Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/081—DNA viruses
  • C07K16/085—Orthoherpesviridae (F), e.g. pseudorabies virus or Epstein-Barr virus
  • C07K16/087—Herpes simplex virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • 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
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
  • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
• • 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
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • 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)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/75—Agonist effect on antigen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • 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
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
  • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
  • C12N2710/16643—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
  • C12N2710/16661—Methods of inactivation or attenuation
  • C12N2710/16662—Methods of inactivation or attenuation by genetic engineering

Definitions

• the present disclosure relates, in part, to recombinant nucleic acids (e.g., recombinant herpes viral genomes) comprising one or more polynucleotides encoding an antibody (or a portion thereof); to viruses (e.g., herpes viruses) comprising the recombinant nucleic acids; to compositions comprising the recombinant nucleic acids and/or viruses; to methods of their use (e.g., for localized, virus-mediated delivery and expression of the encoded antibody); and to articles of manufacture or kits thereof.
• recombinant nucleic acids e.g., recombinant herpes viral genomes
• viruses e.g., herpes viruses
• compositions comprising the recombinant nucleic acids and/or viruses
• methods of their use e.g., for localized, virus-mediated delivery and expression of the encoded antibody
• therapeutic antibodies have become one of the commercially most successful classes of biopharmaceutical drugs. While antibodies have shown success in the treatment of several major diseases including autoimmune, cardiovascular, and infectious diseases, cancer, and inflammation, systemic administration of therapeutic antibodies has a number of functional limitations, including inadequate pharmacokinetics and tissue accessibility. In addition, systemic exposure to certain antibodies has been shown to repress the immune system, exposing the patient to significant risk of infections and other complications. Accordingly, there is need for alternative strategies to administer therapeutic antibodies to patients in need thereof.
• recombinant nucleic acids e.g., recombinant herpes viral genomes
• antibodies e.g., full-length antibodies, antibody fragments, etc.
• viruses e.g., herpes viruses
• compositions, formulations, medicaments, and/or methods useful for delivering e.g., topically, intraarticularly, intravitreally, etc.
• the present inventors have shown that the recombinant, attenuated viruses described herein were capable of 1) expressing both full-length antibodies and antibody fragments (scFv-Fcs), 2) encoding and delivering mouse, chimeric, and fully human antibodies (of various IgG isotypes), and 3) inducing dose-dependent secretion of the encoded antibodies from human cells, which were functional (see e.g., Example 2).
• the present inventors have shown that the viruses described herein may be used to successfully express their encoded antibodies in vivo after localized administration (see e.g., Example 3).
• viruses described herein may be used to successfully express a therapeutic antibody to treat one or more signs of an inflammatory skin condition (atopic dermatitis) after topical administration (see e.g., Example 3).
• atopic dermatitis an inflammatory skin condition
• the recombinant nucleic acids e.g., recombinant viral genomes
• viruses, pharmaceutical compositions, medicaments, and/or methods described herein provide a novel system for delivering therapeutic antibodies to a patient.
• the recombinant herpes viruses described herein provide a unique system to locally administer a therapeutic antibody to a subject in order to: 1) improve antibody pharmacokinetics at the site of interest; 2) increase antibody tissue accessibility and/or infiltration; 3) reduce the total dose of the antibody administered to the subject; 4) provide a less invasive or non-invasive method of administering an antibody to the subject; and/or 5) reduce or eliminate systemic exposure of the subject to the antibody (e.g., to avoid one or more side-effects (such as global immune suppression) observed after systemic administration of certain antibodies).
• the antibody is an antibody fragment.
• the antibody fragment is a Fab, Fab’ Fab’-SH, F(ab’)2, Fv, scFv, or scFv-Fc fragment.
• the antibody fragment is an scFv-Fc fragment.
• the scFv-Fc comprises the Fc region of an IgG antibody (e.g., the Fc region of an IgGl, IgG2, IgG3, or IgG4 antibody).
• the scFv-Fc comprises the Fc region of an IgGl antibody. In some embodiments, the scFv-Fc comprises the Fc region of an IgG4 antibody. In some embodiments, the antibody is a full-length antibody. In some embodiments that may be combined with any of the preceding embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, a monoclonal antibody, or a multispecific antibody. In some embodiments that may be combined with any of the preceding embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody.
• the antibody is an IgG antibody.
• the IgG antibody is an IgGl, IgG2, IgG3, or IgG4 antibody.
• the IgG antibody is an IgGl antibody.
• the IgG antibody is an IgG4 antibody.
• the antibody is an agonist antibody or an antagonist antibody.
• the antibody is an agonist antibody.
• the antibody is an antagonist antibody.
• the antibody comprises a heavy chain variable region comprising an F1VR-F11, an F1VR-F12, and an F1VR-F13, wherein the F1VR-F11 comprises a sequence selected from SEQ ID NOS: 1-59, the F1VR-F12 comprises a sequence selected from SEQ ID NOS: 60-122, and/or the F1VR-F13 comprises a sequence selected from SEQ ID NOS: 123-185.
• the antibody comprises a light chain variable region comprising an F1VR-L1, an F1VR-L2, and an F1VR- L3, wherein the F1VR-L1 comprises a sequence selected from SEQ ID NOS: 186-242, the F1VR-L2 comprises a sequence selected from SEQ ID NOS: 243-294, and/or the F1VR-L3 comprises a sequence selected from SEQ ID NOS: 295-354.
• the antibody comprises a heavy chain variable region comprising a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOS: 355-419 or 614-865.
• the antibody comprises a light chain variable region comprising a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOS: 420-482 or 866-1116.
• the antibody comprises: (a) a heavy chain variable region comprising a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOS: 355- 419 or 614-865; and (b) a light chain variable region comprising a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOS: 420-482 or 866-1116.
• the antibody is selected from abagovomab, abciximab, abituzumab, abrezekimab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afasevikumab, afelimomab, afutuzumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab, andecaliximab, anetumab, anifrolumab, anrukinzumab, apolizumab, aprutumab, arcitumomab, ascrinvacumab, aselizumab, atezolizumab, atinumab, atlizumab, ator
• duvortuxizumab duvortuxizumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezanumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emapalumab, emibetuzumab, emicizumab, enapotamab, enavatuzumab, enfortumab, enlimomab, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxom
• plozalizumab pogalizumab, polatuzumab, ponezumab, porgaviximab, prasinezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranevetmab, ranibizumab, raxibacumab, ravagalimab, ravulizumab, refanezumab, regavirumab, remtolumab, reslizumab,
• rilotumumab rinucumab, risankizumab, rituximab, rivabazumab, robatumumab, roledumab, romilkimab, romosozumab, rontalizumab, rosmantuzumab, rovalpituzumab, rovelizumab, rozanolixizumab, ruplizumab, sacituzumab, samalizumab, samrotamab, sapelizumab, sarilumab, satralizumab, satumomab, secukinumab, selicrelumab, seribantumab,
• setoxaximab setrusumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, pumpuzumab, siplizumab, sirtratumab, sirukumab, sofituzumab, solanezumab, solitomab, sonepcizumab, thankuzumab, spartalizumab, stamulumab, sulesomab, suptavumab, sutimlimab, suvizumab, suvratoxumab, tabalumab, tacatuzumab, tadocizumab, talacotuzumab, talizumab, tamtuvetmab, tanezumab, taplitumomab, tarextumab, tavolimab, tefibazumab, telimom
• timigutuzumab timolumab, tiragotumab, tislelizumab, tisotumab, tocilizumab,
• tomuzotuximab toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, tregalizumab, tremelimumab, trevogrumab, tucotuzumab, tuvirumab, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, utomilumab,
• vadastuximab vanalimab, vandortuzumab, vantictumab, vanucizumab, vapaliximab, varisacumab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab,
• vesencumab visilizumab, vobarilizumab, volociximab, vonlerolizumab, vopratelimab, vorsetuzumab, votumumab, vunakizumab, xentuzumab, zalutumumab, zanolimumab, zatuximab, zenocutuzumab, ziralimumab, zolbetuximab, and zolimomab.
• the recombinant herpes virus genome is replication competent. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes virus genome is replication defective.
• the recombinant herpes virus genome is selected from a recombinant herpes simplex virus genome, a recombinant varicella zoster virus genome, a recombinant human cytomegalovirus genome, a recombinant herpesvirus 6A genome, a recombinant herpesvirus 6B genome, a recombinant herpesvirus 7 genome, a recombinant Kaposi’s sarcoma-associated herpesvirus genome, and any derivatives thereof.
• the recombinant herpes virus genome is a recombinant herpes simplex virus genome.
• the recombinant herpes simplex virus genome is a recombinant type 1 herpes simplex virus (HSV-l) genome, a recombinant type 2 herpes simplex virus (HSV-2) genome, or any derivatives thereof.
• the recombinant herpes simplex virus genome is a recombinant type 1 herpes simplex virus (HSV-l) genome.
• the recombinant herpes simplex virus genome comprises an inactivating mutation.
• the inactivating mutation is in a herpes simplex virus gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the herpes simplex virus gene. In some embodiments, the herpes simplex virus gene is selected from Infected Cell Protein (ICP) 0, ICP4, ICP22, ICP27,
• ICP Infected Cell Protein
• ICP47 thymidine kinase (tk), Long Unique Region (UL) 41, and UL55.
• tk thymidine kinase
• UL Long Unique Region
• the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP4 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP0 gene.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the Joint region. In some embodiments, the recombinant herpes simplex virus genome comprises a deletion of the Joint region.
• the recombinant herpes simplex virus genome comprises the one or more polynucleotides within one or both of the ICP4 viral gene loci.
• the recombinant herpes virus genome has reduced cytotoxicity when introduced into a target cell, as compared to a corresponding wild-type herpes virus genome.
• the target cell is a human cell.
• herpes virus comprising any of the recombinant herpes virus genomes described herein.
• the herpes virus is replication competent.
• the herpes virus is replication defective.
• the herpes virus is attenuated.
• the herpes virus has reduced cytotoxicity as compared to a corresponding wild-type herpes virus.
• the herpes virus is selected from a herpes simplex virus, a varicella zoster virus, a human cytomegalovirus, a herpesvirus 6A, a herpesvirus 6B, a herpesvirus 7, and a Kaposi’s sarcoma-associated herpesvirus.
• the herpes virus is a herpes simplex virus.
• the herpes simplex virus is a type 1 herpes simplex virus (HSV-l), a type 2 herpes simplex virus (HSV-2), or any derivatives thereof.
• the herpes simplex virus is a type 1 herpes simplex virus (HSV-l).
• compositions comprising: (a) any of the recombinant herpes virus genomes described herein and/or any of the herpes viruses described herein; and (b) a pharmaceutically acceptable excipient.
• the pharmaceutical composition is suitable for topical, transdermal, subcutaneous, intradermal, transmucosal, oral, intranasal, intratracheal, sublingual, nasal, buccal, rectal, vaginal, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, intraorbital, intravitreal, subconjunctival, suprachoroidal, subretinal, intraarticular, peri-articular, local, epicutaneous, and/or inhaled administration.
• the pharmaceutical composition is suitable for topical administration.
• the pharmaceutical composition is suitable for inhaled administration.
• the pharmaceutical composition is suitable for injection.
• aspects of the present disclosure relate to the use of any of the recombinant herpes virus genomes, herpes viruses, and/or pharmaceutical compositions described herein as a medicament.
• Other aspects of the present disclosure relate to the use of any of the recombinant herpes virus genomes, herpes viruses, and/or pharmaceutical compositions described herein in a therapy.
• the disease is an inflammatory skin disease (e.g., atopic dermatitis).
• the disease is selected from psoriasis, atopic dermatitis, pyoderma gangrenosum, a blistering disease, pemphigus, pemphigus vulgaris, pemphigus foliaceus, an autoimmune bullous skin disorder, bullous pemphigoid, Behi j et’s disease, cancer, hidradenitis suppurativa, arthritis, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, juvenile idiopathic arthritis, ankylosing spondylitis, axial spondylarthritis, reactive arthritis, enteropathic arthritis, an autoimmune disease, asthma, thyroid eye disease, an infectious disease, and a neurological disease.
• aspects of the present disclosure relate to a method of administering an antibody to a subject comprising administering to the subject an effective amount of any of the herpes viruses described herein and/or any of the pharmaceutical compositions described herein.
• the herpes virus or pharmaceutical composition is
• transdermally administered topically, transdermally, subcutaneously, intradermally, transmucosally, orally, intranasally, intratracheally, sublingually, nasally, buccally, rectally, vaginally, intravenously, intraarterially, intramuscularly, intracardially, intraosseously, intraperitoneally, intraorbitally, intravitreally, subconjunctivally, suprachoroidally, subretinally, intraarticularly, peri- articularly, locally, epicutaneously, or via inhalation.
• aspects of the present disclosure relate to a method of providing prophylactic, palliative, and/or therapeutic relief of one or more signs or symptoms of a disease in a subject comprising administering to the subject an effective amount of any of the herpes viruses described herein and/or any of the pharmaceutical compositions described herein.
• the herpes virus or pharmaceutical composition is
• transdermally administered topically, transdermally, subcutaneously, intradermally, transmucosally, orally, intranasally, intratracheally, sublingually, nasally, buccally, rectally, vaginally, intravenously, intraarterially, intramuscularly, intracardially, intraosseously, intraperitoneally, intraorbitally, intravitreally, subconjunctivally, suprachoroidally, subretinally, intraarticularly, peri- articularly, locally, epicutaneously, or via inhalation.
• the disease is selected from psoriasis, atopic dermatitis, pyoderma gangrenosum, a blistering disease, pemphigus, pemphigus vulgaris, pemphigus foliaceus, an autoimmune bullous skin disorder, bullous pemphigoid, Behcet’s disease, cancer, hidradenitis suppurativa, arthritis, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, juvenile idiopathic arthritis, ankylosing spondylitis, axial spondylarthritis, reactive arthritis, enteropathic arthritis, asthma, an autoimmune disease, thyroid eye disease, an infectious disease, and a neurological disease.
• aspects of the present disclosure relate to a method of an antibody to the epidermis and/or dermis of a subject comprising topically, transdermally, subcutaneously, or intradermally administering to the subject an effective amount of any of the herpes viruses described herein and/or any of the pharmaceutical compositions described herein.
• the skin of the subject is abraded or made more permeable prior to administration.
• Other aspects of the present disclosure relate to a method of administering an antibody to one or more joints of a subject comprising intraarticularly and/or peri-articularly administering to the subject an effective amount any of the herpes viruses described herein and/or any of the pharmaceutical compositions described herein.
• the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the subject is not exposed to the antibody systemically.
• HSV herpes simplex virus
• the antibody is an antibody fragment.
• the antibody fragment is a Fab, Fab’ Fab’-SFl, F(ab’)2, Fv, scFv, or scFv-Fc fragment.
• the antibody is a full-length antibody.
• the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, a monoclonal antibody, or a multispecific antibody.
• the antibody is an IgA, IgD, IgE, IgG, or IgM antibody.
• the antibody is an IgG antibody.
• the IgG antibody is an IgGl, IgG2, IgG3, or IgG4 antibody.
• the antibody is an agonist antibody.
