively, with one or more mutations (see FIGS. 3 A (heavy chain) and 3B (light chain)).
  • the anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 antigenbinding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequences encoding the six CDRs which are, for example, underlined in the heavy and light chain variable domain sequences of Table 8, which are spaced between framework regions, generally human framework regions, and associated with constant domains depending upon the form of the antigenbinding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 antibody or antigen-binding fragment thereof.
  • AAV vectors comprising a viral capsid that is at least 95% identical to the amino acid sequence of an AAV8 capsid (SEQ ID NO:2), AAVS3 capsid (SEQ ID NO:8) or AAV-LK03 capsid (SEQ ID NO:7); and an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding an anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 mAb, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in human liver or muscle cells.
  • ITRs AAV inverted terminal repeats
  • the regulatory sequence is the ApoE.HAAT regulatory sequence (SEQ ID NO:21) or one of LSPX1, LSPX2, LTP1, LTP2, or LTP3, SEQ ID NOS: 9, 10, 11, 12, or 13, respectively).
  • the regulatory sequence is one of the liver-specific CRE sequence selected from Table 16 or a CRE sequence of Table 16 operable linked with a hAAT sequence.
  • the promoter is an LMTP6 promoter (SEQ ID NO: 14).
  • kits for preventing human subjects of and treating human subjects for Ebola, mononucleosis, hepatitis B, or relapsed or refractory mycosis fungoides and Sezary disease by administration of a viral vector containing a transgene encoding an anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 antibody, respectively, or antigen binding fragment thereof.
  • the antibody may be AM001, mogamulizumab, VIR-3434, REGN-EB3 (atoltivimab (REGN3470), maftivimab (REGN3479), and odesivimab (REGN3471)), mAbll4, or ZMapp, and is, for example, a full length antibody or Fab fragment thereof, or other antigen-binding fragment thereof.
  • the patient has been diagnosed with and/or has symptoms associated with Ebola, mononucleosis, hepatitis B, or relapsed or refractory mycosis fungoides and Sezary disease.
  • Recombinant vector used for delivering the transgene are described in Section 5.1.
  • such vectors should have a tropism for human liver cells and can include non-replicating rAAV, particularly those bearing an AAV8, AAVS3, or AAV-LK03 capsid.
  • the recombinant vectors can be administered in any manner such that the recombinant vector enters the liver or muscle tissue, e.g. by introducing the recombinant vector into the bloodstream. See Section 5.2 for details regarding the methods of treatment.
  • Subjects to whom such gene therapy is administered can be those responsive to anti- EBOV therapy.
  • the methods encompass treating patients who have been diagnosed with Ebola, mononucleosis, hepatitis B, or relapsed or refractory mycosis fungoides and Sezary disease, or have one or more symptoms associated therewith, and identified as responsive to treatment with an anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 antibody or considered a good candidate for therapy with an anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 antibody.
  • the patients have previously been treated with AM001, mogamulizumab, VIR-3434, REGN-EB3, mAbll4, or ZMapp, and have been found to be responsive to AMOOl, mogamulizumab, VIR-3434, REGN-EB3, mAbll4, or ZMapp.
  • the anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 antibody or antigen-binding fragment transgene product may be administered directly to the subject.
  • the production of the anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 HuPTM mAh or HuPTM Fab should result in a “biobetter” molecule for the treatment of Ebola, mononucleosis, hepatitis B, or relapsed or refractory mycosis fungoides and Sezary disease accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding the anti- EBOV, anti-EBV, anti-HBV, or anti-CCR4 HuPTM Fab, subcutaneously, intramuscularly, or intravenously to human subjects (patients) diagnosed with or having one or more symptoms of Ebola, to create a permanent depot in the liver or muscle tissue that continuously supplies the fully-human post-translationally modified, such as human-glycosylated, sulfated transgene product produced by transduced liver or muscle cells.
  • the cDNA construct for the anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 HuPTMmAb or anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 HuPTM Fab should include a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced liver or muscle cells.
  • the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO:50).
  • the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Tables 2 or 3 that correspond to the proteins secreted by myocytes or hepatocytes, respectively.
  • the anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 HuPTM mAb or HuPTM Fab can be produced in human cell lines by recombinant DNA technology, and administered to patients diagnosed with Ebola, mononucleosis, hepatitis B, or relapsed or refractory mycosis fungoides and Sezary disease or for whom therapy or prohylaxis for Ebola, mononucleosis, hepatitis B, or relapsed or refractory mycosis fungoides and Sezary disease, e.g. patients undergoing an organ transplant or a weakened immune system.
  • the anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 HuPTM mAb or antigen-binding fragment thereof does not contain detectable NeuGc moieties and/or does not contain detectable alpha-Gal moieties.
  • the HuPTM mAb is a full length or substantially full length mAb with an Fc region.
  • the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1% or 2% glycosylated and/or sulfated and may be at least 5%, 10% or even 50% or 100% glycosylated and/or sulfated.
  • the goal of gene therapy treatment provided herein is to prevent or treat Ebola, mononucleosis, hepatitis B, or relapsed or refractory mycosis fungoides and Sezary disease. Efficacy may be monitored by scoring the Ebola, mononucleosis, hepatitis B, or relapsed or refractory mycosis fungoides and Sezary disease.
  • Section 5.1 describes recombinant vectors that contain a transgene encoding a HuPTM mAb or HuPTM Fab (or other antigen binding fragment of the HuPTM mAb) that binds to EBOV, EBV, HBV, or CCR4.
  • Therapeutically effective doses of any such recombinant vector should be administered in any manner such that the recombinant vector enters the liver or muscle (e.g., skeletal muscle), e.g. by introducing the recombinant vector into the bloodstream.
  • the vector may be administered directly to the liver through hepatic blood flow, e.g., via the suprahepatic veins or via the hepatic artery.
  • the vector is administered subcutaneously, intramuscularly or intravenously.
  • Intramuscular, subcutaneous, intravenous or hepatic administration should result in expression of the soluble transgene product in cells of the liver or muscle.
  • the vector may be administered directly to the liver through hepatic blood flow, e.g., via the suprahepatic veins or via the hepatic artery.
  • the expression of the transgene encoding an anti- EBOV, anti-EBV, anti-HBV, or anti-CCR4 antibody creates a permanent depot in liver and/or muscle of the patient that constinuously supplies the anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 HuPTM mAb, or antigen binding fragment of the anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 mAb to the circulation of the subject.
  • the range of a therapeutically or prophylactically effective amount of an AAV gene therapy vector or pharmaceutical composition is 1E11 to 1E14 genome copies (gc)/kg, preferably between 1E11 to 1E13, and even more preferably 1E12.
  • the dose ranges described herein are exemplary only and do not limit the dose ranges that can be selected.
  • intraveneous administration of an AAV gene therapy vector encoding an anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 antibody results in at least 1.5 pg/mL, 2 g/mL, 5 pg/mL, 10 pg/mL, or at least 15 pg/mL transgene product expression in human serum at least 30, 40, 50 or 60 days after administration.
