WO2022072829A1 - Compositions pour une immunisation passive administrée par voie intranasale contre des pathogènes en suspension dans l'air - Google Patents

Compositions pour une immunisation passive administrée par voie intranasale contre des pathogènes en suspension dans l'air Download PDF

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Publication number
WO2022072829A1
WO2022072829A1 PCT/US2021/053169 US2021053169W WO2022072829A1 WO 2022072829 A1 WO2022072829 A1 WO 2022072829A1 US 2021053169 W US2021053169 W US 2021053169W WO 2022072829 A1 WO2022072829 A1 WO 2022072829A1
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antibody
csr
sars
recombinant
regimen
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PCT/US2021/053169
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English (en)
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James M. Wilson
Christian HINDERER
Makoto Horiuchi
Joshua Joyner SIMS
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The Trustees Of The University Ofpennsylvania
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    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • AAVs are nonpathogenic parvoviruses that circulate broadly in humans and other species. Replacing all viral coding sequences with a gene of interest yields an AAV vector capable of efficient in vivo gene delivery without the insertional mutagenesis risks or robust inflammatory responses observed with lentiviral or adenoviral vectors.
  • AAV vectors have demonstrated an acceptable safety profile in thousands of human subjects, and two products are now approved in the US with dozens more in late-stage clinical development. The stability of AAV vectors make them practical for widespread distribution as prophylactic vaccines.
  • SARS-CoV-2 coronavirus "SARS-CoV-2” outbreak that emerged in China and has rapidly spread across the world has been declared a public health emergency of international concern by the World Health Organization (WHO). Efforts are ongoing to discover effective therapeutic and prophylactic agents that will help to mitigate the spread of the disease.
  • ACE2 angiotensin-converting enzyme 2
  • Soluble ACE2 has been administered intravenously to healthy volunteers and demonstrated an acceptable safety and immunogenicity profile.
  • a pilot efficacy study with soluble human recombinant ACE2 is ongoing in patients with COVID- 19 (Clinicaltrials.gov #NCT04287686).
  • compositions and methods for preventing one or more COVID- 19 symptoms and/or infection are a variety of needs in this area, including compositions and methods for preventing one or more COVID- 19 symptoms and/or infection, and/or methods for reducing viral replication following infection.
  • a passive immunization regimen for delivering at least one antibody intranasally to a patient in need thereof for protecting against infection with a selected airborne pathogen, e.g., SARSCoV-2 or influenza.
  • the composition comprising the antibody may be formulated for aerosolization and delivery to the nasal epithelium and the lung.
  • the at least one antibody construct is a class switched recombinant (CSR) antibody which is an immunoglobulin A or immunoglobin M isotype antibody which neutralizes the target airborne pathogen and/or an scFv which neutralizes the target airborne pathogen.
  • CSR class switched recombinant
  • the composition comprises at least one recombinant vector comprising a nucleic acid sequence encoding the at least one CSR antibody under control of expression control sequences directing its expression.
  • the recombinant vector is independently selected from one or more of a recombinant, replication-defective, adeno-associated virus (rAAV), a recombinant, replication-defective, adenovirus, a recombinant, replication-defective, herpes simplex virus, a recombinant, replication-defective, vaccinia virus, or a recombinant, replication-defective, lentivirus.
  • rAAV adeno-associated virus
  • the composition comprises at least one rAAV having an AAV capsid and a vector genome encoding at least one CSR Mab and/or an scFv.
  • the at least rAAV capsid is selected from AAV9, AAVhu68, AAV5, or AAV6.
  • composition neutralizes more than one target airborne pathogen or more than one subtype of the target airborne pathogen.
  • the target airborne pathogen is a virus.
  • the virus is selected from SARS-CoV2, SARS-CoV, influenza, or Ebola virus.
  • two or more different anti-SARS-CoV2 antibodies are provided in a composition suitable for intranasal administration.
  • the composition is free of IgG4 and/or free of any IgG isotype heavy chain regions.
  • a composition is provided which comprises at least one CSR antibody and at least a second, different neutralizing antibody construct.
  • a composition is provided which comprises at least a bifunctional antibody which binds to at least SARS-CoV2 and is other than an IgG antibody.
  • a method for preventing SARS-CoV-2 infection comprising delivering anti-SARS-CoV2 immunoglobulin A proteins and/or immunoglobulin M protein to a human subject in an amount effective to prevent or reduce anti-SARS-CoV2 infection.
  • the method may comprise administering at least one stock of recombinant adeno- associated virus (rAAV) which expresses the anti-SARS-CoV2 immunoglobulin A and/or immunoglobulin M in a nasal epithelial and/or lung cells.
  • rAAV recombinant adeno- associated virus
  • the rAAV are in a composition comprising at least one stock of rAAV expressing two or more different anti-SARS-CoV2 immunoglobulins, wherein the composition does not comprise any IgG immunoglobulin.
  • the method comprises co-delivering two or more separate compositions comprising the anti-SARS-CoV2 immunoglobulin A and anti-SARS- CoV2 immunoglobulin M.
  • an airborne pathogen includes an infectious agent which infects a subject (e.g., a human) through cells in the nasal or/or nasopharynx region of a subject.
  • non-IgG antibody is an antibody which is of an isotype other than IgG (e.g., IgA or IgM). Such a non-IgG antibody is capable of providing passive immunity against the selected pathogenic agent or a cross-reactive strain of the pathogenic agent.
  • the non-IgG antibody is a neutralizing antibody construct against the pathogenic agent, e.g., a virus, bacterium, fungus, or a pathogenic toxin of said agent (e.g., anthrax toxin).
  • antibody and “immunoglobulin” may be used interchangeably herein and while for convenience only one term may be used, it will be readily understood that the term is not limited to a full-length monoclonal antibody unless otherwise specified.
  • An “immunoglobulin” or an “antibody” may be obtained using an antigen or by isolation of an antibody from a patient.
  • polypeptide sequence derived therefrom whether monoclonal, multiple (dimeric) or single chain, recombinant, or constructs thereof, that include antigen binding portions, domains or regions (including Fab, Fab’, F(ab’)2, Fv dAb and CDR fragments), single chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides, and bifunctional antibodies.
  • suitable antibodies may be single-chain variable fragment antibody (scFV).
  • a monomeric scFv has a molecular mass of only about 30 kDa, which is expressed in a variety of systems as a single VL-VH pair linked by a Gly /Ser-rich synthetic linker.
  • tandem repeats of VL-VH may be selected for expression from a recombinant vector or delivered via a non-viral delivery vehicle.
  • Antibodies having at least the Fc portion of the heavy chain include at least five (iso)types (e.g., IgG, IgE, IgM, IgD, IgA and IgY) and various subclasses (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2).
  • a “Fc region” and or “Fc domain” is a region and/or domain of a constant region of an antibody, wherein the Fc region may contain two domains (CH2 - CH3) as for IgG and IgA. Alternatively, Fc region may contain three domains (CH2-CH3-CH4) as for IgM.
  • a “parent Fc” is an Fc region wherein the Fc is of a same isotype identity as the rest of an antibody molecule, i.e., IgG Fc.
  • a “variant Fc” is an Fc region wherein the Fc is of a different isotype identity as the rest of an antibody molecule, i.e., different from IgG, IgA Fc or IgM Fc.
  • a “polypeptide J chain” is referred to a joining chain, which is a polypeptide comprising in IgA and IgM antibodies, wherein the polypeptide J chain regulates the antibody formation for isotypes IgA and IgM.
  • the J chain is of sequence identified by sequence with NCBI accession Gene ID: 3512.
  • this chain may not be expressed when the antibody is delivered via a viral vector and expressed in vivo.
  • a “class-switched antibody” or “a class-switched immunoglobulin” is an antibody which has been designed or engineered so that it has a different isotype or class. Suitably, this is accomplished without affecting the antigen binding domains or regions of the antibody.