• the antibody is an antagonist antibody.
• the antibody comprises a heavy chain variable region comprising an F1VR-F11, an F1VR-F12, and an F1VR-F13, wherein the F1VR-F11 comprises a sequence selected from the group consisting of SEQ ID NOS: 1-59, the F1VR-F12 comprises a sequence selected from the group consisting of SEQ ID NOS: 60-122, and/or the F1VR-F13 comprises a sequence selected from the group consisting of SEQ ID NOS: 123-185.
• the heavy chain variable region comprises a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 355-419.
• the antibody comprises a light chain variable region comprising an F1VR-L1, an F1VR-L2, and an F1VR-L3, wherein the F1VR-L1 comprises a sequence selected from the group consisting of SEQ ID NOS: 186-242, the F1VR-L2 comprises a sequence selected from the group consisting of SEQ ID NOS: 243-294, and/or the F1VR-L3 comprises a sequence selected from the group consisting of SEQ ID NOS: 395-354.
• the light chain variable region comprises a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NOS: 420-482.
• the recombinant genome is a recombinant HSV-1 genome, a recombinant HSV-2 genome, or any derivatives thereof.
• the recombinant genome comprises an inactivating mutation in a herpes simplex virus gene.
• the herpes simplex virus gene is selected from the group consisting of Infected Cell Protein (ICP) 0, ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), Long Unique Region (UL) 41, and UL55.
• the recombinant genome comprises an inactivation mutation in one or both copies of the ICP4 gene. In some embodiments, the recombinant genome comprises an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant genome comprises an inactivating mutation in the UL41 gene. In some embodiments, the recombinant genome comprises an inactivating mutation in the ICP0 gene. In some embodiments, the recombinant genome comprises an inactivating mutation in the ICP27 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the gene(s).
• the recombinant genome has reduced cytotoxicity when introduced into a target cell as compared to a wild-type herpes simplex virus genome.
• the target cell is a human cell.
• the target cell is a keratinocyte or fibroblast.
• the recombinant genome comprises the one or more polynucleotides within one or more viral gene loci. In some embodiments, the recombinant genome comprises the one or more polynucleotides within one or both of the ICP4 viral gene loci. In some embodiments, the recombinant genome comprises the one or more polynucleotides within the ICP22 viral gene locus. In some embodiments, the recombinant genome comprises the one or more polynucleotides within the UL41 viral gene locus.
• HSV herpes simplex virus
• the HSV is replication competent.
• the HSV is replication defective.
• the HSV has reduced cytotoxicity as compared to a wild-type herpes simplex virus.
• the HSV is a herpes simplex type 1 virus, a herpes simplex type 2 virus, or any derivatives thereof.
• compositions comprising any of the recombinant genomes and/or viruses described herein and a pharmaceutically acceptable excipient.
• the pharmaceutical composition is suitable for topical, transdermal, subcutaneous, intradermal, transmucosal, sublingual, nasal, buccal, intraorbital, intravitreal, subconjunctival, suprachoroidal, intraarticular, and/or inhaled administration.
• the pharmaceutical composition is suitable for topical administration.
• the pharmaceutical composition comprises a hydroxypropyl methylcellulose gel.
• the pharmaceutical composition comprises a phosphate buffer. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition comprises glycerol. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition comprises a lipid carrier. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition comprises a nanoparticle carrier.
• viruses or pharmaceutical compositions described herein relate to a method of administering an antibody to a subject comprising administering to the subject an effective amount of any of the viruses or pharmaceutical compositions described herein.
• the virus or composition is administered topically, transdermally, subcutaneously, intradermally, transmucosally, sublingually, nasally, buccally, intravitreally, subconjunctivally,
• the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the subject is not exposed to the antibody systemically.
• viruses or pharmaceutical compositions described herein relate to a method of providing prophylactic, palliative, and/or therapeutic relief of one or more signs or symptoms of a disease in a subject comprising administering to the subject and effective amount of any of the viruses or pharmaceutical compositions described herein.
• the virus or composition is administered topically, transdermally, subcutaneously, intradermally, transmucosally, sublingually, nasally, buccally, intravitreally, subconjunctivally, suprachoroidally, intraarticularly, or via inhalation.
• the disease is selected from the group consisting of psoriasis, atopic dermatitis, pyoderma gangrenosum, a blistering disease, pemphigus, pemphigus vulgaris, pemphigus foliaceus, an autoimmune bullous skin disorder, bullous pemphigoid, Behi j et’s disease, cancer, hidradenitis suppurativa, arthritis, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, juvenile idiopathic arthritis, ankylosing spondylitis, axial spondylarthritis, reactive arthritis, enteropathic arthritis, autoimmune disease, melanoma, uveal melanoma, and thyroid eye disease.
• the disease is not cancer.
• the subject is a human. In some embodiments that may be used
• Other aspects of the present disclosure relate to a method of administering an antibody to the epidermis and/or dermis of a subject comprising topically, transdermally, or intradermally administering to the subject an effective amount of any of the viruses or pharmaceutical compositions described herein.
• the skin of the subject is abraded prior to administration.
• the subject is a human.
• the subject is not exposed to the antibody systemically.
• the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the subject is not exposed to the antibody systemically.
• aspects of the present disclosure relate to a method of administering an antibody to one or more joints of a subject comprising intraarticularly administering to the subject an effective amount of any of the viruses or pharmaceutical compositions described herein.
• the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the subject is not exposed to the antibody systemically.
• Other aspects of the present disclosure relate to a method of administering an antibody to one or both eyes of a subject comprising topically, intraorbitally, intravitreally, subconjunctivally, or suprachoroidally administering to the subject an effective amount of any of the viruses or pharmaceutical compositions described herein.
• the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the subject is not exposed to the antibody systemically.
• FIGS. 1A-N show schematics of wild-type and modified herpes simplex virus genomes.
• FIG. 1A shows a wild-type herpes simplex virus genome.
• FIG. IB shows a modified herpes simplex virus genome comprising deletions of the coding sequences of ICP4 (both copies) and ICP22, with a polynucleotide containing the coding sequence of a single chain antibody (an scFv-Fc) operably linked to a heterologous promoter integrated at each of the ICP4 loci.
• FIG. 1A shows a wild-type herpes simplex virus genome.
• FIG. IB shows a modified herpes simplex virus genome comprising deletions of the coding sequences of ICP4 (both copies) and ICP22, with a polynucleotide containing the coding sequence of a single chain antibody (an scFv-Fc) operably linked to a heterologous promoter integrated at each of the ICP4 loci
• FIG. 1C shows a modified herpes simplex virus genome comprising deletions of the coding sequence of ICP4 (both copies), with a polynucleotide containing the coding sequence of an scFv-Fc operably linked to a heterologous promoter integrated at each of the ICP4 loci.
• FIG. ID shows a modified herpes simplex virus genome comprising deletions of the coding sequences of ICP4 (both copies) and ICP22, with a polynucleotide containing 1) the coding sequence of an antibody heavy chain operably linked to a first heterologous promoter, and 2) the coding sequence of an antibody light chain operably linked to a second heterologous promoter, integrated at each of the ICP4 loci.
• FIG. IE shows a modified herpes simplex virus genome comprising deletions of the coding sequence of ICP4 (both copies), with a polynucleotide containing 1) the coding sequence of an antibody heavy chain operably linked to a first heterologous promoter, and 2) the coding sequence of an antibody light chain operably linked to a second heterologous promoter, integrated at each of the ICP4 loci. Both the antibody heavy and light chains are encoded on the same strand of DNA.
• FIG. IE shows a modified herpes simplex virus genome comprising deletions of the coding sequence of ICP4 (both copies), with a polynucleotide containing 1) the coding sequence of an antibody heavy chain operably linked to a first heterologous promoter, and 2) the coding sequence of an antibody light chain operably linked to a second heterologous promoter, integrated at each of the ICP4 loci. Both the antibody heavy and light chains are encoded on the same strand of DNA.
• FIG. IE shows a modified herpe
• IF shows a modified herpes simplex virus genome comprising deletions of the coding sequences of ICP4 (both copies) and ICP22, with a polynucleotide containing 1) the coding sequence of an antibody heavy chain operably linked to a first heterologous promoter, and 2) the coding sequence of an antibody light chain operably linked to a second heterologous promoter, integrated at each of the ICP4 loci.
• the antibody heavy and light chains are encoded on opposite strands of DNA.
• 1G shows a modified herpes simplex virus genome comprising deletions of the coding sequence of ICP4 (both copies), with a polynucleotide containing 1) the coding sequence of an antibody heavy chain operably linked to a first heterologous promoter, and 2) the coding sequence of an antibody light chain operably linked to a second heterologous promoter, integrated at each of the ICP4 loci.
• the antibody heavy and light chains are encoded on opposite strands of DNA.
• 1H shows a modified herpes simplex virus genome comprising deletions of the coding sequences of ICP4 (both copies) and ICP22, with a polynucleotide encoding a polycistronic mRNA operably linked to a heterologous promoter integrated at each of the ICP4 loci.
• the polycistronic mRNA contains the coding sequence of an antibody heavy chain and an antibody light chain separated by an internal ribosomal entry site (IRES).
• ICP4 both copies
• ICP22 internal ribosomal entry site
• FIG. II shows a modified herpes simplex virus genome comprising deletions of the coding sequence of ICP4 (both copies), with a polynucleotide encoding a polycistronic mRNA operably linked to a heterologous promoter integrated at each of the ICP4 loci.
• the polycistronic mRNA contains the coding sequence of an antibody heavy chain and an antibody light chain separated by an internal ribosomal entry site (IRES).
• 1J shows a modified herpes simplex virus genome comprising deletions of the coding sequences of ICP4 (both copies), ICP22, and UL41, with a first polynucleotide containing the coding sequence of an antibody heavy chain operably linked to a heterologous promoter integrated at each of the ICP4 loci, and a second polynucleotide containing the coding sequence of an antibody light chain operably linked to a heterologous promoter integrated at the UL41 and ICP22 loci.
• IK shows a modified herpes simplex virus genome comprising deletions of the coding sequences of ICP4 (both copies), ICP22, and UL41, with a first polynucleotide containing the coding sequence of an antibody light chain operably linked to a heterologous promoter integrated at each of the ICP4 loci, and a second polynucleotide containing the coding sequence of an antibody heavy chain operably linked to a heterologous promoter integrated at the UL41 and ICP22 loci.
• 1L shows a modified herpes simplex virus genome comprising deletions of the coding sequences of ICP4 (both copies) and UL41, with a first polynucleotide containing the coding sequence of an antibody heavy chain operably linked to a heterologous promoter integrated at each of the ICP4 loci, and a second polynucleotide containing the coding sequence of a polycistronic mRNA operably linked to a heterologous promoter integrated at the UL41 locus.
• the polycistronic mRNA contains two copies of the coding sequence of an antibody light chain separated by an internal ribosomal entry site (IRES).
• 1M shows a modified herpes simplex virus genome comprising deletions of the coding sequences of ICP4 (both copies) and ICP22, with a first polynucleotide containing the coding sequence of an antibody heavy chain operably linked to a heterologous promoter integrated at each of the ICP4 loci, and a second polynucleotide containing the coding sequence of a polycistronic mRNA operably linked to a heterologous promoter integrated at the ICP22 locus.
• the polycistronic mRNA contains two copies of the coding sequence of an antibody light chain separated by an internal ribosomal entry site (IRES).
• IN shows a modified herpes simplex virus genome comprising deletions of the coding sequences of ICP4 (both copies), ICP22, and UL41, with a first polynucleotide containing the coding sequence of an antibody heavy chain operably linked to a heterologous promoter integrated at the ICP22 locus, and a second polynucleotide containing the coding sequence of an antibody light chain operably linked to a heterologous promoter integrated at the UL41 locus.
• FIGS. 2A-B show the antibody concentration in cell supernatants harvested from mock infected (MOI-0) immortalized human keratinocytes (HaCaTs), or HaCaTs infected at the indicated multiplicities of infection (MOI) with engineered HSV vectors encoding the indicated antibodies, as assessed by ELISA.
• FIG. 2A shows the antibody concentration in cell supernatants harvested from HaCaT cells infected with HSV encoding a human (AblFcl or AblFc2) or chimeric (Ab2Fc2) single-chain antibody.
• FIG.2B shows the antibody concentration in cell supernatants harvested from HaCaT cells infected with HSV encoding a mouse (Ab66Fcl or Ab66Fc2) single-chain antibody. For each condition, data is presented for two replicates ⁇ SEM.
• FIG. 3 shows the detectable levels of recombinant human TNFa spiked into cell supernatants harvested from mock infected (MOI 0) immortalized human keratinocytes (HaCaTs), or HaCaTs infected at the indicated multiplicities of infection (MOI) with an engineered HSV vector encoding an anti-TNFa human single-chain antibody (AblFcl), as assessed by ELISA. For each condition, data is presented for two replicates ⁇ SEM.
• FIGS. 4A-B show the relative fold change vs. ethanol (EtOH) control in transcript levels of certain markers of atopic dermatitis-like lesions in mouse ear and dorsal skin treated topically for five days with the vitamin D3 synthetic analog calcipotriol (MC903), as assessed by qRT-PCR analysis.
• FIG. 4A shows the relative fold change in TSLP transcripts vs. EtOH control in mouse ear and dorsal skin treated topically with MC903 on Days 1-5, with tissues being harvested on Day 5 or Day 7.
• FIG. 4B shows the relative fold change in IL-4 transcripts vs. EtOH control in mouse ear and dorsal skin treated topically with MC903 on Days 1-5, with tissues being harvested on Day 5, Day 7, or Day 9.
• FIG. 5 show the histology of representative mouse ear skin treated with MC903 or EtOH control, as assessed by hematoxylin and eosin (H&E) staining.
• FIGS. 6A-B show representative immunofluorescence images of human single chain antibody (AblFcl) expression in ear and dorsal skin biopsies harvested from MC903- exposed C57BL/6J mice treated topically with either HSV-AblFcl or a negative control (vehicle). DAPI staining was used to visualize nuclei.
• FIG. 6A shows AblFcl expression in mouse ear and dorsal skin treated with MC903 on Days 1-5 and topical HSV-AblFcl (or vehicle control) on Day 5, with tissues being harvested on Day 7.
• FIG. 6B shows AblFcl expression in mouse ear and dorsal skin treated with MC903 on Days 1-5 and topical HSV- AblFcl (or vehicle control) on Day 7, with tissues being harvested on Day 9.
• FIGS. 7A-C show mouse anti-mouse IF-4Ra antibody (Ab66Fcl) nucleic acid analyses of ear and dorsal skin in an MC903-induced atopic dermatitis model after HSV- Ab66Fcl infection.
• FIG. 7A shows the levels of Ab66Fcl DNA present in ear tissue biopsies harvested from MC903- or ethanol (EtOH) treated- animals after repeated topical application of HSV-Ab66Fcl or vehicle control, as determined by qPCR analysis.
• FIG. 7B shows the levels of Ab66Fcl DNA present in dorsal skin tissue biopsies harvested from MC903- or ethanol (EtOH) treated- animals after repeated topical application of HSV-Ab66Fcl or vehicle control, as determined by qPCR analysis.