  • the target human serum concentration (Cmin) of the transgene product is about 1.5 pg/mL to about 15 pg/mL mAb and associated with a neutralization titer of 500 to 5000.
  • doses that maintain a serum concentration of the anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 antibody transgene product at a Cmin of at least 1.5 pg/mL or at least 15 pg/mL e.g., Cmin of 1.5 to 5 pg/ml, 5 to 10 pg/ml or 10 to 15 pg/mL) at least 30, 40, 50 or 69 days after administration are provided.
  • a dose of 1E11 maintains a serum concentration of the anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 antibody transgene product of at least 1.5 pg/mL.
  • a dose of 1E12 maintains a serum concentration of the anti- EBOV, anti-EBV, anti-HBV, or anti-CCR4 antibody transgene product of at least 1.5 pg/mL.
  • doses that maintain a serum concentration of the anti-EBOV, anti-EBV, anti-HBV, anti-CCR4 antibody transgene product reach a neutralization titer of at least 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000 or 5000.
  • the transgene product is continuously produced, maintenance of lower concentrations can be effective. Notwithstanding, because the transgene product is continuously produced, maintenance of lower concentrations can be effective.
  • the concentration of the transgene product can be measured in patient blood serum samples.
  • compositions suitable for intravenous, intramuscular, subcutaneous or hepatic administration comprise a suspension of the recombinant vector comprising the transgene encoding the anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 antibody, or antigen-binding fragment thereof, in a formulation buffer comprising a physiologically compatible aqueous buffer.
  • the formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
  • Combinations of delivery of the anti-EBOV, anti-EBV, anti-HBV, or anti-CCR4 HuPTM mAb or antigen-binding fragment thereof, to the liver or muscles accompanied by delivery of other available treatments are encompassed by the methods provided herein.
  • the additional treatments may be administered before, concurrently, or subsequent to the gene therapy treatment.
  • anti-EBOV anti-EBOV
  • anti-EBV anti-EBV
  • anti-HBV anti-CCR4 agents
  • AM001 mogamulizumab
  • VIR-3434 VIR-3434
  • REGN-EB3 mAbll4
  • ZMapp ZMapp
  • compositions and methods are described for the delivery of HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fabs, that bind to Rabies lyssavirus (RBV) and indicated for prevention and/or treatment of patients diagnosed with Rabies.
  • HuPTM mAbs and antigen-binding fragments thereof such as HuPTM Fabs, that bind to Rabies lyssavirus (RBV) and indicated for prevention and/or treatment of patients diagnosed with Rabies.
  • RBV Rabies lyssavirus
  • the HuPTM mAb has the amino acid sequence of CR57 (SEQ ID NOS:388 and 389), CR4098 (SEQ ID NOS:390 and 391), RABI (SEQ ID NOS:392 and 393), RVC20 (SEQ ID NOS: 394 and 395), RVC58 (SEQ ID NOS:396 and 397), SYN023, CTB011, or CTB012, or an antigen binding fragment of the foregoing.
  • Delivery may be accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding an RBV-binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to patients (human subjects) diagnosed with, or having one or more symptoms of Rabies in a human subject to create a permanent depot that continuously supplies the human PTM, e.g., human-glycosylated, transgene product.
  • a viral vector or other DNA expression construct encoding an RBV-binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to patients (human subjects) diagnosed with, or having one or more symptoms of Rabies in a human subject to create a permanent depot that continuously supplies the human PTM, e.g., human-glycosylated, transgene product.
  • transgene encoding a HuPTM mAb or HuPTM Fab (or other antigen binding fragment of the HuPTM mAb) that binds to RBV that can be administered to deliver the HuPTM mAb or antigen binding fragment in a patient.
  • the transgene is a nucleic acid comprising the nucleotide sequences encoding an antigen binding fragment of an antibody that binds to RBV such as CR57, CR4098, RABI, RVC20, RVC58, SYN023, CTB011, or CTB012, or variants thereof as detailed herein.
  • the transgene may also encode a RBV binding fragment that contains additional glycosylation sites (e.g., see Courtois et al.).
  • the anti-RBV antigen-binding fragment transgene comprises the nucleotide sequences encoding the heavy and light chains of the Fab portion of an anti-RBV antibody.
  • the nucleotide sequences may be codon optimized for expression in human cells.
  • the heavy and light chain sequences both have a signal or leader sequence at the N-terminus appropriate for expression and secretion in human cells, in particular, human liver cells (e.g., hepatocytes) or muscle cells.
  • the signal sequence may have the amino acid sequence of MYRMQLLLLIALSLALVTNS (SEQ ID NO:50).
  • the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Tables 2 or 3 that correspond to the proteins secreted by myocytes or hepatocytes, respectively.
  • the transgenes may comprise, at the C-terminus of the heavy chain CHI domain sequence, all or a portion of the hinge region.
  • the anti-RBV antigen binding domain has a heavy chain Fab domain with additional hinge region sequence starting after the C-terminal valine (V), contains all or a portion of the amino acid sequence EPKSCDKTHTCPPCPAPEAAGG (SEQ ID NO:123), and specifically, EPKSCDKTHL (SEQ ID NO:117), EPKSCDKTHT (SEQ ID NO:118), EPKSCDKTHTCPPCPA (SEQ ID NO: 119), EPKSCDKTHLCPPCPA (SEQ ID NO: 120), EPKSCDKTHTCPPCPAPEAAGGPSVFL (SEQ ID NO: 124) or EPKSCDKTHLCPPCPAPEAAGGPSVFL (SEQ ID NO: 125).
  • the transgenes comprise the amino acid sequences encoding the full length (or substantially full length) heavy and light chains of the antibody, comprising the Fc domain at the C terminus of the heavy chain, e.g., having an amino acid sequence of encoding an IgGl Fc domain of SEQ ID NO: 141 (Table 6), or a mutant or variant thereof.
  • the Fc domain may be engineered for altered binding to one or more Fc receptors and/or effector function as disclosed in Section 5.1.9, infra.
  • the anti-RBV-binding fragment transgene encodes an RBV antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS: 389, 391, 393, 395, or 397.
  • the RBV antigen-binding fragment transgene encodes an anti-RBV antigen-binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS:388, 390, 392, 394, or 396.
  • the RBV antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS:389, 391, 393, 395, or 397 and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NOS:374, 376, 378, 380, 382, or 384.
  • the anti-RBV antigen binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NOS: 388, 390, 392, 394, or 396with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, for example, in the framework regions (e.g., those regions outside of the CDRs) or are substitutions with an amino acid present at that position in the heavy chain of one or more of the other therapeutic antibodies, for example, as identified by the alignment in FIG. 3 A.
  • the RBV antigen binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NOS: 389, 391, 393, 395, or 397 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs) or are substitutions with an amino acid present at that position in the light chain of one or more of the other therapeutic antibodies, for example, as identified by the alignment in FIG. 3B.
  • the anti-RBV antigen-binding fragment transgene encodes a hyperglycosylated Fab, comprising a heavy chain and a light chain of SEQ ID NOS: 388, 390, 392, 394, or 396and 389, 391, 393, 395, or 397, respectively, with one or more mutations (see FIGS. 3A (heavy chain) and 3B (light chain)).