  • a parent IgG Fc region of an antibody molecule has been switched using recombination techniques known in the art, wherein the parent IgG Fc region has been replaced by a variant Fc region, wherein the variant Fc region is IgA Fc.
  • a parent IgG Fc region of an antibody has been replaced by a variant Fc region, wherein the variant Fc region is IgM Fc.
  • the CSR antibody may contain a polypeptide J chain.
  • the methods used to construct any embodiment of this invention are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.
  • an Fc region may be selected form a constant region of a heavy chain of a human IgM antibody.
  • the constant region of a heavy chain of human IgM is of sequence identified by sequence with UniProt accession number P01871, and/or NCBI accession Gene ID: 3507.
  • the IgM class switched antibody presents 10 copies of the antigen binding regions, wherein IgM is pentavalent. In certain embodiments, the IgM class switched antibody presents 12 copies of the antigen binding regions, wherein IgM is hexavalent.
  • the IgM class switched antibody may be pentavalent. In certain embodiments, the IgM class switched antibody may be hexavalent. In certain embodiment, the IgM class switched antibody, wherein IgM is pentavalent, presents 10 copies of the antigen binding domains or regions of an immunoglobulin, wherein optionally presenting copies of same antigen binding domains or regions of an immunoglobulin or a mixture of different antigen binding domains or regions of an immunoglobulin.
  • the IgM class switched antibody presents 12 copies of the antigen binding domains or regions of an immunoglobulin, wherein optionally presenting copies of same antigen binding domains or regions of an immunoglobulin or a mixture of different antigen binding domains or regions of an immunoglobulin.
  • a “hexavalent structure” is referred to an Fc region of the fusion protein which comprises six monomers of IgM Fc region.
  • a “pentavalent structure” is referred to an Fc region of the fusion protein which comprises five monomers of IgM Fc region and a joining polypeptide J chain.
  • an Fc region may be selected form a constant region of a heavy chain of a human IgA antibody.
  • the constant region of a heavy chain of a human IgA 1 is of sequence identified by sequence with UniProt accession number P01876, and/or NCBI accession Gene ID: 3493.
  • the constant region of a heavy chain of a human IgA2 is of sequence identified by sequence with UniProt accession number P01877, and/or NCBI accession Gene ID: 3494.
  • a “neutralizing antibody” is an antibody which defends a cell from an antigen or infectious body by inhibiting or neutralizing its biological effect.
  • "neutralizes” and grammatical variations thereof, refer to an activity of an antibody that prevents entry or translocation of the pathogen into the cytoplasm of a cell susceptible to infection.
  • compositions described herein are designed to deliver at least one antibody lacking an IgG4 or any IgG isotype Fc region which binds a targeted airborne pathogen (or a toxin) and effectively reduces or prevents infection of the subject with the targeted pathogen.
  • Such antibodies may be delivered in protein form, may be admixed or encapsulated with a carrier (e.g., a lipid nanoparticle), delivered via at least one suitable vector, or mixtures and combinations thereof.
  • a carrier e.g., a lipid nanoparticle
  • the regimens and methods provided herein may utilize a single composition containing a single antibody (e.g., a class switched IgA, or an scFv), or a combination or cocktail of antibodies.
  • the regimens and methods may utilized separate compositions for co-administration of different antibodies.
  • Such compositions may be delivered via the same route of administration (intranasally) at the same time or sequentially.
  • the regimens may involve a combination of intranasal administration and delivery via another route.
  • compositions and methods are particularly well suited for viral vector-mediated delivery of antibody constructs to nasal epithelial cells and/or the cells of the nasopharynx and/or the lung.
  • the antibody constructs do not contain an immunoglobulin G isotype Fc region (i.e., a non-IgG Ab).
  • the antibody constructs do not comprise an Fc region of IgG4 subclass.
  • suitable viral vectors may include, e.g., adeno-associated viruses and other parvoviruses, herpes simplex virus, vaccinia virus, adenovirus, or lentivirus.
  • the compositions, methods and regimens may involve intranasal administration of a class-switched antibody or scFV alone or in combination with a viral vector (e.g., recombinant adeno-associated virus).
  • a regimen for passive immunization against an airborne pathogen comprises intranasally administering to a patient in need thereof a composition for delivery to the nasal epithelium and the lung comprising at least one antibody construct comprising a class switched recombinant (CSR) antibody which is an immunoglobulin A or immunoglobin M isotype antibody which neutralizes the target airborne pathogen and/or an scFv which neutralizes the target airborne pathogen.
  • CSR class switched recombinant
  • both the upper respiratory system nose, sinuses, nasopharynx, and/or throat
  • lower respiratory system airways and/or lungs
  • a single composition is designed target both regions by dosing intranasally (e.g., two sprays to each nostril). If needed, dosing may be repeated periodically, e.g., at 3-4 month intervals, at 6 month intervals, at 6-9 month intervals, at 6-12 month intervals, or at other desired intervals.
  • the repeat doses may be the same composition or a different composition as provided herein containing the same or different vectors (e.g., using the same rAAV capsid or different capsids) and/or the same or different antibodies.
  • at least one of the antibodies is delivered via an rAAV having a capsid selected from AAV9, AAVhu68, AAV5, or AAV6.
  • the rAAV may be single-stranded or may have self-complementary (sc) inverted terminal repeats (ITRs).
  • the regimen and optionally the composition comprises at least a second antibody which targets the same or a different airborne pathogen.
  • the antibody is a bifunctional antibody.
  • the regimen may further comprise coadministering an IgG isotype antibody by a route other than intranasal delivery.
  • a method for preventing SARS-CoV-2 infection in humans comprises delivering anti-SARS-CoV2 immunoglobulin A proteins and/or immunoglobulin M protein to a human subject in an amount effective to prevent or reduce anti-SARS-CoV2 infection.
  • the delivering comprises administering at least one stock of recombinant adeno-associated virus (rAAV) which expresses the anti-SARS-CoV2 immunoglobulin A and/or immunoglobulin M in a nasal epithelial and/or lung cells.
  • the composition may contain at least one stock of rAAV expressing two or more different anti- SARS-CoV2 immunoglobulins, wherein the composition does not comprise any IgG immunoglobulin.
  • the method may involve co-delivering two or more separate compositions comprising the anti-SARS-CoV2 immunoglobulin A and anti- SARS-CoV2 immunoglobulin M.
  • compositions provided herein may contain different subunits of the same non-IgG antibody, different antibodies, and/or the same antibodies in vectors which differ in one or more element, e.g., capsid, promoter, enhancer, polyA, marker gene, etc.
  • the non-IgG antibody neutralizes more than one subtype of a viral pathogen.
  • a passive immunization regimen comprises a combination of vectors (e.g., rAAV with different capsids and/or with different transgenes) which comprise different non-IgG antibodies are delivered to the subject.
  • a single composition contains more than one different type of non-IgG antibody.
  • the antibody selected based on the causative agent (pathogen) for the disease against which protection is sought.
  • pathogens may be of viral, bacterial, or fungal origin, and may be used to prevent infection in humans against human disease, or in non-human mammals or other animals to prevent veterinary disease.
  • SARS-CoVl severe acute respiratory syndrome
  • COVID- 19 the causative agent of COVID- 19 and antibodies specific for this virus have been described.
  • IgG antibodies which have been described as being useful for binding the receptor binding domain (RBD) of human ACE2 of SARS-COV2 and having neutralizing activity include, e.g., COV2-2196, COV2-2130, COV2-2165 (Zost et al., Nature, 584, 443-465 (2020)); BD-361, BD-368, BD-368-2 (Cao et al., Cell, 182, 73-84 (2020)); B38, H4 (Y.
  • RBD receptor binding domain
  • IgG antibodies which have been described as being useful for binding either RBD or spike protein of human ACE2 of both SARS-COV1 and SARS-CoV2 and having neutralizing activity include, e.g., CR3022 and 47D11 (Wang et al., Nature Communications, 11, www.nature.com/naturecommunications (2020)).