• FIG. 7C shows the levels of Ab66Fcl transcripts present in ear tissue biopsies harvested from MC903- or ethanol (EtOH) treated- animals after repeated topical application of HSV-Ab66Fcl or vehicle control, as determined by qRT-PCR analysis. For each condition in the qPCR and qRT-PCR analysis, data is presented for two replicates ⁇ SEM.
• FIGS. 8A-B show the effect of HSV-Ab66Fcl or vehicle control on the development of certain ear phenotypes in an MC903-induced atopic dermatitis model.
• FIG. 8A shows average ear thickness on Days 1-10 of MC903- or ethanol treated- animals after repeated topical application of HSV-Ab66Fcl or vehicle control. Asterisks indicate statistically significant differences between the MC903/Veh and MC903/Ab66 groups at each timepoint.
• FIG. 8B shows average ear weight on Day 10 of MC903- or ethanol treated- animals after repeated topical application of HSV-Ab66Fcl or vehicle control. For each timepoint, data is presented for the average of four ears ⁇ SEM. Statistics were calculated using an unpaired student’s t-test: *p ⁇ 0.05; **r ⁇ 0.01 ; ***p ⁇ 0.005.
• the term“and/or” may include any and all combinations of one or more of the associated listed items.
• the term“a and/or b” may refer to“a alone”,“b alone”,“a or b”, or“a and b”;
• the term“a, b, and/or c” may refer to“a alone”,“b alone”,“c alone”,“a or b”,“a or c”,“b or c”,“a, b, or c”,“a and b”,“a and c”,“b and c”, or “a, b, and c”; etc.
• the term“about” refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to“about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.
• aspects and embodiments of the present disclosure include “comprising”,“consisting”, and“consisting essentially of’ aspects and embodiments.
• polynucleotide As used herein, the terms“polynucleotide”, “nucleic acid sequence”, “nucleic acid” , and variations thereof shall be generic to polydeoxyribonucleotides (containing 2- deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and to other polymers containing non-nucleotidic backbones, provided that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, as found in DNA and RNA.
• these terms include known types of nucleic acid sequence modifications, for example, substitution of one or more of the naturally occurring nucleotides with an analog, and inter nucleotide modifications.
• a nucleic acid is“operatively linked” or“operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
• a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
• “operatively linked” or“operably linked” means that the DNA or RNA sequences being linked are contiguous.
• an expression vector refers to discrete elements that are used to introduce heterologous nucleic acids into cells for either expression or replication thereof.
• An expression vector includes vectors capable of expressing nucleic acids that are operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such nucleic acids.
• an expression vector may refer to a DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the nucleic acids.
• Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and those that remain episomal or those which integrate into the host cell genome.
• an“open reading frame” or“ORF” refers to a continuous stretch of nucleic acids, either DNA or RNA, that encode a protein or polypeptide.
• the nucleic acids comprise a translation start signal or initiation codon, such as ATG or AUG, and a termination codon.
• an“untranslated region” or“UTR” refers to untranslated nucleic acids at the 5’ and/or 3’ ends of an open reading frame.
• the inclusion of one or more UTRs in a polynucleotide may affect post-transcriptional regulation, mRNA stability, and/or translation of the polynucleotide.
• transgene refers to a polynucleotide that is capable of being transcribed into RNA and translated and/or expressed under appropriate conditions, after being introduced into a cell. In some aspects, it confers a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic or diagnostic outcome.
• polypeptide As used herein, the terms“polypeptide,”“protein,” and“peptide” are used interchangeably and may refer to a polymer of two or more amino acids.
• antibody As used herein, the term“antibody” is used in the broadest sense, and
• antibody encompasses various antibody structures, including, for example monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc. ), and antibody fragments so long as they exhibit the desired biological activity.
• antibody also encompasses hybrid antibodies, altered antibodies, chimeric antibodies, and humanized antibodies.
• antibody includes: hybrid
• Patent Publication No. GB 2,276,169, published 21 Sep. 1994 Patent Publication No. GB 2,276,169, published 21 Sep. 1994; and, any functional fragments obtained from such molecules, wherein such fragments retain specific-binding properties of the parent antibody molecule.
• the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains and two identical heavy chains.
• VH variable heavy
• VL variable light
• the light chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (“k”) ( see e.g., SEQ ID NO: 601 for an exemplary human kappa constant domain sequence) and lambda (“l”) (see e.g., SEQ ID NO: 602 for an exemplary human lambda constant domain sequence), based on the amino acid sequences of their constant domains.
• kappa see e.g., SEQ ID NO: 601 for an exemplary human kappa constant domain sequence
• l lambda
• immunoglobulins can be assigned to different classes or isotypes.
• immunoglobulins There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha (“a”), delta (“d”), epsilon (“e”), gamma (“g”), and mu (“m”), respectively.
• the g and a classes are further divided into subclasses (isotypes) on the basis of relatively minor differences in the CH sequence and function, e.g., humans expressing the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.
• variable region or“variable domain” of an antibody refer to the amino-terminal domains of the heavy or light chain of the antibody. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.
• variable refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies.
• the variable domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen.
• variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) both in the light chain and the heavy chain variable domains.
• HVRs hypervariable regions
• FR framework regions
• hypervariable region refers to the regions of an antibody variable domain that are hypervariable in sequence and/or form structurally defined loops.
• antibodies comprise six HVRs; three in the VH (HVR-H1, HVR- H2, and HVR-H3), and three in the VL (HVR-L1, HVR-L2, and HVR-L3).
• HVR-H3 and HVR-L3 display the most diversity of the six HVRs, and HVR-H3 in particular is believed to play a unique role in conferring fine specificity to antibodies ( see e.g., Xu et al.
• HVR delineations are in use and are encompassed herein.
• the HVRs that are EU or Rabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used. Chothia refers instead to the location of the structure loops (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)).
• the AbM HVRs represent a compromise between the EU or Rabat CDRs and Chothia structural loops and are used by Oxford Molecular’s AbM antibody modeling software.
• The“contact” HVRs are based on an analysis of the available complex crystal structures.
• HVRs may comprise“extended HVRs” as follows: 24-36 or 24-24 (HVR-L1), 46- 56 or 50-46 (HVR-L2), and 89-97 or 89-96 (HVR-L3) in the VL, and 26-35 (HVR-H1), 50- 65 or 49-65 (HVR-H2), and 93-201, 94-102, or 95-102 (HVR-H3) in the VH.
• the variable domain residues are numbered according to EU or Rabat et al. for each of these extended HVR definitions.
• the terms“Framework” or“FR” refer to variable domain residues other than hypervariable region (HVR) residues.
• the FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences of a variable domain generally appear in the following sequence in VH (or VL): FR1-HVR-H1 (Ll)-FR2-HVR-H2 (L2)-FR3-HVR-H3 (L3)-FR4.
• the ter s“full-length antibody”,“intact antibody”, or“whole antibody” are used interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
• the constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.
• the intact antibody has one or more effector functions.
• the term“Fc region” is used to define a C-terminal region of an immunoglobulin heavy chain, including the native sequence Fc regions and variant Fc regions.
• the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
• the C-terminal lysine (residue 447 according to the EU or Rabat numbering system) of the Fc region may be removed, for example, during production of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
• Antibody“effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody and vary with the antibody isotype.
• the term“native antibodies” refers to antibodies that are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
• VH variable domain
• Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
• the term“monoclonal antibody” refers to an antibody obtained from a population of substantially homogenous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against one or more antigenic sites. In some embodiments, a monoclonal antibody of the present disclosure can be multispecific (e.g., a bispecific antibody, a trispecific antibody).
• each monoclonal antibody is directed against a single determinant on the one or more antigenic sites.
• the modifier“monoclonal” indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies and is not to be construed as requiring production of the antibody by and particular method.
• a“chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
• a“humanized” antibody is used as a subset of“chimeric” antibody.
• a“humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs.
• a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
• a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
• A“humanized form” of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
• a“human” antibody refers to an antibody which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition specifically excludes a humanized antibody comprising non-human antigen-binding residues.
• a“human consensus framework” refers to a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
• the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
• the subgroup of sequences is a subgroup as in Kabat at al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD, vols. 1-3.
• antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody.
• antibody fragments may include, without limitation, Fv, Fab, Fab’, Fab’-SH, F(ab’)2, scFv, SMIP, domain antibodies, di-scFv, scFv-Fc, Nanobodies ® (e.g., monovalent nanobodies, bivalent nanobodies, etc.), minibodies, diabodies, triabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
• Papain digestion of antibodies produces two identical antigen-binding fragments, called Fab fragments, and a residual Fc fragment (a designation reflecting the ability to crystallize readily).
• the Fab fragment consists of an entire light chain, along with the variable region of the heavy chain and the first constant domain (C H I) of one heavy chain.
• Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.
• the Fc fragment comprises the carboxy-terminal portions of both heavy chains held together by disulfide bonds.
• the effector functions of antibodies are determined by sequence in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.
• Pepsin treatment of an antibody yields a single large F(ab’)2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen.
• Fab’ fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the C H I domain including one or more cysteines from the antibody hinge region.
• Fab’-SFl is the designation for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group.
• F(ab’)2 antibody fragments originally were produced as pairs of Fab’ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
• The“Fv” is the minimum antibody fragment which contains a complete antigen- recognition and binding site. This fragment consists of a dimer of one heavy and one light chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. Flowever, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
• Single-chain Fv also abbreviated as“sFv” or“scFv”, are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
• the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
• A“nanobody” refers to a single-domain antibody (sdAb) which is able to bind selectively to an antigen.
• a nanobody may comprise heavy chain variable domains and no light chain variable domains, or vice versa.
• a nanobody may be derived from camelids (VHH antibodies) or cartilaginous fishes (VNAR antibodies).
• a nanobody may be derived from splitting the dimeric variable domains from an antibody, for example, an IgG antibody, into monomers.
• Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific.
• the term“diabodies” refers to small antibody fragments prepared by constructing scFv fragments with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites.
• Bispecific diabodies are heterodimers of two“crossover” scFv fragments in which the VF1 and VL domains of the two antibodies are present on different polypeptide chains.
• Diabodies are described in greater detail in, for example, EP404097, W093/11161, Fludson et al. (2003) Nat. Med. 9:129-134, and Hoolinger et al. PNAS USA 90: 6444-48 (1993). Triabodies and tetrabodies are also described in Hudson et al. (2003) Nat. Med. 9:129-134.
• the terms“specifically recognizes” or“specifically binds” refer to measurable and reproducible interactions, such as attraction or binding between a target and an antibody, that is determinative of the presence of the target in the presence of a heterogenous population of molecules including biological molecules.
• an antibody that specifically or preferentially binds to a target or an epitope is an antibody that binds this target or epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets or other epitopes of the target.
• an antibody (or moiety) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target.
• “specific binding” or“preferential binding” does not necessarily require (although it can include) exclusive binding.
• An“agonist” antibody or an“activating” antibody is an antibody that induces ( e.g ., increases) one or more activities or functions of the antigen after the antibody binds the antigen and/or that induces (e.g., increases) antigen binding to one or more ligands after the antibody binds the antigen.
• A“blocking” antibody, an“antagonist” antibody, or an“inhibitory” antibody is an antibody that inhibits or reduces (e.g., decreases) antigen binding to one or more ligands after the antibody binds the antigen and/or that inhibits or reduces (e.g., decreases) one or more activities or functions of the antigen after the antibody binds the antigen.
• an“blocking” antibody, an“antagonist” antibody, or an“inhibitory” antibody is an antibody that inhibits or reduces (e.g., decreases) antigen binding to one or more ligands after the antibody binds the antigen and/or that inhibits or reduces (e.g., decreases) one or more activities or functions of the antigen after the antibody binds the antigen.
• blocking antibodies, antagonist antibodies, or inhibitory antibodies substantially or completely inhibits antigen binding to one or more ligands and/or substantially or completely inhibits one or more activities or functions of the antigen.
• a“subject”,“host”, or an“individual” refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, as well as animals used in research, such as mice, rats, hamsters, rabbits, and non-human primates, etc.
• the mammal is human.
• the terms“pharmaceutical formulation” or“pharmaceutical composition” refer to a preparation which is in such a form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition or formulation would be administered.
• “Pharmaceutically acceptable” excipients e.g., vehicles, additives
• “cutaneous administration” or“cutaneously administering” refers to the delivery of a composition to a subject by contacting, directly or otherwise, a formulation comprising the composition to all (“systemic”) or a portion (“topical”) of the skin of a subject.
• the term encompasses several routes of administration including, but not limited to, topical and transdermal.
• Topical administration may be used as a means to deliver a composition to the epidermis or dermis of a subject, or to specific strata thereof.
• an“effective amount” is at least the minimum amount required to affect a measurable improvement or prevention of one or more symptoms of a particular disorder.
• An“effective amount” may vary according to factors such as the disease state, age, sex, and weight of the patient.
• An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects.
• beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, its
• beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications used to treat symptoms of the disease, delaying the progression of the disease, and/or prolonging survival.
• An effective amount can be administered in one or more administrations.
• an effective amount of a recombinant nucleic acid, virus, and/or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly.
• an effective amount of a recombinant nucleic acid, virus, and/or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition.
• an“effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
• treatment refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease/disorder/defect progression, ameliorating or palliating the disease/disorder/defect state, and remission or improved prognosis.
• the term“delaying progression of’ a disease/disorder/defect refers to deferring, hindering, slowing, retarding, stabilizing, and/or postponing development of the disease/disorder/defect. This delay can be of varying lengths or time, depending on the history of the disease/disorder/defect and/or the individual being treated. As is evident to one of ordinary skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.
• Certain aspects of the present disclosure relate to recombinant nucleic acids (e.g., isolated recombinant nucleic acids) comprising one or more (e.g., one or more, two or more, three or more, four or more, five or more, ten or more, etc.) polynucleotides encoding an antibody.
• the antibody may be any antibody (in any form) described herein or known in the art.
• the antibody is a full-length antibody.
• the antibody is an antibody fragment.
• the antibody is an agonist antibody.
• the antibody is an antagonist antibody.
• the recombinant nucleic acid is a vector. In some embodiments, the recombinant nucleic acid is a viral vector. In some embodiments, the recombinant nucleic acid is a herpes viral vector. In some embodiments, the recombinant nucleic acid is a herpes simplex virus amplicon. In some embodiments, the recombinant nucleic acid is a recombinant herpes virus genome. In some embodiments, the recombinant nucleic acid is a recombinant herpes simplex virus genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant type 1 herpes simplex virus (HSV-l) genome. Polynucleotides encoding antibodies
• the present disclosure relates to a recombinant nucleic acid (e.g., a recombinant herpes virus genome) comprising one or more (e.g., one or more, two or more, three or more, four or more, five or more, ten or more, etc.) polynucleotides encoding an antibody.
• at least one of the polynucleotides encodes a single-chain antibody (e.g., an scFv, an scFv-Fc, etc.).