  • the anti-RBV anti gen -binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequences encoding the six CDRs which are, for example, underlined in the heavy and light chain variable domain sequences of Table 8, which are spaced between framework regions, generally human framework regions, and associated with constant domains depending upon the form of the antigen-binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-RBV antibody or antigen-binding fragment thereof.
  • AAV vectors comprising a viral capsid that is at least 95% identical to the amino acid sequence of an AAV8 capsid (SEQ ID NO:2), AAVS3 capsid (SEQ ID NO:8) or AAV-LK03 capsid (SEQ ID NO:7); and an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding an anti-RBV mAb, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in human liver or muscle cells.
  • ITRs AAV inverted terminal repeats
  • the regulatory sequence is the ApoE.HAAT regulatory sequence (SEQ ID NO:21) or one of LSPX1, LSPX2, LTP1, LTP2, or LTP3, SEQ ID NOS:9, 10, 11, 12, or 13, respectively).
  • the regulatory sequence is one of the liverspecific CRE sequence selected from Table 16 or a CRE sequence of Table 16 operable linked with a hAAT sequence.
  • the promoter is an LMTP6 promoter (SEQ ID NO: 14).
  • a viral vector containing a transgene encoding an anti-RBV antibody, or antigen binding fragment thereof may be CR57, CR4098, RABI, RVC20, RVC58, SYN023, CTB011, or CTB012, and is, for example, a full length antibody or Fab fragment thereof, or other antigen-binding fragment thereof.
  • the patient has been diagnosed with and/or has symptoms associated with Rabies.
  • Recombinant vector used for delivering the transgene are described in Section 5.1.
  • such vectors should have a tropism for human liver cells and can include non-replicating rAAV, particularly those bearing an AAV8, AAVS3, or AAV- LK03 capsid.
  • the recombinant vectors can be administered in any manner such that the recombinant vector enters the liver or muscle tissue, e.g. by introducing the recombinant vector into the bloodstream. See Section 5.2 for details regarding the methods of treatment.
  • Subjects to whom such gene therapy is administered can be those responsive to anti- RBV therapy.
  • the methods encompass treating patients who have been diagnosed with Rabies, or have one or more symptoms associated therewith, and identified as responsive to treatment with an anti-RBV antibody or considered a good candidate for therapy with an anti-RBV antibody.
  • the patients have previously been treated with CR57, CR4098, RABI, RVC20, RVC58, SYN023, CTB011, or CTB012, and have been found to be responsive to CR57, CR4098, RABI, RVC20, RVC58, SYN023, CTB011, or CTB012.
  • the anti-RBV antibody or antigen-binding fragment transgene product may be administered directly to the subject.
  • the production of the anti-RBV HuPTM mAb or HuPTM Fab should result in a “biobetter” molecule for the treatment of Rabies accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding the anti-RBV HuPTM Fab, subcutaneously, intramuscularly, or intravenously to human subjects (patients) diagnosed with or having one or more symptoms of Rabies, to create a permanent depot in the liver or muscle tissue that continuously supplies the fully-human post-translationally modified, such as human-glycosylated, sulfated transgene product produced by transduced liver or muscle cells.
  • the cDNA construct for the anti-RBV HuPTMmAb or anti-RBV HuPTM Fab should include a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced liver or muscle cells.
  • the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO:50).
  • the signal sequence may have an amino acid sequence selected from any one of the signal sequences set forth in Tables 2 or 3 that correspond to the proteins secreted by myocytes or hepatocytes, respectively.
  • the anti-RBV HuPTM mAb or HuPTM Fab can be produced in human cell lines by recombinant DNA technology, and administered to patients diagnosed with Rabies or for whom therapy or prohylaxis for Rabies.
  • the anti-RBV HuPTM mAb or antigen-binding fragment thereof does not contain detectable NeuGc moieties and/or does not contain detectable alpha-Gal moieties.
  • the HuPTM mAb is a full length or substantially full length mAb with an Fc region.
  • the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1% or 2% glycosylated and/or sulfated and may be at least 5%, 10% or even 50% or 100% glycosylated and/or sulfated.
  • the goal of gene therapy treatment provided herein is to prevent or treat Rabies. Efficacy may be monitored by scoring the Rabies symptoms (e.g. fever, headaches, nausea, vomiting, agitation, anxiety, confusion, hyperactivity, difficulty swallowing, excessive salivation, hallucinations, insomnia, or partial paralysis).
  • Section 5.1 describes recombinant vectors that contain a transgene encoding a HuPTM mAb or HuPTM Fab (or other antigen binding fragment of the HuPTM mAb) that binds to RBV.
  • Therapeutically effective doses of any such recombinant vector should be administered in any manner such that the recombinant vector enters the liver or muscle (e.g., skeletal muscle), e.g. by introducing the recombinant vector into the bloodstream.
  • the vector may be administered directly to the liver through hepatic blood flow, e.g., via the suprahepatic veins or via the hepatic artery.
  • the vector is administered subcutaneously, intramuscularly or intravenously.
  • Intramuscular, subcutaneous, intravenous or hepatic administration should result in expression of the soluble transgene product in cells of the liver or muscle.
  • the vector may be administered directly to the liver through hepatic blood flow, e.g., via the suprahepatic veins or via the hepatic artery.
  • the expression of the transgene encoding an anti-RBV antibody creates a permanent depot in liver and/or muscle of the patient that constinuously supplies the anti-RBV HuPTM mAb, or antigen binding fragment of the anti-RBV mAb to the circulation of the subject.
  • the range of a therapeutically or prophylactically effective amount of an AAV gene therapy vector or pharmaceutical composition is 1E11 to 1E14 genome copies (gc)/kg, preferably between 1E11 to 1E13, and even more preferably 1E12.
  • the dose ranges described herein are exemplary only and do not limit the dose ranges that can be selected.
  • intraveneous administration of an AAV gene therapy vector encoding an anti-RBV antibody results in at least 1.5 pg/mL, 2 pg/mL, 5 pg/mL, 10 pg/mL, or at least 15 pg/mL transgene product expression in human serum at least 30, 40, 50 or 60 days after administration.
  • the target human serum concentration (Cmin) of the transgene product is about 1.5 pg/mL to about 15 pg/mL mAb and associated with a neutralization titer of 500 to 5000.
  • doses that maintain a serum concentration of the anti-RBV antibody transgene product at a Cmin of at least 1.5 pg/mL or at least 15 pg/mL e.g., Cmin of 1.5 to 5 pg/ml, 5 to 10 pg/ml or 10 to 15 pg/mL) at least 30, 40, 50 or 69 days after administration are provided.
  • a dose of IE 11 maintains a serum concentration of the anti-RBV antibody transgene product of at least 1.5 pg/mL.
  • a dose of 1E12 maintains a serum concentration of the anti-RBV antibody transgene product of at least 1.5 pg/mL.
  • doses that maintain a serum concentration of the anti-RBV antibody transgene product reach a neutralization titer of at least 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000 or 5000.