  • influenza virus from the Orthomyxoviridae family, which includes: Influenza A, Influenza B, and Influenza C.
  • the type A viruses are the most virulent human pathogens.
  • the serotypes of influenza A which have been associated with pandemics include, H1N1, which caused Spanish Flu in 1918, and Swine Flu in 2009; H2N2, which caused Asian Flu in 1957; H3N2, which caused Hong Kong Flu in 1968; H5N1, which caused Bird Flu in 2004; H7N7; H1N2; H9N2; H7N2; H7N3; and H10N7. Broadly neutralizing antibodies against influenza A have been described.
  • a “broadly neutralizing antibody” refers to a neutralizing antibody which can neutralize multiple strains from multiple subtypes.
  • CR6261 [The Scripps Institute/ Crucell] has been described as a monoclonal antibody that binds to a broad range of the influenza virus including the 1918 "Spanish flu” (SC1918/H1) and to a virus of the H5N 1 class of avian influenza that jumped from chickens to a human in Vietnam in 2004 (Viet04/H5).
  • CR6261 recognizes a highly conserved helical region in the membrane-proximal stem of hemagglutinin, the predominant protein on the surface of the influenza virus.
  • target pathogenic viruses include, arenaviruses (including funin, machupo, and Lassa), filoviruses (including Marburg and Ebola), hantaviruses, picomoviridae (including rhinoviruses, echovirus), coronaviruses, paramyxovirus, morbillivirus, respiratory synctial virus, togavirus, coxsackievirus, parvovirus B19, parainfluenza, adenoviruses, reoviruses, variola (Variola major (Smallpox)) and Vaccinia (Cowpox) from the poxvirus family, and varicella-zoster (pseudorabies).
  • arenaviruses including funin, machupo, and Lassa
  • filoviruses including Marburg and Ebola
  • hantaviruses including rhinoviruses, echovirus
  • coronaviruses paramyxovirus
  • morbillivirus morbil
  • Viral hemorrhagic fevers are caused by members of the arenavirus family (Lassa fever) (which family is also associated with Lymphocytic choriomeningitis (LCM)), filovirus (ebola virus), and hantavirus (puremala).
  • LCM Lymphocytic choriomeningitis
  • filovirus ebola virus
  • hantavirus puremala
  • the members of picomavirus a subfamily of rhinoviruses
  • coronavirus family which includes a number of non-human viruses such as infectious bronchitis virus (poultry), porcine transmissible gastroenteric virus (pig), porcine hemagglutinatin encephalomyelitis virus (pig), feline infectious peritonitis virus (cat), feline enteric coronavirus (cat), canine coronavirus (dog).
  • the paramyxovirus family includes parainfluenza Virus Type 1, parainfluenza Virus Type 3, bovine parainfluenza Virus Type 3, rubellavirus (mumps virus, parainfluenza Virus Type 2, parainfluenza virus Type 4, Newcastle disease virus (chickens), rinderpest, morbillivirus, which includes measles and canine distemper, and pneumovirus, which includes respiratory syncytial virus (RSV).
  • the parvovirus family includes feline parvovirus (feline enteritis), feline panleucopeniavirus, canine parvovirus, and porcine parvovirus.
  • the adenovirus family includes viruses (EX, AD7, ARD, O.B.) which cause respiratory disease.
  • a neutralizing antibody construct against a bacterial pathogen may also be selected for use in the present invention.
  • the neutralizing antibody construct is directed against the bacteria itself.
  • the neutralizing antibody construct is directed against a toxin produced by the bacteria.
  • airborne bacterial pathogens include, e.g., Neisseria meningitidis (meningitis), Klebsiella pneumonia (pneumonia), Pseudomonas aeruginosa (pneumonia), Pseudomonas pseudomallei (pneumonia), Pseudomonas mallei (pneumonia), Acinetobacter (pneumonia), Moraxella catarrhalis, Moraxella lacunata, Alkaligenes, Cardiobacterium, Haemophilus influenzae (flu), Haemophilus parainfluenzae, Bordetella pertussis (whooping cough), Francisella tularensis (pneumonia/fever), Legionella pneumonia (Legi), Neisser
  • the causative agent of anthrax is a toxin produced by Bacillius anthracis.
  • Neutralizing antibodies against protective agent (PA) one of the three peptides which form the toxoid, have been described.
  • the other two polypeptides consist of lethal factor (LF) and edema factor (EF).
  • Anti-PA neutralizing antibodies have been described as being effective in passively immunization against anthrax. See, e.g., US Patent number 7,442,373; R. Sawada-Hirai et al, J Immune Based Ther Vaccines. 2004; 2: 5. (on-line 2004 May 12).
  • Still other anti-anthrax toxin neutralizing antibodies have been described and/or may be generated.
  • neutralizing antibodies against other bacteria and/or bacterial toxins may be used to generate an non-IgG antibody as described herein.
  • infectious diseases may be caused by airborne fungi including, e.g., Aspergillus species, Absidia corymbifera, Rhixpus stolonifer, Mucor plumbeaus, Cryptococcus neoformans, Histoplasm capsulatum, Blastomyces dermatitidis , Coccidioides immitis, Penicillium species, Micropolyspora faeni, Thermoactinomyces vulgaris, Alternaria alternate, Cladosporium species, Helminthosporium, and Stachybotrys species.
  • Aspergillus species e.g., Aspergillus species, Absidia corymbifera, Rhixpus stolonifer, Mucor plumbeaus, Cryptococcus neoformans, Histoplasm capsulatum, Blastomyces dermatitidis , Coccidioides immitis, Penicillium species, Micropolyspora faeni, Therm
  • passive immunization according to the invention may be used to prevent conditions associated with direct inoculation of the nasal passages, e.g., conditions which may be transmitted by direct contact of the fingers with the nasal passages. These conditions may include fungal infections (e.g., athlete’s foot), ringworm, or viruses, bacteria, parasites, fungi, and other pathogens which can be transmitted by direct contact.
  • fungal infections e.g., athlete’s foot
  • ringworm e.g., ringworm
  • viruses e.g., bacteria, parasites, fungi, and other pathogens which can be transmitted by direct contact.
  • a variety of conditions which affect household pets, cattle and other livestock, and other animals For example, in dogs, infection of the upper respiratory tract by canine sinonasal aspergillosis causes significant disease.
  • BRSV Bovine Respiratory Syncytial Virus
  • An antibody, and particularly, a neutralizing antibody, against a pathogen such as those specifically identified herein may be used to generate a class-switched or non-IgG antibody.
  • Monoclonal antibodies (mAbs) with broad neutralizing capacity can be identified using antibody phage display to screen libraries from donors recently vaccinated with the seasonal flu vaccine, from non-immune humans or from survivors of a natural infection.
  • mAbs Monoclonal antibodies with broad neutralizing capacity can be identified using antibody phage display to screen libraries from donors recently vaccinated with the seasonal flu vaccine, from non-immune humans or from survivors of a natural infection.
  • influenza antibodies have been identified which neutralize more than one influenza subtype by blocking viral fusion with the host cell. This technique may be utilized with other infections to obtain a neutralizing monoclonal antibody.
  • the coding sequences for the antibodies described herein may be engineered into expression cassettes.
  • the expression cassette contains control elements necessary which are operably linked to the transgene in a manner which permits its transcription, translation and/or expression in a cell transfected with the expression cassette.
  • operably linked sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • the regulatory control elements typically contain a promoter sequence as part of the expression control sequences, e.g., located between the selected 5’ ITR sequence and the coding sequence.
  • Constitutive promoters, regulatable promoters [see, e.g., WO 2011/126808 and WO 2013/04943], tissue specific promoters, or a promoter responsive to physiologic cues may be used may be utilized in the vectors described herein.