• At least one of the polynucleotides comprises multiple expression cassettes encoding the antibody (e.g., a first expression cassette encoding an antibody heavy chain and a second expression cassette encoding an antibody light chain, etc.). In some embodiments, at least one of the
• polynucleotides encodes a polycistronic mRNA encoding the antibody (e.g., a polycistronic mRNA comprising an ORF encoding an antibody heavy chain and an ORF encoding an antibody light chain separated by an IRES, etc.).
• at least one of the polynucleotides encodes a chimeric polypeptide (e.g., a polypeptide comprising an antibody heavy chain and an antibody light chain separated by a cleavable linker, etc.).
• the recombinant genome comprises one polynucleotide encoding an antibody.
• the recombinant genome comprises two or more polynucleotides encoding an antibody (e.g., a first polynucleotide encoding an antibody heavy chain and a second polynucleotide encoding an antibody light chain, etc.).
• a first recombinant nucleic acid of the present disclosure comprises one or more polynucleotides encoding a portion of an antibody (e.g., an antibody heavy chain), and is used in conjunction with a second recombinant nucleic acid comprising one or more polynucleotides encoding a complementary portion of an antibody (e.g., an antibody light chain).
• the first and second recombinant nucleic acids are in a single composition (e.g., contained in separate herpes simplex viruses formulated as a single pharmaceutical composition).
• the first and second recombinant nucleic acids are in different compositions (e.g., contained in separate herpes simplex viruses formulated as two distinct pharmaceutical compositions).
• the first recombinant nucleic acid is delivered into a target cell prior to, in conjunction with, or after delivery of the second recombinant nucleic acid into the target cell (e.g., in order to produce a single full-length antibody in one or more cells of the subject).
• a recombinant nucleic acid of the present disclosure comprises polynucleotides encoding two or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) antibodies.
• the two or more antibodies are the same. In some embodiments, the two or more antibodies are different.
• Antibodies encoded by one or more of the polynucleotides of the present disclosure may be from any suitable species known in the art, including, for example, human antibodies, mouse antibodies, rat antibodies, rabbit antibodies, camelid antibodies, chicken antibodies, donkey antibodies, feline antibodies, goat antibodies, sheep antibodies, horse antibodies, hamster antibodies, guinea pig antibodies, shark antibodies, and any chimeric antibodies thereof.
• the antibody is a human antibody.
• the antibody is a mouse antibody.
• the antibody is a chimeric antibody (e.g., a human-mouse chimeric antibody).
• the antibody is a humanized antibody.
• Antibodies encoded by one or more of the polynucleotides of the present disclosure may be of any suitable isotype known in the art, including, for example, IgA, IgD, IgE, IgG, IgM, and any combinations thereof.
• the antibody is an IgG antibody.
• the IgG antibody is an IgGl antibody (see e.g., SEQ ID NOS: 596 or 597 for exemplary human IgGl constant region sequences), an IgG2 antibody (see e.g., SEQ ID NO: 598 for an exemplary human IgG2 constant region sequence), an IgG3 antibody (see e.g., SEQ ID NO: 599 for an exemplary IgG3 constant region sequence), an IgG4 antibody (see e.g., SEQ ID NO: 600 for an exemplary human IgG4 constant region sequence), and any chimeric IgG antibodies thereof.
• the IgG antibody is an IgGl antibody.
• an antibody encoded by one or more polynucleotides of the present disclosure is an antibody fragment.
• Any type or form of antibody fragment known in the art may be encoded by a polynucleotide of the present disclosure including, for example, a Fab fragment, a Fab’ fragment, a Fab’-SH fragment, a F(ab’) 2 fragment, an Fv fragment, an scFv fragment, an scFv-Fc fragment, as well as any other type or form of antibody fragment described herein or known in the art.
• the antibody fragment is a Fab fragment.
• the antibody fragment is an scFv.
• the antibody fragment is an scFv-Fc.
• an antibody encoded by one or more polynucleotides of the present disclosure is a chimeric antibody.
• Certain chimeric antibodies are described, e.g., in US 4,816,567, and Morrison et al. (1984) PNAS USA 81:6851-6855.
• a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, non-human primate, etc.) and a human constant region.
• a chimeric antibody is a“class switched” antibody in which the class or subclass has been changed from that of the parental antibody. Chimeric antibodies include antigen-binding fragments thereof.
• the chimeric antibody is a mouse- human chimeric antibody.
• an antibody encoded by one or more polynucleotides of the present disclosure is a humanized antibody.
• a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
• a humanized antibody comprises one or more variable domains in which HVRs (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
• a humanized antibody optionally will also comprise at least a portion of a human constant region.
• some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity and/or affinity.
• a non-human antibody e.g., the antibody from which the HVR residues are derived
• Human framework regions that may be used for humanization include, for example, framework regions selected using the“best-fit” method (see e.g., Sims et al. (1993) J Immunol 151: 2296), framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions ( see e.g., Carter et al. (1992) PNAS USA 89: 4285; see also Presta et al.
• an antibody encoded by one or more polynucleotides of the present disclosure is a human antibody.
• Human antibodies are generally described in van Dijk and van de Winkel (2001) Curr Opin Pharmacol 5: 368-74 and Lonberg (2008) Curr Opin Immunol 20: 450-459. Certain details regarding human antibodies can be found in, e.g., Hoogenboom and Winter, J. Mol. Biol. 227: 381 (1991); Marks et al. J. Mol. Biol. 222: 581 (1991); Cole et al. Monoclonal Antibodies and Cancer Therapy, p. 77 (1985); Boerner et al.
• an antibody encoded by one or more polynucleotides of the present disclosure is a multispecific antibody (e.g., a bispecific antibody, a trispecific antibody, etc.).
• Techniques for making multispecific antibodies include, for example, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see e.g., Mil stein and Cuello, Nature 305: 537 (1983), WO 93/08829, Traunecker et al. EMBO J. 10: 3655 (1991), and“knob-in-hole” engineering (e.g., as described in in US Pat. No. 5,731,168)).
• Multispecific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (see e.g., W02009/089004); cross-linking two or more antibodies or fragments (see e.g., US Pat. No. 4,676,980, Brennan et al. Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see e.g., Kostelny et al. J Immunol, 148(5): 1547-53 (1992)); using “diabody” technology for making bispecific antibody fragments (see e.g., Hollinger et al. PNAS USA 90:6444-8 (1993); using single-chain Fv dimers (see e.g., Gruber et al. J
• Antibodies (or antigen-binding fragments thereof) encoded by one or more polynucleotides of the present disclosure may contain: 1) an HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of any antibody described herein or known in the art; 2) the heavy chain variable region and/or light chain variable region of any antibody described herein or known in the art; and/or 3) the full-length heavy chain and/or full-length light chain of any antibody described herein or known in the art.
• Suitable antibodies that may be encoded by the polynucleotides of the present disclosure include, for example, abagovomab, abciximab (V H -SEQ ID NO: 617; V L -SEQ ID NO: 869), abituzumab (V H -SEQ ID NO: 618; V L -SEQ ID NO: 870), abrezekimab, abrilumab (V H -SEQ ID NO: 619; V L -SEQ ID NO: 871), actoxumab (V H -SEQ ID NO: 620; V L -SEQ ID NO: 872), adalimumab (V H - SEQ ID NO: 355; V L -SEQ ID NO: 420), adecatumumab, aducanumab (V H -SEQ ID NO: 621 ; V L -SEQ ID NO: 873), afasevikumab (V H H
• V H -SEQ ID NO: 880 ascrinvacumab, aselizumab, atezolizumab (V H -SEQ ID NO: 374; V L -SEQ ID NO: 439), atinumab, atlizumab, atorolimumab, avelumab (V H -SEQ ID NO: 630; V L -SEQ ID NO: 882), azintuxizumab (V H -SEQ ID NO: 631 ; V L -SEQ ID NO: 883), bapineuzumab (V H -SEQ ID NO: 632; V L -SEQ ID NO: 884), basiliximab (V H -SEQ ID NO: 389; V L -SEQ ID NO: 454), bavituximab (V H -SEQ ID NO: 633; V L -SEQ ID NO: 885), bectumomab, begeloma
• clazakizumab VH-SEQ ID NO: 665; VL-SEQ ID NO: 918), clenoliximab, clivatuzumab (VH- SEQ ID NO: 666; VL-SEQ ID NO: 919), codrituzumab (VH-SEQ ID NO: 667; VL-SEQ ID NO: 920), cofetuzumab (VH-SEQ ID NO: 668; VL-SEQ ID NO: 921), coltuximab (VH-SEQ ID NO: 669; VL-SEQ ID NO: 922), conatumumab (VH-SEQ ID NO: 670; VL-SEQ ID NO: 923), concizumab (VH-SEQ ID NO: 671; VL-SEQ ID NO: 924), cosfroviximab (VH-SEQ ID NO: 672; VL-SEQ ID NO: 925), crenezumab (
• VH-SEQ ID NO: 963 epitumomab, epratuzumab (VH-SEQ ID NO: 712; VL-SEQ ID NO: 964), eptinezumab (VH-SEQ ID NO: 713; VL-SEQ ID NO: 965), erenumab (VH-SEQ ID NO: 414; VL-SEQ ID NO: 477), erlizumab, ertumaxomab, etaracizumab (VH-SEQ ID NO: 714; VL-SEQ ID NO: 966), etigilimab (VH-SEQ ID NO: 715; VL-SEQ ID NO: 967), etrolizumab (VH-SEQ ID NO: 716; VL-SEQ ID NO: 968), evinacumab (VH-SEQ ID NO:
• VH-SEQ ID NO: 470 VH-SEQ ID NO: 717; VL-SEQ ID NO: 969
• exbivirumab fanolesomab, faralimomab, faricimab, farletuzumab
• VH-SEQ ID NO: 718; VL- SEQ ID NO: 970 fasinumab
• VH-SEQ ID NO: 407; VL-SEQ ID NO: 471 felvizumab fezakinumab (VH-SEQ ID NO: 719; VL-SEQ ID NO: 971)
• fibatuzumab ficlatuzumab
• figitumumab VH-SEQ ID NO: 721 ; VL-SEQ ID NO: 973
• firivumab VH-SEQ ID NO: 722; VL
• galcanezumab (VH-SEQ ID NO: 729; VL-SEQ ID NO: 980), galiximab (VH-SEQ ID NO: 730; VL-SEQ ID NO: 981), gancotamab, ganitumab (VH-SEQ ID NO: 731; VL-SEQ ID NO: 982), gantenerumab (VH-SEQ ID NO: 732; VL-SEQ ID NO: 983), gatipotuzumab, gavilimomab, gedivumab, gemtuzumab (VH-SEQ ID NO: 733; VL-SEQ ID NO: 984), gevokizumab (VH-SEQ ID NO: 734; VL-SEQ ID NO: 985), gilvetmab, gimsilumab, girentuximab (VH-SEQ ID NO: 735; VL-SEQ ID NO: 9
• V H -SEQ ID NO: 990 ifabotuzumab, igovomab, iladatuzumab, imahimah
• V H -SEQ ID NO: 740 V L -SEQ ID NO: 991
• imaprelimab imciromab
• imgatuzumab V H -SEQ ID NO: 741 ; V L -SEQ ID NO: 992
• inclacumab V H -SEQ ID NO: 742; V L -SEQ ID NO: 993
• indatuximab V H -SEQ ID NO: 743; V L -SEQ ID NO: 994
• indusatumab V H -SEQ ID NO: 744; V L -SEQ ID NO: 995
• inebilizumab inflectra
• infliximab V H -SEQ ID NO: 365; V L - SEQ ID NO: 430
• V L -SEQ ID NO: 1032 oleclumab, olendalizumab, olokizumab
• V H -SEQ ID NO: 782 V L -SEQ ID NO: 1033
• omalizumab V H -SEQ ID NO: 418; V L -SEQ ID NO: 481
• onartuzumab V H -SEQ ID NO: 783; V L -SEQ ID NO: 1034
• ontuxizumab V H -SEQ ID NO: 37; V L -SEQ ID NO: 438
• onvatilimab opicinumab
• V H -SEQ ID NO: 383; V L -SEQ ID NO: 448 oportuzumab, oregovomab, ordcumab
• V H -SEQ ID NO: 784 V L -SEQ ID NO: 1035
• otelixizumab V H -SEQ ID
• V H -SEQ ID NO: 1080 solanezumab
• V H -SEQ ID NO: 830 V L -SEQ ID NO: 1081
• solitomab sonepcizumab, thankuzumab, spartalizumab, stamulumab, sulesomab, suptavumab, sutimlimab, suvizumab
• V H -SEQ ID NO: 831 ; V L -SEQ ID NO: 1082 suvratoxumab, tabalumab
• V H -SEQ ID NO: 832 V L -SEQ ID NO: 1083
• tacatuzumab tadocizumab, talacotuzumab, talizumab, tamtuvetmab, tanezumab
• the antibody is not any one or more of the antibodies described above (e.g., does not comprise a heavy chain variable region and/or a light chain variable region of any one or more of the antibodies described above). In some embodiments, the antibody is not an anti-CTLA4 antibody and/or an anti-PD-Ll antibody.
• an antibody of the present disclosure comprises a light chain variable region having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of the light chain variable region of any of the antibodies described herein or known in the art (e.g., an antibody of the present disclosure comprises a light chain variable region having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of any of the light chain variable regions disclosed in the
• an antibody of the present disclosure comprises a heavy chain variable region having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of the heavy chain variable region of any of the antibodies described herein or known in the art (e.g., an antibody of the present disclosure comprises a heavy chain variable region having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of any of the heavy chain variable regions disclosed in the previous paragraph).
• an antibody of the present disclosure comprises a light chain variable region and a heavy chain variable region having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of the light and heavy chain variable regions of any of the antibodies described herein or known in the art (e.g., an antibody of the present disclosure comprises a light and heavy chain variable region having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, respectively, to the sequence of
• one or more polynucleotides of the present disclosure encode an antibody (e.g., a full-length antibody, an antibody fragment, etc.) comprising a heavy chain variable region comprising an HVR-H1, HVR-H2, and/or an HVR-H3 of any of the antibodies described herein or known in the art.
• an antibody e.g., a full-length antibody, an antibody fragment, etc.
• Methods of identifying the HVR-H1, HVR-H2, and/or HVR-H3 in a given heavy chain variable region are generally known to one of ordinary skill in the art ( see e.g., the methods employed at abysis.org; Al-Lazikani et al, (1997) JMB 273,927-948; Martin, A.C.R.
• one or more polynucleotides of the present disclosure encode an antibody comprising a heavy chain variable region comprising one, two, or three HVRs selected from: an HVR-H1 comprising a sequence selected from SEQ ID NOS: 1-59; an HVR-H2 comprising a sequence selected from SEQ ID NOS: 60-122; and/or an HVR-H3 comprising a sequence selected from SEQ ID NOS: 123-185.
• one or more polynucleotides of the present disclosure encode an antibody comprising a heavy chain variable region comprising the HVR-H1, HVR-H2, and HVR-H3 of any of the antibodies depicted in Table 1.
• one or more polynucleotides of the present disclosure encode an antibody comprising a heavy chain variable region comprising the HVR-H1, HVR-H2, and HVR-H3 of any of the heavy chain variable regions depicted in Table 2 or described in SEQ ID NOS: 355-419 or 614-865.