  • the transgene product is continuously produced, maintenance of lower concentrations can be effective. Notwithstanding, because the transgene product is continuously produced, maintenance of lower concentrations can be effective.
  • the concentration of the transgene product can be measured in patient blood serum samples.
  • compositions suitable for intravenous, intramuscular, subcutaneous or hepatic administration comprise a suspension of the recombinant vector comprising the transgene encoding the anti-RBV antibody, or antigen-binding fragment thereof, in a formulation buffer comprising a physiologically compatible aqueous buffer.
  • the formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
  • Combinations of delivery of the anti-RBV HuPTM mAb or antigen-binding fragment thereof, to the liver or muscles accompanied by delivery of other available treatments are encompassed by the methods provided herein.
  • the additional treatments may be administered before, concurrently, or subsequent to the gene therapy treatment.
  • a CC12.1 Fab cDNA-based vector is constructed comprising a transgene comprising nucleotide sequences encoding the Fab portion of the heavy and light chain sequences of CC12.1 (amino acid sequences being SEQ ID NO:305 and 306, respectively).
  • the nucleotide sequence coding for the Fab portion of the heavy and light chain is codon optimized for expression in human liver cells.
  • the transgene also comprises nucleotide sequences that encodes a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO:50).
  • the nucleotide sequences encoding the light chain ad heavy chain are separated by IRES elements or 2A cleavage sites (SEQ ID NOs: 103. 104, 105, or 106) to create a bicistronic vector. See FIG. 2 for amino acid sequence of a transgene product.
  • the vector additionally includes a constitutive promoter, such as CB7, a tissue-specific promoter, such as a liver specific promoter, particularly ApoE.hAAT promoter (SEQ ID NO:21) an inducible promoter, such as a hypoxia-inducible promoter.
  • a CC12.23 Fab cDNA-based vector is constructed comprising a transgene comprising nucleotide sequences encoding the Fab portion of the heavy and light chain sequences of CC 12.23 (amino acid sequences being SEQ ID NOs:307 and 308, respectively).
  • the nucleotide sequence coding for the Fab portion of the heavy and light chain is codon optimized for expression in human liver cells.
  • the transgene also comprises nucleotide sequences that encodes a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO:50).
  • the nucleotide sequences encoding the light chain ad heavy chain are separated by IRES elements or 2A cleavage sites (SEQ ID NOS: 103, 104, 105, or 106) to create a bicistronic vector.
  • the vector additionally includes a constitutive promoter, such as CB7, a tissue-specific promoter, such as a liver specific promoter, particularly ApoE.hAAT promoter (SEQ ID NO:21) an inducible promoter, such as a hypoxia-inducible promoter.
  • a constitutive promoter such as CB7
  • a tissue-specific promoter such as a liver specific promoter, particularly ApoE.hAAT promoter (SEQ ID NO:21)
  • an inducible promoter such as a hypoxia-inducible promoter.
  • EXAMPLE 3 Lanadelumab Fab cDNA-Based Vector
  • a lanadelumab Fab cDNA-based vector comprising a transgene comprising nucleotide sequences encoding the Fab portion of the heavy and light chain sequences of lanadelumab (amino acid sequences being SEQ ID NOs. 144 and 145, respectively).
  • the nucleotide sequence coding for the Fab portion of the heavy and light chain may be the nucleotide sequence of SEQ ID NOs: 146 and 147, respectively.
  • the transgene also comprises nucleotide sequences that encodes a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO:50).
  • the nucleotide sequences encoding the light chain and heavy chain are separated by IRES elements or 2A cleavage sites (SEQ ID NOs: 103-106) to create a bicistronic vector.
  • the vector additionally includes a constitutive promoter, such as CB7, a tissue-specific promoter, such as a liver specific promoter, particularly ApoE.hAAT promoter (SEQ ID NO:21) an inducible promoter, such as a hypoxiainducible promoter.
  • a lanadelumab cDNA-based vector was constructed comprising a transgene comprising a nucleotide sequence encoding the heavy and light chain sequences of lanadelumab (amino acid sequences being SEQ ID NOs: 144 and 145, respectively).
  • the nucleotide sequence coding for the heavy and light chain of lanadelumab was codon optimized to generate the three nucleotide sequences provided in Table 9 below, L01 (SEQ ID NO: 148), L02 (SEQ ID NO: 149), and L03 (SEQ ID NO: 150). L02 and L03 also have reduced incidence of CpG dimers in the sequence.
  • the transgene also comprised a nucleotide sequence that encodes the signal peptide MYRMQLLLLIALSLALVTNS (SEQ ID NO:50).
  • the nucleotide sequences encoding the light chain and heavy chain were separated by a Furin-F2A linker (SEQ ID NOS: 105 or 106 or a Furin T2A linker (SEQ ID NOS: 103 or 104) to create a bicistronic vector.
  • the vector additionally included a constitutive CAG promoter (SEQ ID NO:36). See FIG. 7Afor a schematic showing the genomic configuration and sequences of the constructs are provided in Table 9 (SEQ ID NOS: 151-159).
  • Table 1 and Table 16 provide the sequences of composite nucleic acid regulatory sequences that may be incorporated into expression cassettes and be operably linked to the transgene to promote liver-specific expression (LSPX1, LSPX2, LTP1, LTP2, or LTP3, SEQ ID NOS:9-13, respectively, sequences of Table 16) and liver and muscle expression (LMTP6, LMTP13, LMTP15, LMTP18, LMTP19 or LMTP20, SEQ ID NOS: 14-20 respectively).
  • promoter sequences provided include the ApoE.hAAT (SEQ ID NO:21, Table 1 above) promoter, wherein four copies of the liver-specific apolipoprotein E (ApoE) enhancer were placed upstream of the human alpha 1- antitrypsin (hAAT) promoter.). .).
  • a promoter sequence can include a CRE sequence selected from Table 16 upstream of a hAAT promoter, such as four copies of a liver-specific CRE selected from Table 16 placed upstream of the human alpha 1 -antitrypsin (hAAT) promoter.).
  • HEK293 cells were plated at a density of 7.5xl0 5 cells/well in each well of a standard 6-well dish containing Dulbecco’s modified eagle medium (DMEM) supplied with 10% fetal bovine serum (FBS). The next day, cells were transfected with CAG.L01 (SEQ ID NO: 148), CAG.L02 (SEQ ID NO: 149), and CAG.L03 (SEQ ID NO: 150) AAV constructs using Lifpofectamine 2000 (Invitrogen) according the manufacturer’s protocol). Non-transfected cells were used as negative control. Cell culture medium was changed 24 hours post-transfection to opti-mem I reduced serum media (2 mL/well).
  • DMEM Dulbecco’s modified eagle medium
  • FBS fetal bovine serum
  • Cell culture supernatant was harvested at 48 hours post-transfection, and cell lysates were harvested with RIPA buffer (Pierce) supplemented with EDTA-free protease inhibitor tablets (Pierce). Supernatant and lysates samples were stored at -80C.