  • constitutive promoters suitable for controlling expression of the therapeutic products include, but are not limited to chicken P-actin (CB) promoter, CB7 promoter, human cytomegalovirus (CMV) promoter, ubiquitin C promoter (UbC), the early and late promoters of simian virus 40 (SV40), U6 promoter, metallothionein promoters, EFla promoter, ubiquitin promoter, hypoxanthine phosphoribosyl transferase (HPRT) promoter, dihydrofolate reductase (DHFR) promoter (Scharfmann et al., Proc. Natl. Acad. Sci.
  • adenosine deaminase promoter phosphoglycerol kinase (PGK) promoter, pyruvate kinase promoter phosphoglycerol mutase promoter, the P-actin promoter (Lai et al., Proc. Natl. Acad. Sci. USA 86: 10006-10010 (1989)), the long terminal repeats (LTR) of Moloney Leukemia Virus and other retroviruses, the thymidine kinase promoter of Herpes Simplex Virus and other constitutive promoters known to those of skill in the art.
  • LTR long terminal repeats
  • tissue- or cell-specific promoters suitable for use in the present invention include, but are not limited to, endothelin-I (ET -I) and Flt-I, which are specific for endothelial cells, FoxJl (that targets ciliated cells).
  • Inducible promoters suitable for controlling expression of the therapeutic product include promoters responsive to exogenous agents (e.g., pharmacological agents) or to physiological cues.
  • These response elements include, but are not limited to a hypoxia response element (HRE) that binds HIF-Ia and /?, a metal-ion response element such as described by Mayo et al. (1982, Cell 29:99-108); Brinster et al. (1982, Nature 296:39-42) and Searle et al. (1985, Mol. Cell. Biol. 5: 1480-1489); or a heat shock response element such as described by Nouer et al. (in: Heat Shock Response, ed. Nouer, L., CRC, Boca Raton, Fla., ppI67-220, 1991).
  • HRE hypoxia response element
  • expression of the antibody is controlled by a regulatable promoter that provides tight control over the transcription of the gene encoding the neutralizing antibody construct, e.g, a pharmacological agent, or transcription factors activated by a pharmacological agent or in alternative embodiments, physiological cues.
  • a regulatable promoter that provides tight control over the transcription of the gene encoding the neutralizing antibody construct, e.g, a pharmacological agent, or transcription factors activated by a pharmacological agent or in alternative embodiments, physiological cues.
  • Promoter systems that are non-leaky and that can be tightly controlled are preferred.
  • regulatable promoters which are ligand-dependent transcription factor complexes that may be used in the invention include, without limitation, members of the nuclear receptor superfamily activated by their respective ligands e.g., glucocorticoid, estrogen, progestin, retinoid, ecdysone, and analogs and mimetics thereof) and rTTA activated by tetracycline.
  • the gene switch is an EcR-based gene switch. Examples of such systems include, without limitation, the systems described in US Patent Nos. 6,258,603, 7,045,315, U.S. Published Patent Application Nos. 2006/0014711, 2007/0161086, and International Published Application No. WO 01/70816.
  • chimeric ecdysone receptor systems are described in U.S. Pat. No. 7,091,038, U.S. Published Patent Application Nos. 2002/0110861, 2004/0033600, 2004/0096942, 2005/0266457, and 2006/0100416, and International Published Application Nos. WO 01/70816, WO 02/066612, WO 02/066613, WO 02/066614, WO 02/066615, WO 02/29075, and WO 2005/108617, each of which is incorporated by reference in its entirety.
  • An example of a non-steroidal ecdysone agonist-regulated system is the RheoSwitch® Mammalian Inducible Expression System (New England Biolabs, Ipswich, MA).
  • Still other promoter systems may include response elements including but not limited to a tetracycline (tet) response element (such as described by Gossen & Bujard (1992, Proc. Natl. Acad. Sci. USA 89:5547-551); or a hormone response element such as described by Lee et al. (1981, Nature 294:228-232); Hynes et al. (1981, Proc. Natl. Acad. Sci. USA 78:2038- 2042); Klock et al. (1987, Nature 329:734-736); and Israel & Kaufman (1989, Nucl. Acids Res. 17:2589-2604) and other inducible promoters known in the art.
  • tetracycline response element such as described by Gossen & Bujard (1992, Proc. Natl. Acad. Sci. USA 89:5547-551
  • a hormone response element such as described by Lee et al. (1981, Nature 294:228-232
  • expression of the neutralizing antibody construct can be controlled, for example, by the Tet- on/off system (Gossen et al., 1995, Science 268: 1766-9; Gossen et al., 1992, Proc. Natl. Acad. Sci. USA., 89( 12):5547-51); the TetR-KRAB system (Urrutia R., 2003, Genome Biol., 4(10):231; Deuschle U et al., 1995, Mol Cell Biol. (4): 1907-14); the mifepristone (RU486) regulatable system (Geneswitch; Wang Y et al., 1994, Proc. Natl. Acad. Sci.
  • the gene switch is based on heterodimerization of FK506 binding protein (FKBP) with FKBP rapamycin associated protein (FRAP) and is regulated through rapamycin or its non-immunosuppressive analogs.
  • FKBP FK506 binding protein
  • FRAP FKBP rapamycin associated protein
  • examples of such systems include, without limitation, the ARGENTTM Transcriptional Technology (ARIAD Pharmaceuticals, Cambridge, Mass.) and the systems described in U.S. Pat. Nos. 6,015,709, 6,117,680, 6,479,653, 6,187,757, and 6,649,595, U.S. Publication No. 2002/0173474, U.S. Publication No. 200910100535, U.S. Patent No. 5,834,266, U.S. Patent No.
  • the Ariad system is designed to be induced by rapamycin and analogs thereof referred to as "rapalogs".
  • suitable rapamycins are provided in the documents listed above in connection with the description of the ARGENTTM system.
  • the molecule is rapamycin [e.g., marketed as RapamuneTM by Pfizer],
  • a rapalog known as AP21967 [ARIAD] is used.
  • rapalogs include, but are not limited to such as AP26113 (Ariad), AP1510 (Amara, J.F., et al., 1997, Proc Natl Acad Sci USA, 94(20): 10618-23) AP22660, AP22594, AP21370, AP22594, AP23054, AP1855, AP1856, AP1701, AP1861, AP1692 and AP1889, with designed 'bumps' that minimize interactions with endogenous FKBP. Still other rapalogs may be selected, e.g, AP23573 [Merck],
  • the expression cassette comprises one or more expression enhancers.
  • the expression cassette contains two or more expression enhancers. These enhancers may be the same or may differ from one another.
  • an enhancer may include a CMV immediate early enhancer. This enhancer may be present in two copies which are located adjacent to one another. Alternatively, the dual copies of the enhancer may be separated by one or more sequences.
  • the expression cassette further contains an intron, e.g., the chicken beta-actin intron.
  • suitable introns include those known in the art, e.g., such as are described in WO 2011/126808.
  • polyA sequences examples include, e.g., rabbit binding globulin (rBG), SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyAs.
  • rBG rabbit binding globulin
  • bGH bovine growth hormone
  • one or more sequences may be selected to stabilize mRNA.
  • An example of such a sequence is a modified WPRE sequence, which may be engineered upstream of the polyA sequence and downstream of the coding sequence (see, e.g., MA Zanta-Boussif, et al, Gene Therapy (2009) 16: 605-619).
  • an expression cassette may be bicistronic and may encode each subunit of a protein (e.g, an immunoglobulin domain, an immunoglobulin heavy chain, an immunoglobulin light chain).
  • a cell produces the multi-subunit protein following infected/transfection with the virus containing each of the different subunits.
  • different subunits of a protein may be encoded by the same transgene.
  • a single transgene includes the DNA encoding each of the subunits, with the DNA for each subunit separated by an internal ribozyme entry site (IRES) or a self-cleaving peptide (e.g, 2A).
  • IRES is desirable when the size of the DNA encoding each of the subunits is small, e.g., the total size of the DNA encoding the subunits and the IRES is less than five kilobases.
  • Expression control sequences include appropriate enhancer; transcription factor; transcription terminator; promoter; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE); sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • an intron is included in the expression cassette.