• one or more polynucleotides of the present disclosure encode an antibody comprising a light chain variable region comprising an HVR-L1, HVR- L2, and/or an HVR-L3 of any of the antibodies described herein or known in the art.
• Methods of identifying the HVR-L1, HVR-L2, and/or HVR-L3 in a given light chain variable region are generally known to one of ordinary skill in the art (see e.g., the methods employed at abysis.org; Al-Lazikani et al, (1997) JMB 273,927-948; Martin, A.C.R. (1996) Proteins 25(1): 130-3; etc.).
• one or more polynucleotides of the present disclosure encode an antibody comprising a light chain variable region comprising one, two, or three HVRs selected from: an HVR-L1 comprising a sequence selected from SEQ ID NOS: 186-242; an HVR-L2 comprising a sequence selected from SEQ ID NOS: 243-294; and/or an HVR-L3 comprising a sequence selected from SEQ ID NOS: 295-354.
• one or more polynucleotides of the present disclosure encode an antibody comprising a light chain variable region comprising the HVR-L1, HVR-L2, and HVR-L3 of any of the antibodies depicted in Table 1.
• one or more polynucleotides of the present disclosure encode an antibody comprising a light chain variable region comprising the HVR-L1, HVR-L2, and HVR-L3 of any of the antibodies depicted in Table 1.
• one or more polynucleotides of the present disclosure encode an antibody comprising a light chain variable region compris
• polynucleotides of the present disclosure encode an antibody comprising a light chain variable region comprising the HVR-L1, HVR-L2, and HVR-L3 of any of the light chain variable regions depicted in Table 2 or disclosed in SEQ ID NOS: 420-482 or 866-1116.
• one or more polynucleotides of the present disclosure encode an antibody comprising a heavy chain variable region comprising an HVR-H1, HVR- H2, and/or an HVR-H3 of any of the antibodies described herein or known in the art, and a light chain variable region comprising an HVR-L1, HVR-L2, and/or and HVR-L3 of any of the antibodies described herein or known in the art.
• one or more polynucleotides of the present disclosure encode an antibody comprising: a heavy chain variable region comprising one, two, or three HVRs selected from: an HVR-H1 comprising a sequence selected from SEQ ID NOS: 1-59; an HVR-H2 comprising a sequence selected from SEQ ID NOS: 60-122; and/or an HVR-H3 comprising a sequence selected from SEQ ID NOS: 123-185; and a light chain variable region comprising one, two, or three HVRs selected from: an HVR-L1 comprising a sequence selected from SEQ ID NOS: 186-242; an HVR-L2 comprising a sequence selected from SEQ ID NOS: 243-294; and/or an HVR-L3 comprising a sequence selected from SEQ ID NOS: 295-354.
• one or more polynucleotides of the present disclosure encode an antibody comprising the HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of any of the antibodies depicted in Table 1.
• one or more polynucleotides of the present disclosure encode an antibody comprising the HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of any of the heavy chain variable regions and light chain variable regions depicted in Table 2 or disclosed in SEQ ID NOS: 355-419, 420-482, 614-865, or 86-1116.
• HVRs of exemplary antibodies encode an antibody comprising a heavy chain variable region comprising: an HVR-H1 comprising the sequence of SEQ ID NO: 1; an HVR-H2 comprising the sequence of SEQ ID NO: 60; and an HVR-H3 comprising the sequence of SEQ ID NO: 123.
• an HVR-H1 comprising the sequence of SEQ ID NO: 1
• an HVR-H2 comprising the sequence of SEQ ID NO: 60
• HVR-H3 comprising the sequence of SEQ ID NO: 123.
• one or more polynucleotides of the present disclosure encode an antibody comprising a heavy chain variable region comprising: an HVR-H1 comprising the sequence of SEQ ID NO: 2; an HVR-H2 comprising the sequence of SEQ ID NO: 61; and an HVR-H3 comprising the sequence of SEQ ID NO: 124.
• one or more polynucleotides of the present disclosure encode an antibody comprising a heavy chain variable region comprising: an HVR-H1 comprising the sequence of SEQ ID NO: 3; an HVR-H2 comprising the sequence of SEQ ID NO: 62; and an HVR-H3 comprising the sequence of SEQ ID NO: 125.
• one or more polynucleotides of the present disclosure encode an antibody comprising a light chain variable region comprising: an HVR-L1 comprising the sequence of SEQ ID NO: 186; an HVR-L2 comprising the sequence of SEQ ID NO: 243; and an HVR-L3 comprising the sequence of SEQ ID NO: 295.
• one or more polynucleotides of the present disclosure encode an antibody comprising a light chain variable region comprising: an HVR-L1 comprising the sequence of SEQ ID NO: 187; an HVR-L2 comprising the sequence of SEQ ID NO: 244; and an HVR-L3 comprising the sequence of SEQ ID NO: 296.
• one or more polynucleotides of the present disclosure encode an antibody comprising a light chain variable region comprising: an HVR-L1 comprising the sequence of SEQ ID NO: 188; an HVR-L2 comprising the sequence of SEQ ID NO: 245; and an HVR-L3 comprising the sequence of SEQ ID NO: 2297.
• one or more polynucleotides of the present disclosure encode an antibody comprising: an HVR-H1 comprising the sequence of SEQ ID NO: 1; an HVR-H2 comprising the sequence of SEQ ID NO: 60; an HVR-H3 comprising the sequence of SEQ ID NO: 123; an HVR-L1 comprising the sequence of SEQ ID NO: 186; an HVR-L2 comprising the sequence of SEQ ID NO: 243; and an HVR-L3 comprising the sequence of SEQ ID NO: 295.
• one or more polynucleotides of the present disclosure encode an antibody comprising: an HVR-H1 comprising the sequence of SEQ ID NO: 2; an HVR-H2 comprising the sequence of SEQ ID NO: 61; an HVR-H3 comprising the sequence of SEQ ID NO: 124; an HVR-L1 comprising the sequence of SEQ ID NO: 187; an HVR-L2 comprising the sequence of SEQ ID NO: 244; and an HVR-L3 comprising the sequence of SEQ ID NO: 296.
• one or more polynucleotides of the present disclosure encode an antibody comprising: an HVR-H1 comprising the sequence of SEQ ID NO: 3; an HVR-H2 comprising the sequence of SEQ ID NO: 62; an HVR-H3 comprising the sequence of SEQ ID NO: 125; an HVR-L1 comprising the sequence of SEQ ID NO: 188; an HVR-L2 comprising the sequence of SEQ ID NO: 245; and an HVR-L3 comprising the sequence of SEQ ID NO: 297.
• one or more polynucleotides of the present disclosure encode an antibody comprising a heavy chain variable region of any of the antibodies described herein or known in the art. In some embodiments, one or more polynucleotides of the present disclosure encode an antibody comprising a heavy chain variable region comprising a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NOS: 355-419 or 614-865. In some
• one or more polynucleotides of the present disclosure encode an antibody comprising a heavy chain variable region comprising a sequence selected from SEQ ID NOS: 355-419 or 614-865. In some embodiments, one or more polynucleotides of the present disclosure encode an antibody comprising the heavy chain variable region of any of the antibodies depicted in Table 2.
• one or more polynucleotides of the present disclosure encode an antibody comprising a light chain variable region of any of the antibodies described herein or known in the art. In some embodiments one or more polynucleotides of the present disclosure encode an antibody comprising a light chain variable region comprising a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NOS: 420-482 or 866-1116.
• one or more polynucleotides of the present disclosure encode an antibody comprising a light chain variable region comprising a sequence selected from SEQ ID NOS: 420-482 or 866-1116. In some embodiments, one or more polynucleotides of the present disclosure encode an antibody comprising the light chain variable region of any of the antibodies depicted in Table 2.
• one or more polynucleotides of the present disclosure encode an antibody comprising a heavy chain variable region and a light chain variable region of any of the antibodies described herein or known in the art. In some embodiments, one or more polynucleotides of the present disclosure encode an antibody comprising: a heavy chain variable region comprising a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least
• sequence identity 99%, or 100% sequence identity to a sequence selected from SEQ ID NOS: 355-419 or 614- 865; and a light chain variable region comprising a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least
• one or more polynucleotides of the present disclosure encode an antibody comprising a heavy chain variable region comprising a sequence selected from SEQ ID NOS: 355-419 or 614-865, and a light chain variable region comprising a sequence selected from SEQ ID NOS: 420-482 or 866-1116. In some embodiments, one or more polynucleotides of the present disclosure encode an antibody comprising a heavy chain variable region and a light chain variable region each
• one or more polynucleotides of the present disclosure encode an antibody comprising the heavy chain variable region and the light chain variable region of any of the antibodies depicted in Table 2.
• one or more polynucleotides of the present disclosure encode an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 355, and light chain variable region comprising the sequence of SEQ ID NO: 420. In some embodiments, one or more polynucleotides of the present disclosure encode an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 356, and light chain variable region comprising the sequence of SEQ ID NO: 421. In some embodiments, one or more polynucleotides of the present disclosure encode an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 357, and light chain variable region comprising the sequence of SEQ ID NO: 422.
• a polynucleotide of the present disclosure encoding an antibody may further encode additional coding and non-coding sequences.
• additional coding and non coding sequences may include, but are not limited to, sequences encoding additional polypeptide tags (e.g., encoded in-frame with the antibody in order to produce a fusion protein), introns (e.g., native, modified, or heterologous introns), 5’ and/or 3’ UTRs (e.g., native, modified, or heterologous 5’ and/or 3’ UTRs), and the like.
• suitable polypeptide tags may include, but are not limited, to any combination of purification tags, such as his-tags, flag-tags, maltose binding protein and glutathione- S-transferase tags, detection tags, such as tags that may be detected photometrically (e.g., green fluorescent protein, red fluorescent protein, etc.) and tags that have a detectable enzymatic activity (e.g., alkaline phosphatase, etc.), tags containing secretory sequences, signal sequences, leader sequences, and/or stabilizing sequences, protease cleavage sites (e.g., furin cleavage sites, TEV cleavage sites, Thrombin cleavage sites, etc.), and the like.
• purification tags such as his-tags, flag-tags, maltose binding protein and glutathione- S-transferase tags
• detection tags such as tags that may be detected photometrically (e.g., green fluorescent protein, red fluorescent protein, etc.) and tags
• the 5’ and/or 3’UTRs increase the stability, localization, and/or translational efficiency of the polynucleotides. In some embodiments, the 5’ and/or 3’UTRs improve the level and/or duration of protein expression. In some embodiments, the 5’ and/or 3’UTRs include elements (e.g., one or more miRNA binding sites, etc.) that may block or reduce off-target expression (e.g., inhibiting expression in specific cell types (e.g., neuronal cells), at specific times in the cell cycle, at specific developmental stages, etc.). In some embodiments, the 5’ and/or 3’UTRs include elements (e.g., one or more miRNA binding sites, etc.) that may enhance antibody expression in specific cell types.
• elements e.g., one or more miRNA binding sites, etc.
• a polynucleotide of the present disclosure encodes a leader, signal, and/or secretory sequence (in-frame) at the N-terminus of an encoded antibody. Any leader, signal, and/or secretory sequence known in the art may be encoded by a
• polynucleotide of the present disclosure including, for example, a native antibody signal sequence (e.g., any of the antibody leader sequences described in Retter I et al. VBASE2, an integrative V gene database. Nucleic Acids Res. 2005 Jan l;33: D671-4), or a heterologous or synthetic signal sequence (see e.g., von Heijne G. (1983) Patterns of amino acids near signal- sequence cleavage sites. Eur J Biochem 133 (1) 17-21; Martoglio B. and Dobberstein B. (1998) Signal sequences: More than just greasy peptides. Trends Cell Biol 8 (10), 410-5; Hegde R.S. and Bernstein H.D. (2006) The surprising complexity of signal sequences.
• a native antibody signal sequence e.g., any of the antibody leader sequences described in Retter I et al. VBASE2, an integrative V gene database. Nucleic Acids Res. 2005 Jan l;33: D671-4
• Exemplary secretion sequences include the human CD33 leader sequence (MPLLLLLPLLWAGALA, SEQ ID NO: 483), the human IL2 leader sequence (MYRMQLLSCIALSLALVTNS, SEQ ID NO: 484), the human tissue plasminogen activator leader sequence (MDAMKRGLCCVLLLCGAVFVSP, SEQ ID NO: 485), human antibody leader sequences (Tables 3A-C), and the synthetic secrecon leader sequence
• a polynucleotide of the present disclosure encoding an antibody is operably linked to one or more (e.g., one or more, two or more, three or more, four or more, five or more, ten or more, etc.) regulatory sequences.
• regulatory sequence may include enhancers, insulators, promoters, and other expression control elements (e.g., polyadenylation signals).
• enhancer(s) known in the art may be used, including, for example, enhancer sequences from mammalian genes (such as globin, elastase, albumin, a-fetoprotein, insulin and the like), enhancer sequences from a eukaryotic cell virus (such as SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, and the like), and any combinations thereof.
• mammalian genes such as globin, elastase, albumin, a-fetoprotein, insulin and the like
• enhancer sequences from a eukaryotic cell virus such as SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, and
• any suitable insulator(s) known in the art may be used, including, for example, HSV chromatin boundary (CTRL/CTCF-binding/insulator) elements CTRL1 and/or CTRL2, chicken hypersensitive site 4 insulator (cHS4), human HNRPA2B1— CBX3 ubiquitous chromatin opening element (UCOE), the scaffold/matrix attachment region (S/MAR) from the human interferon beta gene (IFNB1), and any combinations thereof.
• HSV chromatin boundary (CTRL/CTCF-binding/insulator) elements CTRL1 and/or CTRL2
• cHS4 chicken hypersensitive site 4 insulator
• UCOE ubiquitous chromatin opening element
• S/MAR scaffold/matrix attachment region from the human interferon beta gene
• any suitable promoter e.g., suitable for transcription in mammalian host cells
• suitable promoters including, for example, promoters obtained from the genomes of viruses (such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40 (SV40), and the like), promoters from heterologous mammalian genes (such as the actin promoter (e.g., the b-actin promoter, a ubiquitin promoter (e.g., a ubiquitin C (UbC) promoter), a phosphogly cerate kinase (PG) promoter, an immunoglobulin promoter, from heat-shock promoters, and the like), promoters from homologous mammalian genes (e.g., native human immuno
• a polynucleotide of the present disclosure is operably linked to one or more heterologous promoters.
• the one or more heterologous promoters are one or more of constitutive promoters, tissue-specific promoters, temporal promoters, spatial promoters, inducible promoters and repressible promoters.
• the one or more heterologous promoters are one or more of the human cytomegalovirus (HCMV) immediate early promoter, the human elongation factor-l (EF1) promoter, the human b-actin promoter, the human UbC promoter, the human PGK promoter, a human immunoglobulin promoter, the synthetic CAGG promoter, and any combinations thereof.
• HCMV human cytomegalovirus
• EF1 human elongation factor-l
• EF1 human elongation factor-l
• human UbC promoter the human PGK promoter
• a human immunoglobulin promoter the synthetic CAGG promoter, and any combinations thereof.
• a polynucleotide of the present disclosure encoding an antibody is operably linked to an HCMV promoter.