  • Proteins from supernatant or cell lysate samples were separated via the NuPAGE electrophoresis system (Thermo Fisher Scientific). For samples derived from cell lysates, 40 pg of protein was loaded unless indicated otherwise. Purified human IgG or Lanadelumab IgG (produced by Genscript) were used as loading controls (50-100 ng). Samples were heated with LDS sample buffer and NuPAGE reducing agent at 70C for 10 minutes and then loaded into NuPAGE 4-12% Bis-Tris protein gels. Separated proteins were transferred to PVDF membranes using the iBlot2 dry blotting system according to manufacturer’s instructions (P3 default setting was used for the protein transfer).
  • Membranes were immediately washed in phosphate buffer saline with 0.1% v/v Tween-20 (PBST). Membranes were then incubated in blocking solution containing PBST and 1% Clear Milk Blocking Buffer (Thermo Scientific) for 1 hour at room temperature. Membranes were then incubated in fresh blocking solution supplemented with goat anti-human kappa light chain-HRP antibody (Bethyl Laboratories; 1 :2000 dilution) and goat anti-human IgG Fc-HRP antibody (1 :2000 dilution). Following antibody incubation, membranes were washed three times in PBST for 5 minutes per wash. Finally, membranes were incubated in SuperSignal West Pico PLUS chemiluminescent substrate for 5 minutes and imaged on the BioRad Universal Hood II gel doc system for detection of horseradish peroxidase (HRP) signal.
  • HRP horseradish peroxidase
  • AAV8 or AAV9 containing an AAV construct comprising the L01 sequence (SEQ ID NO: 148), which contains the Furin and F2A sequence (SEQ ID NO: 106).
  • ELISA enzyme-linked immunosorbent assay
  • mice serum was obtained before treatment and at 1, 3, 5 and 7 weeks post in vivo gene transfection and stored at -80°C.
  • 96-well plate was coated with 1 pg/ml human IgG-Fc fragment antibody (Bethyl, Montgomery, TX) in carbonate bicarbonate buffer (0.05M, pH 9.6, Sigma-Aldrich, St. Louis, MO) and incubated overnight at 4°C.
  • Tween 20 washing buffer PBST, 0.05%, Alfa Aesar, Haverhill, MA
  • blocking buffer 3% BSA in PBS, ThermoFisher Scientific, Waltham, MA
  • Mouse serum samples diluted in sample dilution buffer (0.1% Tween 20 and 3% BSA in PBS) was added to the plate (50pl/well) and incubated for 2 h at 37°C.
  • a standard curve of known lanadelumab concentrations ranging from 360 to 0.001 ng/mL was included in each plate. Plate was washed with PBST for five times after incubation.
  • the levels of lanadelumab was detected by incubation with horseradish peroxidase- conjugated goat anti-human IgG (H+L) (200 ng/mL; Bethyl, Montgomery, TX) for 1 h at 37°C.
  • the optical density was assessed using KPL TMB Microwell Peroxidase Substrate System (Seracare, Milford, MA) following the manufacturer’s specifications. Data analysis was performed with SoftMax Pro version 7.0.2 software (Molecular Devices, Sunnyvale, CA).
  • A. Results from a representative experiment are shown in FIG. 8. Serum analysis of AAV8-, AAV9-injected and control (vehicle) NSG mice at 7 weeks post gene transfer showed expression and serum accumulation of Lanadelumab following AAV9 delivery (2E 11 gc). Serum Lanadelumab concentration was 100-fold higher in AAV9-injected mice compared to AAV8-injected mice and slightly higher in IV-AAV9-injected compared to IM-AAV9-injected mice. Serum human antibody levels in control mice were undetectable at 7 week time point. [0382] B. In an analogous experiment, a time course of lanadelumab serum levels in NSG mice post-AAV9 administration (n 5 per group) was performed.
  • AAV9 vectors (2E 11 gc) were injected either IV or IM (as above, in experiment A), and serum antibody levels were determined by ELISA at day 7 (D7), day 21 (D21), day 35 (D35), and day 49 (D49).
  • Serum Lanadelumab expression is detectable as early as 1 week (D7) after AAV9 administration in NSG mice. The expression levels increased at 3 weeks (D2), peaked at 5 weeks (D35) and then sustained up to 7 week post-injection (D49). It was observed that serum lanadelumab concentration is higher in IV vs. IM injected mice over the entire time course. See FIG. 9.
  • EXAMPLE 6 Analysis of in vitro Transduction and Expression of Tandem Liver- and Tandem Liver/Muscle-Specific Promoters Driving Expression of Lanadelumab
  • Cis plasmids expressing vectorized lanadelumab were packaged in AAV, then rAAV particles evaluated for potency of the transduction by AAV.
  • Each cis plasmid contained lanadelumab (Mabl) antibody light chain and heavy chain which are multicistrons driven by the CAG, ApoE.hAAT (SEQ ID NO:21) or LMTP6 (SEQ ID NO: 14) promoter.
  • Full-length lanadelumab antibody light chain and antibody heavy chain genes were separated by a furin 2A linker to ensure separate expression of each antibody chain.
  • the entire cassette is flanked by AAV2 ITRs, and the genome is encapsidated in an AAV8 capsid for delivery to C2C12 cells (IE 10 vg per well).
  • the cells are treated with FITC conjugated anti-Fc (IgG) antibody.
  • the AAV8.CAG.Mabl and AAV8.LMTP6.Mabl infected cells show high expression in muscle cells, whereas the AAV8.hAAT.Mabl infection does not result in expression of the antibody in muscle cells (FIG. 10).
  • Cells appeared to be equally confluent and viable in all test wells, as seen by DAPI (DNA) staining (FIG. 10).
  • EXAMPLE 7 Antibody Expression And Vector Biodistribution In Mouse Treated With AAV8 Lanadelumab Vectors Driven By Various Promoters
  • Thyroxine binding globulin (TBG) and alpha- 1 antitrypsin (hAAT) promoters have been widely used as liver-specific promoters in previous pre-clinical and clinical gene therapy studies.
  • a panel of designed promoter cassettes derived from multiple promoters and enhancers were generated and tested them in vitro by transfecting Huh7 cells, a human liver cell line.
  • Promoter candidates were selected, which include ApoE.hAAT (SEQ ID NO:21), LSPX1 (SEQ ID NO:9), LSPX2 (SEQ ID NO: 10), LTP1 (SEQ ID NO: 11) and LMTP6 (SEQ ID NO: 14).
  • AAV8 vectors encoding vectorized lanadelumab regulated by these promoter candidates were then generated.
  • CAG (SEQ ID NO: 36) and TBG (SEQ ID NO:40) promoters served as controls for ubiquitous and liver-specific promoters, respectfully. Strength of these promoters and vector biodistribution were tested in vivo by measuring lanadelumab protein expression compared to vector genome copy in each wild type mouse.
  • Vectors were administered intravenously to C57B1/6 mice at equivalent doses (2.5xl0 12 vg/kg).
  • Mouse serum was collected biweekly, and lanadelumab protein expression levels were determined by ELISA.
  • Liver samples were harvested at 49 days post vector administration.
  • the presence of viral genomes in each sample was quantified using Lanadelumab probe and primer by Droplet Digital PCR (ddPCR) (the NAICATM system from Stilla).