  • Suitable introns include chicken beta-actin intron, the human beta globin IVS2 (Kelly et al, Nucleic Acids Research, 43(9):4721-32 (2015)); the Promega chimeric intron (Almond, B. and Schenbom, E. T. A Comparison of pCI-neo Vector and pcDNA4/HisMax Vector); and the hFIX intron.
  • Various introns suitable herein are known in the art and include, without limitation, those found at bpg.utoledo.edu/ ⁇ afedorov/lab/eid.html, which is incorporated herein by reference. See also, Shepelev V., Fedorov A. Advances in the Exon-Intron Database. Briefings in Bioinformatics 2006, 7: 178-185, which is incorporated herein by reference.
  • the expression cassette may contain dorsal root ganglion detargetting sequences described in WO 2020/132455, published June 5, 2020 and incorporated herein by reference, may be incorporated into an antibody-expressing expression cassette.
  • the expression cassettes can be carried on any suitable genetic element, e.g., a plasmid, which is delivered to a packaging host cell.
  • the plasmids may be engineered such that they are suitable for replication and packaging in vitro in prokaryotic cells, insect cells, mammalian cells, among others. Suitable transfection techniques and packaging host cells are known and/or can be readily designed by one of skill in the art.
  • a viral vector selected for carrying an expression cassette is desirable selected for its ability to target the lower respiratory (e.g., nasal epithelium cells and nasopharynx) and the airways and/or lungs.
  • suitable viral vectors may include recombinant, replicationdefective: adenovirus, lentivirus, herpes simplex virus, vaccinia virus, or an adeno-associated virus.
  • Suitable AAV capsid sources include, e.g., AAVhu68 [see, e.g, WO 2018/160582, which is incorporated herein by reference]; AAV9 capsid and chimeric capsids derived from AAV9 have been described. See, e.g., US 7,906,111, which is incorporated by reference herein.
  • AAV serotypes which transduce nasal cells and lung, or another suitable target may be selected as sources for capsids of AAV viral vectors (DNase resistant viral particles) including, e.g., rh90 [PCT/US20/30273, filed April 28, 2020], rh91 [PCT/US20/30266, filed April 28, 2020], rh 92, rh93, rh91.93 [PCT/US20/30281, filed April 28, 2020] AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, rhlO, AAVrh64Rl, AAVrh64R2, rh8 (See, e.g...
  • an AAV capsid (cap) for use in the viral vector can be generated by mutagenesis (i.e., by insertions, deletions, or substitutions) of one of the aforementioned AAV caps or its encoding nucleic acid.
  • An expression cassette to be packaged into an AAV capsid is flanked at its extreme 5 ’ end and its extreme 3 ’ end with AAV inverted terminal repeats (ITRs) in order to permit packaging into an AAV capsid.
  • ITRs AAV inverted terminal repeats
  • These are the only AAV sequences in the vector genome. Both single-stranded AAV and self-complementary (sc) AAV are encompassed with the rAAV.
  • the vector genome typically comprises the cis-acting 5' and 3' inverted terminal repeat sequences (See, e.g., B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168 (1990)).
  • the native AAV ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al, “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J. Virol., 70:520 532 (1996)).
  • an example of such a molecule employed in the present invention is a “cis-acting” plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5' and 3' AAV ITR sequences.
  • the ITRs are from an AAV different than that supplying a capsid.
  • the ITR sequences from AAV2.
  • ITRs from other AAV sources may be selected.
  • a shortened version of the 5 ’ ITR, termed AITR has been described in which the D-sequence and terminal resolution site (trs) are deleted.
  • the vector genome includes a shortened AAV2 ITR of 130 base pairs, wherein the external A elements is deleted.
  • the shortened ITR is reverts back to the wild type length of 145 base pairs during vector DNA amplification using the internal (A’) element as a template.
  • A internal
  • full-length AAV 5’ and 3’ ITRs are used.
  • the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be termed pseudotyped.
  • pseudotyped the pseudotyped.
  • other configurations of these elements may be suitable.
  • AAV-based viral vectors Methods of preparing AAV-based viral vectors are known. See, e.g., US Published Patent Application No. 2007/0036760 (February 15, 2007), which is incorporated by reference herein.
  • the sequences of any of the AAV capsids provided herein can be readily generated using a variety of techniques. Suitable production techniques are well known to those of skill in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY).
  • peptides can also be synthesized by the well-known solid phase peptide synthesis methods (Merrifield, (1962) J. Am. Chem.
  • the components required to be cultured in the host cell to package an AAV vector genome in an AAV capsid may be provided to the host cell in trans.
  • any one or more of the required components e.g., vector genome with the expression cassette flanked by the AAV ITR sequences, rep sequences, AAV cap sequences, and/or helper functions
  • a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art.
  • a stable host cell will contain the required component(s) under the control of an inducible promoter.
  • the required component(s) may be under the control of a constitutive promoter.
  • a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters.
  • a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contains the rep and/or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.
  • the expression cassettes described herein are engineered into a genetic element (e.g., a shuttle plasmid) which transfers the immunoglobulin construct sequences carried thereon into a packaging host cell for production a viral vector.
  • a genetic element e.g., a shuttle plasmid
  • the selected genetic element may be delivered to an AAV packaging cell by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. Stable AAV packaging cells can also be made.
  • the expression cassettes may be used to generate a viral vector other than AAV, or for production of mixtures of antibodies in vitro.
  • AAV intermediate or “AAV vector intermediate” refers to an assembled rAAV capsid which lacks the desired genomic sequences packaged therein. These may also be termed an “empty” capsid. Such a capsid may contain no detectable genomic sequences of an expression cassette, or only partially packaged genomic sequences which are insufficient to achieve expression of the gene product. These empty capsids are non-functional to transfer the gene of interest to a host cell.
  • the recombinant AAV described herein may be generated using techniques which are known. See, e.g., WO 2003/042397; WO 2005/033321, WO 2006/110689; US 7588772 B2.
  • Such a method involves culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette composed of, at a minimum, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the expression cassette into the AAV capsid protein.
  • ITRs AAV inverted terminal repeats
  • cells are manufactured in a suitable cell culture (e.g., HEK 293 cells).
  • suitable cell culture e.g., HEK 293 cells.
  • Methods for manufacturing the gene therapy vectors described herein include methods well known in the art such as generation of plasmid DNA used for production of the gene therapy vectors, generation of the vectors, and purification of the vectors.
  • the gene therapy vector is an AAV vector and the plasmids generated are an AAV cis-plasmid encoding the AAV genome and the gene of interest, an AAV trans-plasmid containing AAV rep and cap genes, and an adenovirus helper plasmid.
  • the vector generation process can include method steps such as initiation of cell culture, passage of cells, seeding of cells, transfection of cells with the plasmid DNA, post-transfection medium exchange to serum free medium, and the harvest of vector-containing cells and culture media.
  • the harvested vector-containing cells and culture media are referred to herein as crude cell harvest.
  • the gene therapy vectors are introduced into insect cells by infection with baculovirus-based vectors.
  • the crude cell harvest may thereafter be subject method steps such as concentration of the vector harvest, diafdtration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, fdtration of microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and/or formulation and filtration to prepare bulk vector.
  • a two-step affinity chromatography purification at high salt concentration followed anion exchange resin chromatography are used to purify the vector drug product and to remove empty capsids. These methods are described in more detail in International Patent Application No. PCT/US2016/065970, filed December 9, 2016 and its priority documents, US Patent Application Nos. 62/322,071, filed April 13, 2016 and 62/226,357, filed December 11, 2015 and entitled “Scalable Purification Method for AAV9”, each of which is incorporated by reference herein. Purification methods for AAV8, International Patent Application No. PCT/US2016/065976, filed December 9, 2016 and is priority documents US Patent Application Nos.
  • the number of particles (pt) per 20 pL loaded is then multiplied by 50 to give particles (pt) /mL.
  • Pt/mL divided by GC/mL gives the ratio of particles to genome copies (pt/GC).