• a polynucleotide of the present disclosure does not comprise the coding sequence of (e.g., a transgene encoding) a Collagen alpha- 1 (VII) chain polypeptide (COL7).
• a polynucleotide of the present disclosure does not comprise the coding sequence of (e.g., a transgene encoding) a Lysyl hydroxylase 3 polypeptide (LH3).
• a polynucleotide of the present disclosure does not comprise the coding sequence of (e.g., a transgene encoding) a Keratin type I cytoskeletal 17 polypeptide (KRT17).
• a polynucleotide of the present disclosure does not comprise the coding sequence of (e.g., a transgene encoding) a transglutaminase (TGM) polypeptide (e.g., a human transglutaminase polypeptide such as a human TGM1 polypeptide).
• TGM transglutaminase
• a polynucleotide of the present disclosure does not comprise the coding sequence of (e.g., a transgene encoding) a cosmetic protein (e.g., collagen proteins, fibronectins, elastins, lumicans, vitronectins/vitronectin receptors, laminins, neuromodulators, fibrillins, additional dermal extracellular matrix proteins, etc.).
• a cosmetic protein e.g., collagen proteins, fibronectins, elastins, lumicans, vitronectins/vitronectin receptors, laminins, neuromodulators, fibrillins, additional dermal extracellular matrix proteins, etc.
• a polynucleotide of the present disclosure does not comprise the coding sequence of (e.g., a transgene encoding) a Collagen alpha- 1 (VII) chain polypeptide, a Lysyl hydroxylase 3 polypeptide, a Keratin type I cytoskeletal 17 polypeptide, and/or any chimeric polypeptides thereof.
• a polynucleotide of the present disclosure does not comprise the coding sequence of (e.g., a transgene encoding) a Collagen alpha- 1 (VII) chain polypeptide, a Lysyl hydroxylase 3 polypeptide, a Keratin type I cytoskeletal 17 polypeptide, a transglutaminase (TGM) polypeptide (e.g., a human transglutaminase polypeptide such as a human TGM1 polypeptide), a cosmetic protein, and/or any chimeric polypeptides thereof.
• TGM transglutaminase
• a recombinant nucleic acid e.g., a recombinant herpes virus genome
• a recombinant nucleic acid comprises one or more polynucleotides encoding an antibody, where the antibody is a single-chain antibody.
• Any suitable form of single-chain antibody known in the art may be encoded by a polynucleotide of the present disclosure.
• the single-chain antibody comprises a heavy chain variable region and a light chain variable region.
• the single-chain antibody comprises a heavy chain variable region and a light chain variable region separated by a linker polypeptide.
• the single-chain antibody comprises, from N-terminus to C-terminus, 1) a heavy chain variable region, 2) a linker polypeptide, and 3) a light chain variable region. In some embodiments, the single-chain antibody comprises, from N-terminus to C-terminus, 1) a light chain variable region, 2) a linker polypeptide, and 3) a heavy chain variable region. In some embodiments, the single-chain antibody further comprises an antibody hinge region (e.g., an IgGl hinge region). An exemplary IgGl hinge region is provided as SEQ ID NO: 603. In some embodiments, the single -chain antibody further comprises an antibody Fc region (e.g., an IgGl Fc region).
• the single-chain antibody is an scFv-Fc antibody (e.g., an scFv fused to the hinge and Fc region of an IgGl antibody heavy chain).
• Any suitable linker polypeptide known in the art may be used in a single-chain antibody of the present disclosure, including, for example, a GGGGSGGGGSGGGGS (SEQ ID NO: 566) linker, a GGSSRSSSSGGGGSGGGG (SEQ ID NO: 567) linker, a
• GGGGSGGGGSGGGGSGGGGS linker, a CGGGSGGGGSGGGGS (SEQ ID NO: 569) linker, a SHGGHGGGGSGGGGS (SEQ ID NO: 570) linker, a
• MGGMSGGGGSGGGGS (SEQ ID NO: 571) linker, a Y GGY SGGGGSGGGGS (SEQ ID NO: 572) linker, a WGGYSGGGGSGGGGS (SEQ ID NO: 573) linker, a SVSVGMKPSPRP (SEQ ID NO: 574) linker, a VISNH AGS SRRL (SEQ ID NO: 575) linker, a PWIPTPRPTFTG (SEQ ID NO: 576) linker, a RGRGRGRGRGR (SEQ ID NO: 577) linker, etc.
• Exemplary polynucleotides encoding single-chain antibodies comprising a leader sequence, an antibody heavy chain variable region, a linker polypeptide, an antibody light chain variable region, and an antibody hinge and Fc region are provided as SEQ ID NOS: 578-583.
• a recombinant nucleic acid e.g., recombinant herpes virus genome
• the polynucleotide comprises, from 5’ to 3’, a first expression cassette encoding a polypeptide comprising an antibody heavy chain variable region (e.g., a full-length antibody heavy chain), and a second expression cassette encoding a polypeptide comprising an antibody light chain variable region (e.g., a full-length antibody light chain).
• the polynucleotide comprises, from 5’ to 3’, a first expression cassette encoding a polypeptide comprising an antibody light chain variable region (e.g., a full-length antibody light chain), and a second expression cassette encoding a polypeptide comprising an antibody heavy chain variable region (e.g., a full-length antibody heavy chain).
• the first and second expression cassettes have independent regulatory sequences (e.g., promoters, enhancers, polyadenylation signals, etc.).
• the first and second expression cassettes are in the same orientation in the DNA. In some embodiments, the first and second expression cassettes are in opposite orientations to one another in the DNA. Without wishing to be bound by theory, incorporating two expression cassettes in an antisense orientation (opposite strands of DNA) may help to avoid read-through and ensure proper expression of each cassette.
• a recombinant nucleic acid e.g., recombinant herpes virus genome
• the polycistronic rnRNA comprises: 1) a first open reading frame (ORF) encoding a polypeptide comprising an antibody heavy chain variable region (e.g., an antibody heavy chain), and 2) a second open reading frame (ORF) encoding a polypeptide comprising an antibody light chain variable region (e.g., an antibody light chain).
• the polycistronic mRNA comprises: 1) a first open reading frame (ORF) encoding a polypeptide comprising an antibody light chain variable region (e.g., an antibody light chain), and 2) a second open reading frame (ORF) encoding a polypeptide comprising an antibody heavy chain variable region (e.g., an antibody heavy chain).
• ORF open reading frame
• ORF second open reading frame
• the polycistronic mRNA further comprises an internal ribosomal entry site (IRES) separating the first ORF and the second ORF.
• IRS internal ribosomal entry site
• the polycistronic mRNA comprises, from 5’ to 3’, the first ORF encoding the polypeptide comprising the antibody heavy chain variable region-the IRES-the second ORF encoding the polypeptide comprising the antibody light chain variable region. In some embodiments, the polycistronic mRNA comprises, from 5’ to 3’, the first ORF encoding the polypeptide comprising the antibody light chain variable region-the IRES-the second ORF encoding the polypeptide comprising the antibody heavy chain variable region.
• any suitable IRES known in the art may be used in the polycistronic mRNAs of the present disclosure, including, for example, a virally-derived IRES (e.g. an IRES derived from a poliovirus, rhinovirus, encephalomyocarditis virus (EMCV), foot-and-mouth disease virus, hepatitis C virus, classic swine fever virus, rous sarcoma virus, human
• a virally-derived IRES e.g. an IRES derived from a poliovirus, rhinovirus, encephalomyocarditis virus (EMCV), foot-and-mouth disease virus, hepatitis C virus, classic swine fever virus, rous sarcoma virus, human
• mRNA-derived IRES e.g. an IRES derived from growth factor mRNAs, such as fibroblast growth factor 2, platelet-derived growth factor B, and vascular endothelial growth factor; an IRES derived from transcription factor mRNAs, such as antennapedia, ultrabithorax, and NF-kB repressing factor; an IRES derived from oncogene mRNAs, such as c-myc, pim-l, and protein kinase p58 PITSLRE , etc.), a synthetic IRES (e.g., a CP148 IRES), and others (see e.g., Mokrejs et al.
• growth factor mRNAs such as fibroblast growth factor 2, platelet-derived growth factor B, and vascular endothelial growth factor
• transcription factor mRNAs such as antennapedia, ultrabithorax, and NF-kB repressing factor
• oncogene mRNAs such as c-myc,
• the IRES is a CP148 IRES.
• An exemplary nucleic acid sequence encoding a CP148 IRES is provided as SEQ ID NO: 584.
• the IRES is an EMCV IRES.
• An exemplary nucleic acid sequence encoding an EMCV IRES is provided as SEQ ID NO: 585.
• the nucleic acid sequence encoding the IRES has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleic acid sequence selected from SEQ ID NO: 584 or SEQ ID NO: 585.
• the nucleic acid sequence encoding the IRES has the sequence of SEQ ID NO: 584 or SEQ ID NO: 585.
• Exemplary polynucleotides encoding polycistronic mRNAs comprising 1) a first ORF encoding a leader sequence and an antibody light chain (Kappa), 2) an IRES, and 3) second ORF encoding a leader sequence and an antibody heavy chain (IgGl) are provided as SEQ ID NOS: 586-588.
• a recombinant nucleic acid e.g., recombinant herpes virus genome
• a recombinant nucleic acid comprises one or more polynucleotides encoding a chimeric polypeptide comprising an antibody heavy chain variable region and an antibody light chain variable region separated by a cleavable linker polypeptide.
• the chimeric polypeptide comprises a first amino acid sequence comprising an antibody heavy chain and a second amino acid sequence comprising an antibody light chain separated by a cleavable linker polypeptide.
• the chimeric polypeptide comprises, from N-terminus to C-terminus, 1) a first amino acid sequence comprising an antibody light chain variable region (e.g., an antibody light chain), 2) a cleavable linker polypeptide, and 3) a second amino acid sequence comprising an antibody heavy chain variable region (e.g., an antibody heavy chain).
• the chimeric polypeptide comprises, from N-terminus to C-terminus, 1) a first amino acid sequence comprising an antibody heavy chain variable region (e.g., an antibody heavy chain), 2) a cleavable linker polypeptide, and 3) a second amino acid sequence comprising an antibody light chain variable region (e.g., an antibody light chain).
• cleavable linker polypeptide known in the art may be used in the chimeric polypeptides of the present disclosure, including, for example, a T2A linker
• the linker polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOS: 589-592.
• the linker polypeptide comprises a sequence selected from SEQ ID NOS: 589-592.
• Exemplary polynucleotides encoding chimeric polypeptides comprising a leader sequence, an antibody light chain, a linker polypeptide, a leader sequence, and an antibody heavy chain are provided as SEQ ID NOS: 593-595.
• a recombinant nucleic acid (e.g., recombinant herpes virus genome) of the present disclosure comprises two or more polynucleotides encoding an antibody.
• the recombinant nucleic acid comprises a first
• the recombinant nucleic acid comprises two copies of a first polynucleotide encoding a polypeptide comprising an antibody heavy chain variable region (e.g., a full-length antibody heavy chain), and two copies of a second polynucleotide encoding a polypeptide comprising an antibody light chain variable region (e.g., a full-length antibody light chain).
• the recombinant nucleic acid comprises two copies of a first polynucleotide encoding a polypeptide comprising an antibody heavy chain variable region (e.g., a full-length antibody heavy chain), and a single copy of a second polynucleotide encoding a polycistronic mRNA comprising a first and second ORF each encoding a polypeptide comprising an antibody light chain variable region (e.g., a full-length antibody light chain) separated by an IRES (e.g., any of the IRESs described herein).
• an antibody heavy chain variable region e.g., a full-length antibody heavy chain
• IRES e.g., any of the IRESs described herein
• the recombinant nucleic acid comprises a single copy of a first polynucleotide encoding a polycistronic mRNA comprising a first and second ORF each encoding a polypeptide comprising an antibody heavy chain variable region (e.g., a full-length antibody heavy chain) separated by an IRES (e.g., any of the IRESs described herein), and two copies of a second polynucleotide encoding a polypeptide comprising an antibody light chain variable region (e.g., a full-length antibody light chain).
• an IRES e.g., any of the IRESs described herein
• the recombinant nucleic acid comprises a first polynucleotide encoding a first antibody and a second polynucleotide encoding a second antibody.
• the first and second antibody are the same. In some embodiments, the first and second antibodies are different.
• the present disclosure relates to recombinant nucleic acids comprising any one or more of the polynucleotides described herein.
• the recombinant nucleic acid is a vector (e.g., an expression vector, a display vector, etc.).
• the vector is a DNA vector or an RNA vector.
• vectors suitable to maintain, propagate, and/or express polynucleotides to produce one or more polypeptides in a subject may be used.
• suitable vectors may include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, Sindbis-viral vectors, measles vectors, herpes viral vectors, lentiviral vectors, retroviral vectors, etc.).
• the vector is a herpes viral vector.
• the vector is capable of autonomous replication in a host cell.
• the vector is incapable of autonomous replication in a host cell.
• the vector can integrate into a host DNA.
• the vector cannot integrate into a host DNA (e.g., is episomal).
• Methods of making vectors containing one or more polynucleotides of interest are well known to one of ordinary skill in the art, including, for example, by chemical synthesis, or by artificial manipulation of isolated segments of nucleic acids (e.g., by genetic engineering techniques).
• a recombinant nucleic acid of the present disclosure is a herpes simplex virus (HSV) amplicon.
• HSV herpes simplex virus
• Herpes virus amplicons including the structural features and methods of making the same, are generally known to one of ordinary skill in the art (see e.g., de Silva S. and Bowers W.“Herpes Virus Amplicon Vectors”. Viruses 2009, 1, 594-629).
• the herpes simplex virus amplicon is an HSV-l amplicon.
• the herpes simplex virus amplicon is an HSV-l hybrid amplicon.
• HSV-l hybrid amplicons may include, but are not limited to, HSV/AAV hybrid amplicons, HSV/EBV hybrid amplicons, HSV/EBV/RV hybrid amplicons, and/or
• the amplicon is an
• a recombinant nucleic acid of the present disclosure is a recombinant herpes virus genome.
• the recombinant herpes virus genome may be a recombinant genome from any member of the Herpesviridae family of DNA viruses known in the art, including, for example, a recombinant herpes simplex virus genome, a recombinant varicella zoster virus genome, a recombinant human cytomegalovirus genome, a recombinant herpesvirus 6A genome, a recombinant herpesvirus 6B genome, a recombinant herpesvirus 7 genome, a recombinant Kaposi’s sarcoma-associated herpesvirus genome, and any combinations thereof or any derivatives thereof.
• the recombinant herpes virus genome comprises more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) inactivating mutations.
• the one or more inactivating mutations are in one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) herpes virus genes.
• the recombinant herpes virus genome is attenuated (e.g., as compared to a corresponding, wild-type herpes virus genome). In some embodiments, the recombinant herpes virus genome is replication competent. In some embodiments, the recombinant herpes virus genome is replication defective.
• the recombinant nucleic acid is a recombinant herpes simplex virus (HSV) genome.
• the recombinant herpes simplex virus genome is a recombinant type 1 herpes simplex virus (HSV-l) genome, a recombinant type 2 herpes simplex virus (HSV-2) genome, or any derivatives thereof.