  • ddPCR Droplet Digital PCR
  • the genome copy number of glucagon was also measured simultaneously in each sample, the viral genomes were then normalized and demonstrated as vector genome copy number per cell (assuming 2 glucagon/cell).
  • Statistical analysis was performed using one-way ANOVA in GraphPad Prism 8.
  • EXAMPLE 8 Lanadelumab expression in rat serum following administration of vectorized antibody
  • the levels of antibody in rat serum were detectable at 7 days post treatment. It increased over time and reached the peak level at 17 (lower dose) and 21 (higher dose) days post treatment in IV groups and 28 days in IM group. The antibody levels gradually decreased and sustains up to 48 days post treatment in all groups. For animals treated with lower dose (IxlO 13 vg/kg) vector, the antibody expression levels in IV groups are significantly higher than that in IM group at 7, 14 and 21 days post vector administration. For animals received IV administration, the antibody expression levels were dose-dependent at all time points. The highest level of lanadelumab expression was 252.6 ⁇ 149.4 pg/ml, which was detected in animals treated with higher dose (1 xlO 14 vg/kg) at 21 days post IV administration. See FIG. 12A.
  • the aim of this experiment was to investigate the rat strain and the vector dose that will be used for a rat efficacy study.
  • Vectors were administered via IV injection at a dose of 5xl0 13 vg/kg.
  • the highest antibody levels were 173.1 ⁇ 78.8 pg/ml and 109.57 ⁇ 18.9 pg/ml at 35 and 49 days respectively in control CAG- Lanadelumab and hAAT-Lanadelumab vector-treated animals. In SD rats, however, the levels of antibody reached peaks at 14 and 21 days in control and lead vector-treated animals, respectively, and decreased gradually afterward in both groups.
  • the highest antibody concentrations were 48.23 ⁇ 3.1 pg/ml and 22.33 ⁇ 8.98 pg/ml in CAG.L02 (SEQ ID NO: 153) and ApoE.hAAT.L02 (SEQ ID NO: 155) vector groups, respectively. See Table 13 and FIG. 12B.
  • EXAMPLE 9 Characterization of vectorized Lanadelumab regulated by tissue-specific promoters following intramuscular administration
  • Vectors regulated by the hAAT and LMTP6 promoters demonstrated significantly increased antibody concentrations in serum compared to CAG at all time points (FIG. 13 A).
  • the hAAT and LMTP6 were not significantly different from each other in this experiment.
  • Vector genome copies per cell of vectorized lanadelumab was detected and quantified in GA, liver and heart (FIG. 13B) with a notable difference of higher quantity of genome detected in heart for the dual muscle/liver promoter, LMTP6 vector.
  • Increased liver RNA expression was also detected for all test vectors directly injected into GA muscle at 49 days (relative fold gene expression compared to a reference gene) (FIG. 13C).
  • EXAMPLE 11 Vectorized human anti-pKal antibody, Lanadelumab, derived from mouse serum suppressed human pKal function
  • the signal-to-noise ratio for each pKal concentration RFU (last RFU fluorescent value chosen) was calculated by dividing its RFU by background PFR-AMC substrate fluorescence.
  • the two lowest pKal concentrations with a signal-to-noise ratio > 2 (6.25nM and 12.5nM) were then chosen to evaluate the suppressive effect and range of lanadelumab antibody of pKal function in a lanadelumab dose response.
  • Lanadelumab (GenScript) or human IgG control antibody was diluted in SDB to top concentration of 200nM and two-fold serially diluted to 0.39nM.
  • 25pL pKal (each of two chosen concentrations) was incubated with 25 pL lanadelumab or human IgG at 30°C for 1 hour.
  • Antibody-pKal mixture was then given PFR-AMC and immediately run in kinetic mode for AMC fluorescence at excitation/ emission wavelengths of 380/460 nm, respectively, for 3 hours using a SpectraMax fluorescent plate reader.
  • mouse serum was diluted in sample dilution buffer and incubated 1 : 1 with 6.25nM (1.56nM in-well) pKal for 30°C/l hour.
  • AMC standard curve was generated by a two-fold downward dilution series of AMC (500nM, eleven dilutions and blank subtracted) diluted in assay buffer.
  • AMC was read as end point fluorescence at excitation/ emission wavelengths of 380/460 nm, respectively.
  • Specific plasma kallikrein activity was calculated as: (adjusted experimental sample Vmax, RFU/sec) x (Conversion factor, AMC standard curve pM/RFU)/ (pKal concentration, nM). Percent reduction in pKal activity was derived from calculating day 49 by day -7 pKal activity.
  • the assay showed noticeable lanadelumab-mediated suppression of pKal activity down to 0.1 nM (in-well concentration) (FIG. 15C) at a defined enzyme concentration.
  • Serum from mice 49 days post-administration was diluted 1 :25 (in range predicted to be suppressive), incubated with pKal in vitro, and pKal activity was assayed.
  • Example 12 A Effects of AAV-Lanadelumab in the carrageenan paw edema model in
  • Carrageenan is a strong chemical agent that functions in stimulating the release of inflammatory and proinfl ammatory mediators, including bradykinin, histamine, tachykinins, reactive oxygen, and nitrogen species.
  • Typical signs of inflammation include edema, hyperalgesia, and erythema, which develop immediately following the treatment of carrageenan. This example evaluated the effect of AAV-mediated gene delivery of Lanadelumab on carrageenan-induced paw edema in mice.
  • Vector 1 AAV8-GFP
  • ApoE.hAAT.L02 SEQ ID NO: 155 treatment significantly reduced the paw volume at 2, 4, 6 and 8 hours post carrageenan injection in 1.0% Cg model when compared with the vehicle control group (group 1, vector formulation buffer) (FIG. 17A and 17B).
  • group 1, vector formulation buffer group 1, vector formulation buffer
  • These vectors differ in their promoter sequences which includes: a) a ubiquitous CAG promoter (SEQ ID NO:36) b) the liver-specific hAAT promoter with upstream ApoE enhancer (SEQ ID NO:21) c), the muscle-specific CK8 promoter cassette composed of the CK core promoter and three copies of a modified MCK enhancer (SEQ ID NO: 37), and d) liver-muscle tandem promoter 6 (LMTP6, SEQ ID NO: 14) that contains sequence elements derived from hAAT and CK8.
  • IV intravenous
  • Study endpoints will include characterization of humoral and cell-mediated immune responses against the mOVA transgene product.
  • tissues will be harvested for vector biodistribution and transgene expression analysis.
  • EXAMPLE 14 Plasma expression of Vectorized Lanadelumab in Cynomolgus Monkeys Methods
  • Plasma kinetics of lanadelumab expression in non-human primates administered AAV vectors encoding lanadelumab antibodies were assessed.
  • the goal of this study was to assess and select the dose of AAV8.ApoE.hAAT.Lan vector that results in sustained lanadelumab expression of at least 200 pg/ml lanadelumab by three months or more.
  • the cynomolgus monkey were chosen as the test system because of its established usefulness and acceptance as a model for AAV biodistribution studies in a large animal species and for further translation to human. All animals on this study were naive with respect to prior treatment.