  • Pt/mL- GC/mL gives empty pt/mL.
  • Empty pt/mL divided by pt/mL and x 100 gives the percentage of empty particles.
  • the methods include subjecting the treated AAV stock to SDS-polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel containing 3-8% Tris-acetate in the buffer, then running the gel until sample material is separated, and blotting the gel onto nylon or nitrocellulose membranes, preferably nylon.
  • Anti-AAV capsid antibodies are then used as the primary antibodies that bind to denatured capsid proteins, preferably an anti-AAV capsid monoclonal antibody, most preferably the Bl anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281- 9293).
  • a secondary antibody is then used, one that binds to the primary antibody and contains a means for detecting binding with the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound to it, most preferably a sheep antimouse IgG antibody covalently linked to horseradish peroxidase.
  • a method for detecting binding is used to semi-quantitatively determine binding between the primary and secondary antibodies, preferably a detection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit.
  • a detection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit.
  • samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing reducing agent e.g., DTT), and capsid proteins were resolved on pre-cast gradient polyacrylamide gels (e.g., Novex).
  • Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions or other suitable staining method, i.e. SYPRO ruby or coomassie stains.
  • the concentration of AAV vector genomes (vg) in column fractions can be measured by quantitative real time PCR (Q-PCR).
  • Samples are diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the samples are further diluted and amplified using primers and a TaqManTM Anorogenic probe specific for the DNA sequence between the primers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 Sequence Detection System. Plasmid DNA containing identical sequences to that contained in the AAV vector is employed to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) values obtained from the samples are used to determine vector genome titer by normalizing it to the Ct value of the plasmid standard curve. End-point assays based on the digital PCR can also be used.
  • DNase I or another
  • Sedimentation velocity as measured in an analytical ultracentrifuge (AUC) can detect aggregates, other minor components as well as providing good quantitation of relative amounts of different particle species based upon their different sedimentation coefficients.
  • AUC analytical ultracentrifuge
  • This is an absolute method based on fundamental units of length and time, requiring no standard molecules as references.
  • Vector samples are loaded into cells with 2-channel charcoal-epon centerpieces with 12mm optical path length.
  • the supplied dilution buffer is loaded into the reference channel of each cell.
  • the loaded cells are then placed into an AN- 60Ti analytical rotor and loaded into a Beckman-Coulter ProteomeLab XL-I analytical ultracentrifuge equipped with both absorbance and RI detectors.
  • the rotor After full temperature equilibration at 20 °C the rotor is brought to the final run speed of 12,000 rpm. A280 scans are recorded approximately every 3 minutes for ⁇ 5.5 hours (110 total scans for each sample). The raw data is analyzed using the c(s) method and implemented in the analysis program SEDFIT. The resultant size distributions are graphed and the peaks integrated. The percentage values associated with each peak represent the peak area fraction of the total area under all peaks and are based upon the raw data generated at 280nm; many labs use these values to calculate empty: full particle ratios. However, because empty and full particles have different extinction coefficients at this wavelength, the raw data can be adjusted accordingly. The ratio of the empty particle and full monomer peak values both before and after extinction coefficient-adjustment is used to determine the empty-full particle ratio.
  • an optimized q-PCR method which utilizes a broad spectrum serine protease, e.g., proteinase K (such as is commercially available from Qiagen). More particularly, the optimized qPCR genome titer assay is similar to a standard assay, except that after the DNase I digestion, samples are diluted with proteinase K buffer and treated with proteinase K followed by heat inactivation. Suitably samples are diluted with proteinase K buffer in an amount equal to the sample size.
  • the proteinase K buffer may be concentrated to 2 fold or higher. Typically, proteinase K treatment is about 0.2 mg/mL, but may be varied from 0. 1 mg/mL to about 1 mg/mL.
  • the treatment step is generally conducted at about 55 °C for about 15 minutes, but may be performed at a lower temperature (e.g., about 37 °C to about 50 °C) over a longer time period (e.g., about 20 minutes to about 30 minutes), or a higher temperature (e.g., up to about 60 °C) for a shorter time period (e.g., about 5 to 10 minutes).
  • heat inactivation is generally at about 95 °C for about 15 minutes, but the temperature may be lowered (e.g., about 70 to about 90 °C) and the time extended (e.g., about 20 minutes to about 30 minutes).
  • Samples are then diluted (e.g., 1000 fold) and subjected to TaqMan analysis as described in the standard assay. Quantification also can be done using ViroCyt or flow cytometry.
  • droplet digital PCR may be used.
  • ddPCR droplet digital PCR
  • methods for determining single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2): 115-25. doi: 10. 1089/hgtb.2013. 131. Epub 2014 Feb 14.
  • a composition may include one or more vector which contain elements necessary for the inducible/regulatable promoter.
  • an AAV carrying a neutralizing antibody construct may be co-administered with a different AAV carrying a transcription factor which forms a part of the regulatable expression system.
  • examples of such systems include those described, e.g., in International Patent Application No. PCT/US 11/20213, filed March 28, 2011, which is incorporated by reference herein.
  • an anti-pathogen antibody or a portion thereof e.g., a light chain, heavy chain, or another fragment
  • Such fusion proteins combine to form an “non-IgG antibody” as defined herein following activation with a transcription factor or induction by a pharmacologic agent.
  • compositions may carry a single type of viral vector (e.g., rAAV) carrying at least one antibody.
  • a single type of viral vector e.g., rAAV
  • two or more viral vectors may be co-administered.
  • a composition may carry two or more viral vectors which combine in vivo to form a single antibody.
  • a composition may be delivered which contains two or more different viral vectors.
  • Such different viral vectors may contain different subunits of the same antibody, different antibodies, a bi-functional antibody and/or the same antibodies in viral vectors (e.g., AAV) which differ in one or more element, e.g., capsid, promoter, enhancer, polyA, marker gene, etc., or one of the AAV viral vector may contain another transgene desired to be coexpressed with the non-IgG antibody.
  • the antibody neutralizes more than one strain and/or more than one subtype of said pathogen.
  • the replication-defective virus compositions can be formulated in dosage units to contain an amount of replication-defective virus that is in the range of about 10 9 GC to about 10 16 GC (to treat an average subject of 70 kg in body weight) including all integers or fractional amounts within the range, and preferably 10 12 GC to 10 14 GC for a human patient.
  • the compositions are formulated to contain at least 10 9 , 2xl0 9 , 3xl0 9 , 4xl0 9 , 5xl0 9 , 6xl0 9 , 7xl0 9 , 8xl0 9 , or 9xl0 9 GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 10 10 , 2xlO 10 , 3xl0 10 , 4xlO 10 , 5xl0 10 , 6xlO 10 , 7xlO 10 , 8xl0 10 , or 9xlO 10 GC per dose including all integers or fractional amounts within the range.
  • the compositions are formulated to contain at least 10 11 , 2xlO n , 3xl0 n , 4xlO n , 5xl0 n , 6xlO n , 7xlO n , 8xl0 n , or 9xlO n GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 10 12 , 2xl0 12 , 3xl0 12 , 4xl0 12 , 5xl0 12 , 6xl0 12 , 7xl0 12 , 8xl0 12 , or 9xl0 12 GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 10 13 , 2xl0 13 , 3xl0 13 , 4xl0 13 , 5xl0 13 , 6xl0 13 , 7xl0 13 , 8xl0 13 , or 9x10 13 GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 10 14 , 2xl0 14 , 3xl0 14 , 4xl0 14 , 5xl0 14 , 6xl0 14 , 7xl0 14 , 8xl0 14 , or 9xl0 14 GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 10 15 , 2 x 10 15 , 3xl0 15 , 4xl0 15 , 5xl0 15 , 6xl0 15 , 7xl0 15 , 8xl0 15 , or 9xl0 15 GC per dose including all integers or fractional amounts within the range.