• the recombinant herpes simplex virus genome is a recombinant HSV-l genome.
• the recombinant herpes simplex virus genome is replication competent.
• the recombinant herpes simplex virus genome is replication defective.
• the recombinant herpes simplex virus genome comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) inactivating mutations.
• the one or more inactivating mutations are in one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) herpes simplex virus genes.
• an “inactivating mutation” may refer to any mutation that results in the gene product (RNA or protein) having reduced, undetectable, or eliminated quantity and/or function (e.g., as compared to a corresponding sequence lacking the inactivating mutation).
• inactivating mutations may include, but are not limited to, deletions, insertions, point mutations, and rearrangements in transcriptional control sequences (promoters, enhancers, insulators, etc.) and/or coding sequences of a given gene or regulon. Any suitable method of measuring the quantity of a gene or regulon product known in the art may be used, including, for example, qPCR, Northern blots, RNAseq, western blots, ELISAs, etc.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all eight of the Infected Cell Protein (or Infected Cell
• the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the ICP34.5 (one or both copies) and/or ICP47 herpes simplex virus genes (e.g., to avoid production of an immune-stimulating virus). In some embodiments, the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the ICP34.5 (one or both copies) herpes simplex virus gene.
• the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the ICP47 herpes simplex virus gene. In some embodiments, the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the ICP34.5 (one or both copies) and ICP47 herpes simplex virus genes. In some embodiments, the recombinant herpes simplex virus genome is not oncolytic.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies). In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), and further comprises an initiating mutation in the ICP4 (one or both copies) ICP22, ICP27, ICP47, UL41, and/or UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), and an inactivating mutation in the ICP4 gene (one or both copies).
• the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), and an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), and an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICPO gene (one or both copies), an inactivating mutation in the ICP4 gene (one or both copies), and an inactivating mutation in the ICP22 gene.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICPO gene (one or both copies), an inactivating mutation in the ICP4 gene (one or both copies), and an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICPO gene (one or both copies), an inactivating mutation in the ICP22 gene, and an inactivating mutation in the UL41 gene.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICPO gene (one or both copies), an inactivating mutation in the ICP4 gene (one or both copies), an inactivating mutation in the ICP22 gene, and an inactivating mutation in the UL41 gene.
• the inactivating mutation is a deletion of the coding sequence of the ICPO (one or both copies), ICP4 (one or both copies), ICP22, and/or UL41 genes.
• the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP27, ICP47, and/or UL55 genes.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies).
• the recombinant herpes complex virus genome comprises an inactivating mutation in the ICP4 (one or both copies, and further comprises an inactivating mutation in the ICPO (one or both copies), ICP22, ICP27, ICP47, UL41, and/or UL55 genes.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies), and an inactivating mutation in the ICP22 gene.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies), and an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies), an inactivating mutation in the ICP22 gene, and an inactivating mutation in the UL41 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP4 (one or both copies), ICP22, and/or UL41 genes. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICPO, ICP27, ICP47, and/or UL55 genes.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene, and further comprises an inactivating mutation in the ICPO (one or both copies), ICP4 (one or both copies), ICP27, ICP47, UL41, and/or UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene, and an inactivating mutation UL41 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP22 and/or UL41 genes. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICPO (one or both copies), ICP4 (one or both copies), ICP27, ICP47, and/or UL55 genes.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene, and further comprises an inactivating mutation in the ICPO (one or both copies), ICP4 (one or both copies), ICP22, ICP47, UL41, and/or UL55 genes. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP27 gene.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP47 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP47 gene, and further comprises an inactivating mutation in the ICPO (one or both copies), ICP4 (one or both copies), ICP22, ICP27, UL41, and/or UL55 genes. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP47 gene.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene, and further comprises an inactivating mutation in the ICPO (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, and/or UL55 genes. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the UL41 gene.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene, and further comprises an inactivating mutation in the ICPO (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, and/or UL41 genes. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the UL55 gene.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in (e.g., a deletion of) the internal repeat (Joint) region comprising the internal repeat long (IRL) and internal repeat short (IRS) regions.
• inactivation (e.g., deletion) of the Joint region eliminates one copy each of the ICP4 and ICP0 genes.
• inactivation (e.g., deletion) of the Joint region further inactivates (e.g., deletes) the promoter for the ICP22 and ICP47 genes.
• inactivating e.g., deleting
• the Joint region may contribute to the stability of the recombinant herpes simplex virus genome and/or allow for the recombinant herpes simplex virus genome to accommodate more and/or larger transgenes.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 (one or both copies), ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 (one or both copies), ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 (one or both copies), ICP22, ICP27, ICP47, and UL55 genes.
• the inactivating mutation in the ICP4 (one or both copies), ICP27, and/or UL55 genes is a deletion of the coding sequence of the ICP4 (one or both copies), ICP27, and/or UL55 genes.
• the inactivating mutation in the ICP22 and ICP47 genes is a deletion in the promoter region of the ICP22 and ICP47 genes (e.g., the ICP22 and ICP47 coding sequences are intact but are not transcriptionally active).
• the recombinant herpes simplex virus genome comprises a deletion in the coding sequence of the ICP4 (one or both copies), ICP27, and UL55 genes, and a deletion in the promoter region of the ICP22 and ICP47 genes. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP0 (one or both copies) and/or UL41 genes.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 (one or both copies) gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 (one or both copies) and ICP4 (one or both copies) genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, and ICP27 genes.
• the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27 and UL55 genes.
• the inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27 and/or UL55 genes comprises a deletion of the coding sequence of the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27 and/or UL55 genes.
• the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP47 and/or the UL41 genes.
• a recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within one, two, three, four, five, six, seven or more viral gene loci.
• suitable viral loci may include, without limitation, the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, tk, UL41 and UL55 herpes simplex viral gene loci.
• a recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within one or both of the viral ICP4 gene loci (e.g., a recombinant virus carrying a polynucleotide encoding an antibody (or a portion thereof) in one or both of the ICP4 loci).
• a recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral ICP22 gene locus (e.g., a recombinant virus carrying a polynucleotide encoding an antibody (or a portion thereof) in the ICP22 locus).
• a recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral UL41 gene locus (e.g., a recombinant virus carrying a polynucleotide encoding an antibody (or a portion thereof) in the UL41 locus).
• a recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral ICP47 gene locus (e.g., a recombinant virus carrying a polynucleotide encoding an antibody (or a portion thereof) in the ICP47 locus).
• a recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within one or both of the viral ICP4 gene loci, and one or more polynucleotides of the present disclosure within the viral ICP22 gene locus (e.g., a recombinant virus carrying a polynucleotide encoding an antibody heavy chain in one or both of the ICP4 loci, and a polynucleotide encoding an antibody light chain in the ICP22 locus; a recombinant virus carrying a polynucleotide encoding an antibody heavy chain in one or both of the ICP4 loci, and a polynucleotide encoding a polycistronic mRNA encoding two copies of an antibody light chain in the ICP22 locus; etc.).
• a recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within one or both of the viral ICP4 gene loci, and one or more polynucleotides of the present disclosure within the viral UL41 gene locus (e.g., a recombinant virus carrying a polynucleotide encoding an antibody heavy chain in one or both of the ICP4 loci, and a polynucleotide encoding an antibody light chain in the UL41 locus; a recombinant virus carrying a polynucleotide encoding an antibody heavy chain in one or both of the ICP4 loci, and a polynucleotide encoding a polycistronic mRNA encoding two copies of an antibody light chain in the UL41 locus; etc.).
• a recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral UL41 gene locus, and one or more polynucleotides of the present disclosure within the viral ICP22 gene locus (e.g., a recombinant virus carrying a polynucleotide encoding an antibody heavy chain in the UL41 locus, and a polynucleotide encoding an antibody light chain in the ICP22 locus; a recombinant virus carrying a polynucleotide encoding an antibody light chain in the UL41 locus, and a polynucleotide encoding an antibody heavy chain in the ICP22 locus; etc.).
• a recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within one or more of the viral ICP4 gene loci, one or more polynucleotides of the present disclosure within the viral ICP22 gene locus, and one or more polynucleotides of the present disclosure within the viral UL41 gene locus (e.g., a recombinant virus carrying a polynucleotide encoding an antibody heavy chain in one or both of the ICP4 loci, a polynucleotide encoding an antibody light chain in the ICP22 locus, and a polynucleotide encoding an antibody light chain in the UL41 locus; a recombinant virus carrying a polynucleotide encoding an antibody light chain in one or both of the ICP4 loci, a polynucleotide encoding an antibody heavy chain in the ICP22 locus, and a polynucleotides
• the recombinant herpes virus genome (e.g., a recombinant herpes simplex virus genome) has been engineered to decrease or eliminate expression of one or more toxic herpes virus genes (such as one or both copies of the HSV ICP4 gene, the ICP22 gene, the UL41 gene, and/or the ICP27 gene).
• one or more toxic herpes virus genes such as one or both copies of the HSV ICP4 gene, the ICP22 gene, the UL41 gene, and/or the ICP27 gene.
• the recombinant herpes virus genome (e.g., recombinant herpes simplex virus genome) has been engineered to reduce cytotoxicity of the recombinant genome (e.g., when introduced into a target cell) as compared to a corresponding wild-type herpes virus genome (e.g., a wild-type herpes simplex virus genome).
• cytotoxicity (e.g., in human keratinocytes and/or fibroblast cells) of the recombinant virus genome is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% as compared to a corresponding wild- type herpes virus genome (e.g., measuring the relative cytotoxicity of a recombinant AICP4 (one or both copies) herpes simplex virus genome vs.
• AICP4 one or both copies
• a wild-type herpes simplex virus genome in human keratinocytes or fibroblasts primary cells or cell lines
• cytotoxicity (e.g., in human keratinocytes and/or fibroblast cells) of the recombinant herpes virus genome is reduced by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about lOO-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, at least about 1000-fold, or more as compared to a corresponding wild- type herpes virus genome (e.g., measuring the relative cytotoxicity of a recombinant AICP4 (one or both copies) herpes simplex
• a wild-type herpes simplex virus genome in human keratinocytes or fibroblasts primary cells or cell lines
• cytotoxicity Methods of measuring cytotoxicity are known to one of ordinary skill in the art, including, for example, through the use of vital dyes (formazan dyes), protease biomarkers, an MTT assay (or an assay using related tetrazolium salts such as XTT, MTS, water-soluble tetrazolium salts, etc.), measuring ATP content, etc.
• vital dyes formazan dyes
• protease biomarkers an MTT assay (or an assay using related tetrazolium salts such as XTT, MTS, water-soluble tetrazolium salts, etc.), measuring ATP content, etc.
• the recombinant herpes virus genome (e.g., a recombinant herpes simplex virus genome) has been engineered to reduce its impact on host cell proliferation after exposure of a target cell to the recombinant genome, as compared to a corresponding wild-type herpes virus genome (e.g., a wild-type herpes simplex virus genome).
• the target cell is a human cell.
• the target cell is a cell of the epidermis and/or dermis.
• the target cell is a cell of the eye.
• the target cell is a cell of the joint.
• the target cell is a cell of the lungs.
• host cell proliferation (e.g., of human keratinocytes and/or fibroblast cells) after exposure to the recombinant genome is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% faster as compared to host cell proliferation after exposure to a corresponding wild-type herpes virus genome (e.g., measuring the relative cellular proliferation after exposure to a recombinant AICP4 (one or both copies) herpes simplex virus genome vs.
• AICP4 one or both copies
• host cell proliferation (e.g., of human keratinocytes and/or fibroblast cells) after exposure to the recombinant genome is at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9- fold, at least about lO-fold, at least about l5-fold, at least about 20-fold, at least about 25- fold, at least about 50-fold, at least about 75-fold, at least about lOO-fold, at least about 250- fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold faster as compared to host cell proliferation after exposure to a corresponding wild-type herpes virus genome (e.g., measuring the relative cellular proliferation after exposure to a recombinant AICP4 (one or both copies) herpes simplex virus genome vs.
• AICP4 one or both copies
• cellular proliferation after exposure to a wild-type herpes simplex virus genome in human keratinocytes or fibroblasts primary cells or cell lines
• Methods of measuring cellular proliferation are known to one of ordinary skill in the art, including, for example, through the use of a Ki67 cell proliferation assay, a BrdU cell proliferation assay, etc.
• a vector may include one or more polynucleotides of the present disclosure in a form suitable for expression of the polynucleotide in a host cell.
• Vectors may include one or more regulatory sequences operatively linked to the
• polynucleotide to be expressed e.g., as described above.
• a recombinant nucleic acid e.g., a recombinant herpes simplex virus genome
• a recombinant nucleic acid of the present disclosure comprises one or more of the
• polynucleotides described herein inserted in any orientation in the recombinant nucleic acid. If the recombinant nucleic acid comprises two or more polynucleotides described herein (e.g., two or more, three or more, etc.), the polynucleotides may be inserted in the same orientation or opposite orientations to one another. Without wishing to be bound be theory, incorporating two polynucleotides (e.g., two transgenes) into a recombinant nucleic acid (e.g., a vector) in an antisense orientation may help to avoid read-through and ensure proper expression of each polynucleotide.
• two polynucleotides e.g., two transgenes
• a recombinant nucleic acid e.g., a vector
• viruses comprising any of the polynucleotides and/or recombinant nucleic acids described herein.
• the virus is capable of infecting one or more target cells of a subject (e.g., a human).
• the virus is suitable for delivering the polynucleotides and/or recombinant nucleic acid into one or more target cells of a subject (e.g., a human subject).
• the one or more target cells are one or more cells of the mucosa or skin (e.g., one or more cells of the epidermis, dermis, and/or subcutis).
• the one or more cells are selected from keratinocytes, melanocytes, Langerhans cells, Merkel cells, mast cells, fibroblasts, and/or adipocytes. In some embodiments, the one or more cells are keratinocytes. In some embodiments, the one or more cells reside in the stratum corneum, stratum granulosum, stratum spinulosum, stratum basale, and/or basement membrane. In some embodiments, the one or more target cells are one or more epidermal cells. In some embodiments, the one or more target cells are one or more dermal cells. In some
• the one or more target cells are one or more cells of a joint. In some embodiments, the one or more target cells are one or more cells of the eye. In some embodiments, the one or more target cells are one or more cells of the airway and/or lung.
• any suitable virus known in the art may be used, including, for example, adenovirus, adeno-associated virus, retrovirus, lentivirus, sendai virus, herpes virus (e.g., a herpes simplex virus), vaccinia virus, and/or any hybrid or derivative viruses thereof.
• the virus is attenuated.
• the virus is replication defective.
• the virus is replication competent.
• the virus has been modified to alter its tissue tropism relative to the tissue tropism of a corresponding unmodified, wild-type virus.
• the virus has reduced cytotoxicity as compared to a corresponding wild-type virus.
• the virus is a member of the Herpesviridae family of DNA viruses, including, for example, a herpes simplex virus, a varicella zoster virus, a human cytomegalovirus, a herpesvirus 6A, a herpesvirus 6B, a herpesvirus 7, and a Kaposi’s sarcoma-associated herpesvirus, etc.
• the herpes virus is attenuated.