  • Clinical signs were recorded at least once daily beginning approximately two weeks prior to initiation of dosing and continuing throughout the study period. The animals were observed for signs of clinical effects, illness, and/or death. Additional observations were recorded based upon the condition of the animal at the discretion of the Study Director and/or technicians.
  • Blood samples were collected from a peripheral vein for bioanalytical analysis prior to dose administration and then at weekly intervals for 10 weeks. The samples were collected in clot tubes and the times were recorded. The tubes were maintained at room temperature until fully clotted, then centrifuged at approximately 2400 rpm at room temperature for 15 minutes. The serum was harvested, placed in labeled vials, frozen in liquid nitrogen, and stored at -60°C or below.
  • plasma samples were assayed for lanadelumab concentration by ELISA and/or western blot, to be reported at least as pg lanadelumab per ml plasma; and lanadelumab activity, for example, kallikrein inhibition, by fluorogenic assay.
  • the optimized expression cassette containing a liver-specific promoter and a codon optimized and CpG depleted transgene with a modified furin-T2A processing signal resulted in dose- depedent serum antibody concentrations when delivered intravenously using an AAV8 vector.
  • Sustained levels of functional anti-kallikrein antibody were achieved in the serum of 7 out of 9 cynomolgus monkeys following IV vector administration at all three doses (1E12 gc/kg, 1E13 gc/kg, and 1E14 gc/kg) (FIG. 18).
  • Functional anti-kallikrein antibody was detected in the serum of all animals regardless of the administered dose.
  • NUNC Maxisorp plates (Thermo Scientific) will be coated with equimolar antigen amounts at 4 °C overnight. Plates will be washed three times with PBS containing 0.05% Tween-20 and blocked with 3% bovine serum albumin (Bio-Connect) in PBS containing 0.1% Tween-20 at room temperature for 2 hours. Four-fold serial dilutions of mAbs starting at 10 pg/ml (diluted in blocking buffer) will be added and plates will be incubated for 1 hour at room temperature.
  • HRP horseradish peroxidase
  • ITK Southern Biotech horseradish peroxidase-conjugated goat anti-human secondary antibody
  • HRP activity will be measured at 450 nanometer using tetramethylbenzidine substrate (BioFX) and an ELISA plate reader (EL-808, Biotek).
  • Halfmaximum effective concentration (EC50) binding values will be calculated by non-linear regression analysis on the binding curves.
  • EXAMPLE 16 In vitro Neutralization Assay using VSV based Pseudoparticles
  • vero cells (ATCC: CCL-81) will be seeded in 96-well black, clear bottom tissue culture treated plated (Coming: 3904) at 20,000 cells/well in DMEM high glucose media containing 10% heat- inactivated fetal bovine serum, and IX Penicillin/Streptomycin/L-Glutamine 24 hours prior to assay. Cells will be allowed to reach approximately 85% confluence before use in assay.
  • Antibodies will be diluted in DMEM high glucose media containing 0.7% Low IgG BSA (Sigma), IX Sodium Pyruvate, and 0.5% Gentamicin (this will be referred to as “Infection Media”) to 2X assay concentration and diluted 3-fold down in Infection media, for an 11-point dilution curve in the assay beginning at 10 ug/mL (66.67 nM).
  • pVSV-SARS-CoV-2-S (mNeon) will be diluted 1 : 1 in Infection media for a fluorescent focus (ffu) count in the assay of -1000 ffu.
  • Antibody dilutions will be mixed 1 :1 with pseudoparticles for 30 minutes at room temperature prior to addition onto Vero cells. Cells will be incubated at 37C, 5% CO2 for 24 hours. Supernatant will be removed from cells and replaced with 100 uL PBS, and fluorescent foci will be quantitated using the SpectraMax i3 plate reader with MiniMax imaging cytometer. Exported values will be analyzed using GraphPad Prism (v8.2.0).
  • VSV-SARS-CoV-2-S virus neutralization assays antibodies will be diluted as described in Example 16 but in VSV media (DMEM high glucose media containing 3% heat-inactivated fetal bovine serum and Penicillin/-Streptomycin-L-Glutamine). An equal volume of media containing 2000 pfu of VSV-SARS-CoV-2-S virus will be mixed with the antibody incubated for 30 minutes at room temperature. The mixture will then be added onto Vero cells and incubated at 37C, 5% CCh for 24 hours.
  • VSV media DMEM high glucose media containing 3% heat-inactivated fetal bovine serum and Penicillin/-Streptomycin-L-Glutamine.
  • An equal volume of media containing 2000 pfu of VSV-SARS-CoV-2-S virus will be mixed with the antibody incubated for 30 minutes at room temperature. The mixture will then be added onto Vero cells and incubated at 37C, 5% CCh for 24 hours.
  • the cells will be fixed (PBS with 2% paraformaldehyde) for 20 minutes, permeabilized (PBS with 5% fetal bovine serum and 0.1% Triton-XlOO) for 15 minutes and blocked (PBS with 3% bovine serum albumin) for 1 hour.
  • Infected cells will be immunostained with a rabbit anti-VSV serum (Imanis Life Sciences) and an Alexa Fluor® 488 secondary antibody in PBS + 3% bovine serum albumin. Fluorescent foci will be quantitated using the SpectraMax i3 plate reader with MiniMax imaging cytometer.
  • IC50 values (M) will be calculated using the following formula: (IgG concentration mg/mL / IgG molecular weight Da) / pVNT50 titer.
  • EXAMPLE 17 In vitro Neutralization Assay using live replicating virus (Plaque assay)
  • Monoclonal antibodies and antibody combinations will be serially diluted in DMEM (Quality Biological), supplemented with 10% (v/v) heat inactivated fetal bovine serum (Sigma), 1% (v/v) penicillin/streptomycin (Gemini Bio-products) and 1% (v/v) L-glutamine (2 mM final concentration, Gibco) (VeroE6 media) to a final volume of 250 pL.
  • 250 pL of VeroE6 media containing SARS-CoV-2 (WA-1) 1000 PFU/mL
  • the virus-antibody mixtures will be incubated for 60 min at 37°C.
  • the levels of neutralizing antibodies will be determined by a SARS-CoV-2 spike protein pseudotyped vesicular stomatitis virus (pVSV)-based neutralization test (pVNT).
  • pVSV pseudotyped vesicular stomatitis virus
  • pVNT pseudotyped vesicular stomatitis virus
  • This pVNT relies on the use of replication-defective VSV particles carrying SARS-CoV-2 S protein to reflect the entry of SARS-CoV-2 into host cells. Particles carrying the G-protein of VSV will be used as a control.
  • VSV particles will be produced by calcium-phosphate transfecting HEK293T cells with expression plasmids for the respective glycoproteins (SARS-2-SA18 for the SARS-CoV2 spike protein). 18 hours later the cells will be infected with VSV* AG-FLuc, a replication deficient recombinant VSV in which the VSV-G open reading frame has been replaced by combined GFP and firefly luciferase expression cassettes. After incubating the transduced cells with the viral particles for 2 hours at 37°C the supernatant will be removed and the cells will be washed twice with PBS.