  • the dose can range from IO 10 to about 10 12 GC per dose including all integers or fractional amounts within the range. In one embodiment, for human application the dose can range from 10 9 to about 7x10 13 GC per dose including all integers or fractional amounts within the range. In one embodiment, for human application the dose ranges from 6.25xl0 12 GC to 5 xlO 13 GC. In a further embodiment, the dose is about 6.25 x 10 12 GC, about 1.25 x 10 13 GC, about 2.5 x 10 13 GC, or about 5 xlO 13 GC. In certain embodiment, the dose is divided into one half thereof equally and administered to each nostril. In certain embodiments, for human application the dose ranges from 6.25xl0 12 GC to 5 xlO 13 GC administered as two aliquots of 0.2 ml per nostril for a total volume delivered in each subject of 0.8mL.
  • the volume of carrier, excipient or buffer is at least about 25 pL. In one embodiment, the volume is about 50 pL. In another embodiment, the volume is about 75 pL. In another embodiment, the volume is about 100 pL. In another embodiment, the volume is about 125 pL. In another embodiment, the volume is about 150 pL. In another embodiment, the volume is about 175 pL.
  • the volume is about 200 pL. In another embodiment, the volume is about 225 pL. In yet another embodiment, the volume is about 250 pL. In yet another embodiment, the volume is about 275 pL. In yet another embodiment, the volume is about 300 pL. In yet another embodiment, the volume is about 325 pL. In another embodiment, the volume is about 350 pL. In another embodiment, the volume is about 375 pL. In another embodiment, the volume is about 400 pL. In another embodiment, the volume is about 450 pL. In another embodiment, the volume is about 500 pL. In another embodiment, the volume is about 550 pL. In another embodiment, the volume is about 600 pL. In another embodiment, the volume is about 650 pL. In another embodiment, the volume is about 700 pL. In another embodiment, the volume is between about 700 and 1000 pL.
  • the recombinant vectors may be dosed intranasally by using two sprays to each nostril.
  • the two sprays are administered by alternating to each nostril, e.g., left nostril spray, right nostril spray, then left nostril spray, right nostril spray.
  • each nostril may receive multiple sprays which are separated by an interval of about 10 to 60 seconds, or 20 to 40 seconds, or about 30 seconds, to a few minutes, or longer.
  • Such sprays may deliver, e.g., about 150 pL to 300 pL, or about 250 pL in each spray, to achieve a total volume dosed of about 200 pL to about 600 pL, 400 pL to 700 pL, or 450 pL to 1000 pL.
  • such suspensions may be volumes doses of about 1 mL to about 25 mL, with doses of up to about 2.5xlO 15 GC.
  • the above-described recombinant vectors may be delivered to host cells according to published methods.
  • the rAAV preferably suspended in a physiologically compatible carrier, may be administered to a human or non-human mammalian patient.
  • the composition includes a carrier, diluent, excipient and/or adjuvant.
  • Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the transfer virus is directed.
  • one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline).
  • exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water.
  • the buffer/carrier should include a component that prevents the rAAV, from sticking to the infusion tubing but does not interfere with the rAAV binding activity in vivo.
  • compositions of the invention may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers.
  • suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
  • Suitable chemical stabilizers include gelatin and albumin.
  • the compositions according to the present invention may comprise a pharmaceutically acceptable carrier, such as defined above.
  • compositions described herein comprise an effective amount of one or more AAV suspended in a pharmaceutically suitable carrier and/or admixed with suitable excipients designed for delivery to the subject via injection, osmotic pump, intrathecal catheter, or for delivery by another device or route.
  • the composition is formulated for intranasal administration.
  • the composition is formulated for intramuscular administration.
  • the composition is formulated for intravenous administration.
  • the composition is formulated for intraperitoneal administration.
  • a composition containing the viral vectors encoding the antipathogen construct is delivered intranasally as liquid (e.g., atomized, aerosol, spray, etc), to the subject at a relatively low instillation volume in order to minimize lung transduction.
  • liquid e.g., atomized, aerosol, spray, etc
  • the intranasal delivery device provides a spay atomizer which delivers a mist of particles having an average size range of about 30 microns to about 100 microns in size. In certain embodiments, the average size range is about 10 microns to about 50 microns.
  • Suitable devices have been described in the literature and some are commercially available, e.g., the LMA MAD NASALTM (Teleflex Medical; Ireland); Teleflex VaxINatorTM (Teleflex Medical; Ireland); Controlled Particle Dispersion® (CPD) from Kurve Technologies. See, also, PG Djupesland, Drug Deliv and Transl. Res (2013) 3: 42-62.
  • the particle size and volume of delivery is controlled in order to preferentially target nasal epithelial cells and minimize targeting to the lung.
  • the mist of particles is about 0. 1 micron to about 20 microns, or less, in order to deliver to lung cells. Such smaller particle sizes may minimize retention in the nasal epithelium.
  • Any suitable method or route can be used to administer an composition as described herein, and optionally, to co-administer other active drugs or therapies in conjunction with the non-IgG antibodies (or viral vector(s) encoding same), described herein.
  • Routes of administration include, for example, systemic, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration.
  • a frozen composition which contains a buffer solution as described herein, in frozen form.
  • one or more surfactants e.g., Pluronic F68
  • stabilizers or preservatives is present in this composition.
  • a composition is thawed and titrated to the desired dose with a suitable diluent, e.g., sterile saline or a buffered saline.
  • useful levels of expression of the non-IgG antibody are detectable in the nasopharynx cells of said subject within about 3 days to about 7 days following administration.
  • the inducer may be delivered to the subject within this time period, i.e., after about 3 days following administration of the AAV vectors. However, in certain embodiments, delivery may be earlier and later following AAV delivery.
  • the AAV viral vector(s) transduces the subject’s nasopharynx cells in the presence of high level serum-circulating AAV neutralizing antibodies.
  • a regulatable promoter permits further control of the expression of the non-IgG antibody(s), through controlling contacting of the cells carrying the AAV carrying the coding sequences for the non-IgG antibody(s).
  • the ligand for the regulatable promoter e.g., rapamycin or a rapalog
  • the ligand for the regulatable promoter may be delivered locally to the AAV -transfected cells of the nasopharynx.
  • the rapamycin or rapalog is capable of regulating expression of the non-IgG antibody if delivered topically to the cells.
  • Topical delivery may be by delivering a bolus containing the rapamycin or rapalog to each nostril/nare.
  • topical delivery may be by formulating the rapamycin or rapalog in a suitable composition for topical delivery (e.g., a cream or gel).
  • a suitable composition for topical delivery e.g., a cream or gel.
  • the compositions and methods may be adapted for use with another ligand for non-IgG antibodies which are under the control of a different regulatable system.
  • gene expression is controlled in a dose dependent manner by the regulating pharmacologic compound. In other words, the level of gene expression is lower when low levels of the compound are delivered and increased by increasing the amount of compound.
  • a rapalog e.g., AP21967
  • Inducing agents e.g., rapamycin or a rapalog
  • Inducing agents have been described as being delivered systemically, e.g., by oral or intraperitoneal administration, e.g., by injection.
  • the present inventors have found that it is possible to induce expression in nasal cells following local administration of the inducer.
  • Such local administration may involve intranasal injection.
  • this local administration involves topical administration.
  • topical administration may be performed through use of a bolus delivery to each nostril/nare of a subject.
  • a liquid suspension or solution containing the inducing agent may be delivered topically, e.g., by blocking and instilling each nostril (nare) and allowing the liquid to remain in the nostril for a period of time and then repeating the procedure in the other nostril.
  • the compound may be formulated for delivery as a gel, cream, or other composition which can be applied to the nostril(s)/nare(s).
  • the volume of the liquid delivered is controlled such that there is an insufficient amount to reach the lung.
  • a rapalog e.g., AP21967
  • a rapalog may be administered at a dose of about 0. 1 to about 100 nM, or about 0.5 to 1 mg, or adjusted as needed or desired.
  • the inducing agent e.g., a pharmacologic compound such as rapamycin or a rapalog
  • the inducing agent is delivered to the subject between 5 days to 12 weeks, or longer, following delivery of the AAV composition to the cell.
  • the inducing agent is dosed periodically in order to provide for short-term expression (e.g., 3 to 7, or about 5 days) of the anti-pathogen agent.
  • prophylactically effective levels of expression of the non-IgG antibody is detectable in the nasopharynx of said subject within about twenty- four hours following delivery of an adequate dose of the inducing compound.
  • expression levels may in certain cases be detectable as quickly as about 8 hours, about 12 hours, or about 18 hours following induction.
  • expression may be deferred, e.g., through administration of a delayed release formulation containing the inducing agent.
  • the inducing agent may be delivered once per week for any of weeks 1 to 12 following delivery of the AAV compositions, optionally with breaks of 7 days (one week) or more between inductions.
  • the amount of inducing agent may change in subsequent inductions. For example, it may be desirable to start with a high dose of the pharmacologic compound and then use lower doses for subsequent inductions.
  • a higher dose of the pharmacologic agent when the induction is performed at a time more remote to the delivery of the AAV e.g., a higher amount of inducing agent may be desired after more than 8, 10, or 12 weeks has passed since delivery of the AAV(s) carrying the sequence encoding the anti-pathogen compound(s).
  • nasal and nasopharynx are the nasal part of the pharynx which lies behind the nose and above the level of the soft palate and which is believed to be the primary site of infection from naturally acquired respiratory infections, e.g., such as influenza virus (causing flu) or SARS- CoV2 virus (COVID -19).
  • target nasal and nasopharynx cells include nasal epithelial cells, which may be ciliated nasal epithelial cells, microvilli coated columnar epithelial cells, goblet cells (which secrete mucous onto the surface of the nasal cavity which is composed of the ciliated and microvilli coated cells), and stratified squamous nasal epithelial cells which line the surface of the nasopharynx.
  • An antibody “hinge region” is a flexible amino acid portion of the heavy chains of IgG and IgA immunoglobulin classes, which links these two chains by disulfide bonds.
  • immunoglobulin heavy chain is a polypeptide that contains at least a portion of the antigen binding domain or region of an immunoglobulin and at least a portion of a variable region of an immunoglobulin heavy chain or at least a portion of a constant region of an immunoglobulin heavy chain.
  • the immunoglobulin derived heavy chain has significant regions of amino acid sequence homology with a member of the immunoglobulin gene superfamily.
  • the heavy chain in a Fab fragment is an immunoglobulin-derived heavy chain.
  • immunoglobulin light chain is a polypeptide that contains at least a portion of the antigen binding domain or region of an immunoglobulin and at least a portion of the variable region or at least a portion of a constant region of an immunoglobulin light chain.
  • the immunoglobulin-derived light chain has significant regions of amino acid homology with a member of the immunoglobulin gene superfamily.
  • immunoadhesin is a chimeric, antibody-like molecule that combines the functional domain of a binding protein, usually a receptor, ligand, or cell-adhesion molecule, with immunoglobulin constant domains, usually including the hinge and fragment crystallizable (Fc) regions.
  • fragment antigen-binding (Fab) fragment is a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain.
  • a “recombinant AAV” or “rAAV” is a DNAse-resistant viral particle containing two elements, an AAV capsid and a vector genome containing at least non-AAV coding sequences packaged within the AAV capsid.
  • the capsid contains about 60 proteins composed of vp 1 proteins, vp2 proteins, and vp3 proteins, which self-assemble to form the capsid.
  • “recombinant AAV” or “rAAV” may be used interchangeably with the phrase “rAAV vector”.
  • the rAAV is a “replication-defective virus” or "viral vector”, as it lacks any functional AAV rep gene or functional AAV cap gene and cannot generate progeny.
  • the only AAV sequences are the AAV inverted terminal repeat sequences (ITRs), typically located at the extreme 5’ and 3’ ends of the vector genome in order to allow the gene and regulatory sequences located between the ITRs to be packaged within the AAV capsid.
  • ITRs AAV inverted terminal repeat sequences
  • nuclease-resistant indicates that the AAV capsid has assembled around the expression cassette which is designed to deliver a transgene to a host cell and protects these packaged genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids which may be present from the production process.
  • a “vector genome” refers to the nucleic acid sequence packaged inside the rAAV capsid which forms a viral particle. Such a nucleic acid sequence contains AAV inverted terminal repeat sequences (ITRs).
  • ITRs AAV inverted terminal repeat sequences
  • a vector genome contains, at a minimum, from 5’ to 3’, an AAV 5’ ITR, coding sequence(s), and an AAV 3’ ITR.
  • ITRs from AAV2, a different source AAV than the capsid, or other than full-length ITRs may be selected, or may be self-complementary AAV.
  • the ITRs are from the same AAV source as the AAV which provides the rep function during production or a transcomplementing AAV. Further, other ITRs may be used.
  • the vector genome contains regulatory sequences which direct expression of the gene products. Suitable components of a vector genome are discussed in more detail herein.
  • non-viral genetic elements used in manufacture of a rAAV will be referred to as vectors (e.g., production vectors).
  • these vectors are plasmids, but the use of other suitable genetic elements is contemplated.
  • Such production plasmids may encode sequences expressed during rAAV production, e.g., AAV capsid or rep proteins required for production of a rAAV, which are not packaged into the rAAV.
  • such a production plasmid may carry the vector genome which is packaged into the rAAV.
  • an “expression cassette” refers to a nucleic acid molecule which comprises a coding sequence, promoter, and may include other regulatory sequences therefor.
  • a vector genome may contain two or more expression cassettes.
  • the term “transgene” may be used interchangeably with “expression cassette”.
  • translation in the context of the present invention relates to a process at the ribosome, wherein an mRNA strand controls the assembly of an amino acid sequence to generate a protein or a peptide.
  • target tissue refers to a tissue, an organ or a cell type, that an embodiment, a regimen or a composition as described herein targets.
  • the target tissue is a respiratory organ or a respiratory tissue.
  • the target tissue is lung.
  • the target tissue is nose.
  • the target tissue is nasopharynx.
  • the target tissue is respiratory epithelium.
  • the target tissue is nasal airway epithelium.
  • the target tissue is nasal cells.
  • the target tissue is nasopharynx cells.
  • the target tissue is nasal epithelial cells, which may be ciliated nasal epithelial cells, columnar epithelial cells, goblet cells (which secrete mucous onto the surface of the nasal cavity which is composed of the ciliated epithelial cells) and stratified squamous nasal epithelial cells which line the surface of the nasopharynx.
  • the target tissue is lung epithelial cells.
  • the target tissue is muscle, e.g., skeletal muscle.
  • a refers to one or more, for example, “an enhancer”, is understood to represent one or more enhancer(s).
  • the terms “a” (or “an”), “one or more,” and “at least one” is used interchangeably herein.

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Abstract

L'invention concerne une posologie d'immunisation passive contre un pathogène en suspension dans l'air qui comprend l'administration intranasale à un patient qui en a besoin d'une composition destinée à être administrée à l'épithélium nasal et au poumon comprenant au moins une construction d'anticorps comprenant un anticorps recombinant à commutation de classe (CSR) qui est un anticorps d'isotype d'immunoglobuline A ou d'immunoglobuline M qui neutralise le pathogène en suspension dans l'air cible et/ou un scFv qui neutralise le pathogène en suspension dans l'air cible. L'invention concerne également un procédé de prévention d'une infection par le SARS-CoV-2 comprenant l'administration de protéines d'immunoglobuline A anti-SARS-CoV-2 et/ou d'une protéine d'immunoglobuline M anti-SARS-CoV-2 à un sujet humain en une quantité efficace pour prévenir ou réduire une infection par le SARS-CoV-2.
PCT/US2021/053169 2020-10-02 2021-10-01 Compositions pour une immunisation passive administrée par voie intranasale contre des pathogènes en suspension dans l'air WO2022072829A1 (fr)

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