  • VSV-G supernatant from mouse hybridoma CRL-2700; ATCC
  • the pseudotype particle containing supernatant will be separated from the cells by centrifugation and used for the neutralization assays.
  • Vero76 cells will be seeded at 1 x 10 4 cells per well in 96-well plates. The next day the complement in test sera will be inactivated by heating samples to 56°C for 30 min. Sera will then be serially diluted, mixed 1 :2 with the pseudotyped VSV and incubated for 30 minutes at 37°C.
  • samples will be heat-inactivated for 30 min at 56°C and serially diluted in 96-well plates starting from a dilution of 1 :8. Samples will be incubated for 1 h at 37°C together with 100 50% tissue culture infective doses (TCID50) SARS-CoV-2. Cytopathic effect on Vero E6 cells (ATCC, Cat#CRL-1586) will be analyzed 4 days post-infection. Neutralization is defined as absence of cytopathic effect compared to virus controls. For each test, a positive control (e.g. neutralizing COVID-19 patient plasma) will be used in duplicates as an inter-assay neutralization standard.
  • TCID50 tissue culture infective doses
  • Binding kinetics and affinities for anti-spike mAbs will be assessed using surface plasmon resonance technology on a Biacore T200 instrument (GE Healthcare, Marlborough, MA) using a Series S CM5 sensor chip in filtered and degassed HBS-EP running buffer (10 mM HEPES, 150 mM NaCl, 3mM EDTA, 0.05% (v/v) polysorbate 20, pH 7.4).
  • a capture sensor surfaces will be prepared by covalently immobilizing with a mouse anti-human Fc mAb on to the chip surface using the standard amine coupling chemistry. Following surface activation, the remaining active carboxyl groups on the CM5 chip surface will be later blocked by injecting IM ethanolamine, pH8.0 for 7 minutes.
  • RBD.mmh 3.33nM - 90nM, three-fold serial dilution
  • RBD.mFc 1.1 InM - 30nM, three-fold serial dilution
  • SARS-CoV2 Spike ECD foldon 0.78nM - 25nM, three-fold serial dilution
  • All of the specific SPR binding sensorgrams will be double-reference subtracted and the kinetic parameters will be obtained by globally fitting the double-reference subtracted data to a 1 : 1 binding model with mass transport limitation using Biacore T200 Evaluation software v 3.1 (GE Healthcare) or Biacore Insight Evaluation software (GE Healthcare).
  • the dissociation rate constant (kd) will be determined by fitting the change in the binding response during the dissociation phase and the association rate constant (ka) will be determined by globally fitting analyte binding at different concentrations.
  • the equilibrium dissociation constant (KD) will be calculated from the ratio of the kd and ka.
  • the dissociative half-life (t 1 A) in minutes will be calculated as ln2/(kd*60).
  • Antibodies displaying dissociation constant values (Kd) of 0.5 to 50 nM (monomeric SARS-CoV-2 RBD), 5 to 50 pM (dimeric SARS-CoV-2 RBD), and 30 to 50 pM (timeric SARS-CoV- 2 spik) are considered specific anti-SARS-CoV-2 antibodies with a high affinity to monomeric, dimeric SARS-CoV-2 RBD and trimeric SARS-CoV-2 spike.
  • listed antibodies will be captured to -50-100 RU via Fc-capture on the active flow cell prior to analyte injection.
  • a concentration series of SARS-CoV-2 RBD will be injected across the antibody and control surface for 2 min, followed by a 5 min dissociation phase using a multi-cycle method. Regeneration of the surface in between inj ections of SARS-CoV-2 RBD will be achieved with a single, 120 s injection of 3M MgC12.
  • Kinetic analysis of each reference subtracted injection series will be performed using the BIAEvaluation software (Cytiva). All sensorgram series will be fit to a 1 : 1 (Langmuir) binding model of interaction.
  • a SPR assay can also be used to assess the competition between SARS-CoV-2 RBD and ACE2 for binding to an anti-SARS-CoV-2 antibody, e.g. CC12.1.
  • the anti-SARS-CoV-2 antibody will be captured to the surface of 3 flow cells to -100 RU via Fc-capture.
  • SARS-CoV-2 RBD will be injected to each flow cell at a concentration of 50 nM to establish a basal level of SARS-CoV-2 RBD binding. This concentration will be held constant for the competition experiments, which will be carried out by varying the ACE2 concentration over eight points from 800 to 6.25 nM.
  • the sensorgram responding to the corresponding ACE2 injection alone will be subtracted from the SARS-CoV-2 RBD plus ACE2 injection series.
  • the average response for the 5s preceding the injection stop will be plotted against the concentration of ACE2 and fit to a dose-response inhibition curve by nonlinear regression [log(inhibitor) vs. response - variable slope (4 parameters)] using GraphPad Prism. Regeneration between injections was carried out as noted above.
  • Cis-regulatory elements are non-coding regions of DNA that regulate transcription of proximal or distal gene regions. Based on the CREs’ specific function, CREs may be further classified as promoters, enhancers, and silencers. Putative CREs are typically identified based upon structural features such as chromatin accessibility. In addition, these regions may be characterized via density of epigenetic marks commonly associated with high transcriptional activity. In this work, genomic locations for candidate CREs found proximal to genes that are highly expressed and specific to liver were obtained from the ENCODE database (www. encodeproi ect.or /) .
  • Sequences were isolated from the current human genome assembly (GRCh38) using the NCBI Gene portal (www.ncbi.nlm.nih. ov/ ene/). These sequences were synthesized and cloned into AAV reporter cassettes upstream of the liver-specific hAAT promoter. In addition, each construct contained a unique 10-bp DNA barcode between the eGFP coding sequence and polyadenylation signal to allow characterization of transgene expression using next generation sequencing. Cis plasmids containing individual CREs were pooled and produced into an AAV8 vector library.
  • FIG. 19 is a schematic of the cassette construct used in the screening study.
  • Each cassette is flanked by the canonical AAV2 inverted terminal repeats (ITRs).
  • the promoter region is composed of the liver-specific hAAT promoter coupled with a Vh4 intron. Upstream of the hAAT promoter is one of the individual CRE candidate sequence (Table 16).
  • a unique 10 basepair DNA barcode is placed between the eGFP coding sequence and rabbit beta globin (RBG) polyadenylation signal inorder to identify which cassette was expressing the eGFP.
  • RBG beta globin
  • Cis plasmid library containing a mixture of up to 55 cassettes was transfected with rep2/cap8 and helper plasmids, and thus packaged in AAV8, resulting in a pool concentration of 1.69el3 vg/mL. Barcodes were identified for even distribution within the pool. [0439] The pool was then applied to Huh7 cells (expressing AAVR) to allow for transduction. The cells were then harvested.
  • a sample of the pool was also administered systemically to C57B16 mice (3 dose groups, 5 mice/group). Study animals were euthanized, and tissues were collected.
  • the AAV8 Liv-CRE vector library will also be produced in a manufacturing process at 2L scale so that material can be administered systemically to two non-human primates for evaluation of expression of each vector in the pool.

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