• the herpes virus is replication defective.
• the herpes virus is replication competent.
• the herpes virus has reduced cytotoxicity as compared to a corresponding wild-type herpes virus.
• the herpes virus is not oncolytic.
• the virus is a herpes simplex virus.
• Herpes simplex viruses comprising recombinant nucleic acids may be produced by a process disclosed, for example, in W02015/009952 and/or WO2017/176336.
• the herpes simplex virus is attenuated.
• the herpes simplex virus is replication defective.
• the herpes simplex virus is replication competent.
• the herpes simplex virus is a herpes simplex type 1 virus (HSV-l), a herpes simplex type 2 virus (HSV-2), or any derivatives thereof.
• the herpes simplex virus is a herpes simplex type 1 virus (HSV-l). In some embodiments, the HSV-l is attenuated. In some embodiments, the HSV-l is replication defective. In some embodiments, the HSV-l is replication competent. In some embodiments, the HSV-l has reduced cytotoxicity as compared to a corresponding wild- type HSV-l. In some embodiments, the HSV-l is not oncolytic. [0168] In some embodiments, the herpes simplex virus has been modified to alter its tissue tropism relative to the tissue tropism of an unmodified, wild-type herpes simplex virus. In some embodiments, the herpes simplex virus comprises a modified envelope.
• HSV-l herpes simplex type 1 virus
• the modified envelope comprises one or more (e.g., one or more, two or more, three or more, four or more, etc.) mutant herpes simplex virus glycoproteins.
• herpes simplex virus glycoproteins may include, but are not limited to, the glycoproteins gB, gC, gD, gH, and gL.
• the modified envelope alters the herpes simplex virus tissue tropism relative to a wild-type herpes simplex virus
• the transduction efficiency (in vitro and/or in vivo ) of a virus of the present disclosure e.g., a herpes virus such as a herpes simplex virus
• a virus of the present disclosure e.g., a herpes virus such as a herpes simplex virus
• target cells e.g., one or more human keratinocytes and/or fibroblasts
• the transduction efficiency of the virus for one or more target cells may be at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, at least about 99.5%, or more.
• the virus is a herpes simplex virus and the transduction efficiency of the virus for one or more target cells (e.g., one or more human keratinocytes and/or fibroblasts) is about 85% to about 100%.
• the virus is a herpes simplex virus and the transduction efficiency of the virus for one or more target cells (e.g., one or more human keratinocytes and/or fibroblasts) is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.
• target cells e.g., one or more human keratinocytes and/or fibroblasts
• Methods of measuring viral transduction efficiency in vitro or in vivo are well known to one of ordinary skill in the art, including, for example, qPCR analysis, deep sequencing, western blotting, fluorometric analysis (such as fluorescent in situ hybridization (FISH), fluorescent reporter gene expression,
• compositions and formulations comprising any of the recombinant nucleic acids (e.g., a recombinant herpes virus genome) and/or viruses (e.g., a herpes virus comprising a recombinant genome) described herein, and a pharmaceutically acceptable excipient or carrier.
• recombinant nucleic acids e.g., a recombinant herpes virus genome
• viruses e.g., a herpes virus comprising a recombinant genome
• the pharmaceutical composition or formulation comprises any one or more of the viruses (e.g., herpes viruses) as described herein.
• viruses e.g., herpes viruses
• the pharmaceutical composition or formulation comprises from about 10 4 to about 10 12 plaque forming units (PFU)/mL of the virus.
• the pharmaceutical composition or formulation may comprise from about 10 4 to about 10 12 , about 10 5 to about 10 12 , about 10 6 to about 10 12 , about 10 7 to about 10 12 , about 10 8 to about 10 12 , about 10 9 to about 10 12 , about 10 10 to about 10 12 , about 10 11 to about 10 12 , about 10 4 to about 10 11 , about 10 5 to about 10 11 , about 10 6 to about 10 11 , about 10 7 to about 10 11 , about 10 8 to about 10 11 , about 10 9 to about 10 11 , about 10 10 to about 10 11 , about 10 4 to about 10 10 , about 10 5 to about
• the pharmaceutical composition or formulation comprises about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 4 to about 10 9 , about 10 5 to about 10 9 , about 10 6 to about 10 9 , about 10 7 to about 10 9 , about 10 8 to about 10 9 , about 10 4 to about 10 8 , about 10 5 to about 10 8 , about 10 6 to about 108, about 10 7 to about 10 8 , about 10 4 to about 10 7 , about 10 5 to about 10 7 , about 10 6 to about 10 7 , about 10 4 to about 10 6 , about 10 5 to about 10 6 , or about 10 4 to about 10 5 PFU/mL of the virus.
• the pharmaceutical composition or formulation comprises about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about
• compositions and formulations can be prepared by mixing the active ingredient(s) (such as a recombinant nucleic acid and/or a virus) having the desired degree of purity with one or more pharmaceutically acceptable carriers or excipients.
• active ingredient(s) such as a recombinant nucleic acid and/or a virus
• Pharmaceutically acceptable carriers or excipients are generally nontoxic to recipients at the dosages and concentrations employed, and may include, but are not limited to: buffers (such as phosphate, citrate, acetate, and other organic acids); antioxidants (such as ascorbic acid and methionine); preservatives (such as octadecyldimethylbenzyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine); low molecular weight (less than about 10 residues) polypeptides; proteins (such as serum albumin, gelatin, or
• immunoglobulins include polyols (such as glycerol, e.g., formulations including 10% glycerol); hydrophilic polymers (such as polyvinylpyrrolidone); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrins); chelating agents (such as EDTA); sugars (such as sucrose, mannitol, trehalose, or sorbitol); salt-forming counter-ions (such as sodium); metal complexes (such as Zn-protein complexes); and/or non-ionic surfactants (such as polyethylene glycol (PEG)).
• PEG polyethylene glycol
• the pharmaceutical composition or formulation comprises one or more lipid (e.g., cationic lipid) carriers.
• the pharmaceutical composition or formulation comprises one or more nanoparticle carriers.
• Nanoparticles are submicron (less than about 1000 nm) sized drug delivery vehicles that can carry encapsulated drugs (such as synthetic small molecules, proteins, peptides, cells, viruses, and nucleic acid- based biotherapeutics for rapid or controlled release.
• encapsulated drugs such as synthetic small molecules, proteins, peptides, cells, viruses, and nucleic acid- based biotherapeutics for rapid or controlled release.
• encapsulated drugs such as synthetic small molecules, proteins, peptides, cells, viruses, and nucleic acid- based biotherapeutics for rapid or controlled release.
• a variety of molecules e.g., proteins, peptides, recombinant nucleic acids, etc.
• a molecule“encapsulated” in a nanoparticle may refer to a molecule (such as a virus) that is contained within the nanoparticle or attached to and/or associated with the surface of the nanoparticle, or any combination thereof.
• Nanoparticles for use in the compositions or formulations described herein may be any type of biocompatible nanoparticle known in the art, including, for example, nanoparticles comprising poly(lactic acid), poly(glycolic acid), PLGA, PLA, PGA, and any combinations thereof (see e.g., Vauthier et al. Adv Drug Del Rev. (2003) 55: 519-48; US2007/0148074; US2007/0092575; US2006/0246139; US5753234; US7081483; and W02006/052285).
• the pharmaceutically acceptable carrier or excipient may be adapted for or suitable for any administration route known in the art, including, for example, intravenous, intramuscular, subcutaneous, cutaneous, oral, intratracheal, sublingual, buccal, topical, transdermal, intradermal, intraperitoneal, intraorbital, intravitreal, subretinal, transmucosal, intraarticular, by implantation, by inhalation, intrathecal, intraventricular, and/or intranasal administration.
• the pharmaceutically acceptable carrier or excipient is adapted for or suitable for topical, transdermal, subcutaneous, and/or intradermal administration.
• the pharmaceutical composition or formulation is adapted for or suitable for topical, transdermal, subcutaneous, and/or intradermal administration.
• the pharmaceutically acceptable carrier or excipient is adapted for or suitable for topical, transdermal, and/or intradermal administration.
• the pharmaceutical composition or formulation is adapted for or suitable for topical, transdermal, and/or intradermal administration.
• the pharmaceutically acceptable carrier or excipient is adapted for or suitable for oral, sublingual, nasal, intranasal, intratracheal, or buccal administration, or administration via inhalation.
• the pharmaceutical composition or formulation is adapted for or suitable for oral, sublingual, nasal, intranasal, intratracheal, or buccal administration, or administration via inhalation.
• the pharmaceutically acceptable carrier or excipient is adapted for or suitable for topical (to the eye), intravitreal, subretinal, or intraorbital administration.
• the pharmaceutical composition or formulation is adapted for or suitable for topical (to the eye), intravitreal, subretinal, or intraorbital administration.
• the pharmaceutically acceptable carrier or excipient is adapted for or suitable for intraarticular administration.
• the pharmaceutical composition or formulation is adapted for or suitable for intraarticular administration.
• compositions or formulations of the present disclosure may include, but are not limited to, ointments, oils, pastes, creams, aerosols, suspensions, emulsions, fatty ointments, gels, powders, liquids, lotions, solutions, sprays, patches (e.g., transdermal patches or microneedle patches), adhesive strips, a microneedle or microneedle arrays, and inhalants.
• ointments oils, pastes, creams, aerosols, suspensions, emulsions, fatty ointments, gels, powders, liquids, lotions, solutions, sprays, patches (e.g., transdermal patches or microneedle patches), adhesive strips, a microneedle or microneedle arrays, and inhalants.
• the carrier or excipient comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more, etc.) of an ointment, oil, paste, cream, aerosol, suspension, emulsion, fatty ointment, gel, powder, liquid, lotion, solution, spray, adhesive strip, and an inhalant.
• the carrier comprises a patch (e.g. a patch that adheres to the skin), such as a transdermal patch or a microneedle patch.
• the carrier comprises a microneedle or microneedle array. Methods for making and using microneedle arrays suitable for composition delivery are generally known in the art (Kim Y. et al.“Microneedles for drug and vaccine delivery”. Advanced Drug Delivery Reviews 2012, 64 (14): 1547-68).
• the pharmaceutical composition or formulation further comprises one or more additional components.
• additional components may include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.) fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.)] lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.)] disintegrants (e.g., starch, sodium starch glycolate, etc.)] wetting agents (e.g., sodium lauryl sulphate, etc.)] salt solutions; alcohols; poly(ethylene glycol)
• the pharmaceutical composition or formulation comprises a hydroxypropyl methylcellulose gel. In some embodiments, the pharmaceutical composition or formulation comprises a phosphate buffer. In some embodiments, the pharmaceutical composition or formulation comprises glycerol (e.g., at about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, etc.).
• glycerol e.g., at about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, etc.
• compositions and formulations e.g., pharmaceutical compositions and formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
• any of the recombinant nucleic acids, viruses, and/or pharmaceutical compositions or formulations described herein may be used to deliver one or more polynucleotides encoding an antibody (e.g., a therapeutic antibody) into one or more cells of a subject and/or may be used to express an antibody (e.g., a therapeutic antibody) in one or more tissues of a subject.
• any of the recombinant nucleic acids, viruses, and/or pharmaceutical compositions or formulations described herein may be used in a therapy.
• any of the recombinant nucleic acids, viruses, and/or pharmaceutical compositions or formulations may be used in the treatment of a disease or condition that would benefit from the administration of an antibody (e.g., a therapeutic antibody).
• any of the recombinant nucleic acids, viruses, and/or pharmaceutical compositions or formulations described herein may be used in the treatment of one or more of psoriasis (e.g., chronic plaque psoriasis), atopic dermatitis, pyoderma gangrenosum, a blistering disease, pemphigus, pemphigus vulgaris, pemphigus foliaceus, an autoimmune bullous skin disorder, bullous pemphigoid, Behcet’s disease, cancer (e.g., skin cancer, breast cancer, lymphoma, colorectal cancer, head and neck cancer, etc.), hidradenitis suppurativa, arthritis, rheumatoid
• graft/tissue/organ rejection graft/tissue/organ rejection, and/or neurological diseases (e.g., Alzheimer’s).
• neurological diseases e.g., Alzheimer’s.
• any of the recombinant nucleic acids, viruses, and/or pharmaceutical compositions or formulations described herein may be used in the preparation or manufacture of a medicament.
• any of the recombinant nucleic acids, viruses, and/or pharmaceutical compositions or formulations described herein may be used in the preparation or manufacture of a medicament useful for delivering one or more polynucleotides encoding an antibody (e.g., a therapeutic antibody) into one or more cells of a subject and/or may be used in the preparation or manufacture of a medicament useful for expressing an antibody (e.g., a therapeutic antibody) in one or more tissues of a subject.
• any of the recombinant nucleic acids, viruses, and/or pharmaceutical compositions or formulations described herein may be used in the preparation or manufacture of a medicament useful for the treatment of a disease or condition that would benefit from the administration of an antibody (e.g., a therapeutic antibody).
• an antibody e.g., a therapeutic antibody
• any of the recombinant nucleic acids, viruses, and/or pharmaceutical compositions or formulations described herein may be used in the preparation or manufacture of a medicament useful for the treatment of one or more of psoriasis (e.g., chronic plaque psoriasis), atopic dermatitis, pyoderma gangrenosum, a blistering disease, pemphigus, pemphigus vulgaris, pemphigus foliaceus, an autoimmune bullous skin disorder, bullous pemphigoid, Behcet’s disease, cancer (e.g., skin cancer, breast cancer, lymphoma, colorectal cancer, head and neck cancer, etc.), hidradenitis suppurativa, arthritis, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, juvenile idiopathic arthritis, ankylosing spondylitis, axial spondylarthritis, reactive arthritis, enteropathic arthritis, psoria
• Certain aspects of the present disclosure relate to methods of delivering an antibody to a subject comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, medicaments, and/or compositions or formulations described herein.
• the present disclosure relates to a method of locally delivering an antibody to one or more specific tissues of interest in a subject (e.g., tissues of the eye, tissues of the joints, tissues of the skin, tissues of the lungs, etc.) comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, medicaments, and/or compositions or formulations described herein.
• the subject is a human.
• localized delivery of an antibody using a recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation described herein reduces or eliminates systemic exposure to the antibody in the subject, e.g., as compared to a subject who received treatment with a purified antibody delivered via a traditional route of antibody administration (such as intravenous or subcutaneous administration).
• a traditional route of antibody administration such as intravenous or subcutaneous administration.
• use of a recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation described herein for localized delivery of an antibody to a subject reduces or eliminates one or more side effects of the antibody, as compared to the side effects observed after systemic exposure of the subject to the same antibody (e.g., comparing one or more side effects of expressing the antibody in the subject after delivery (e.g., topical, intraarticular, intravitreal, etc.) of a recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation described herein vs. the side effects after systemic (e.g., intravenous or subcutaneous) administration of the purified antibody).
• systemic e.g., intravenous or subcutaneous
• Examples of side effects resulting from systemic exposure to a therapeutic antibody include, for example, allergic reactions, chills, weakness, diarrhea, nausea, vomiting, rash, itching, high blood glucose levels, cough, constipation, shortness of breath, peripheral edema, headache, fever, muscle aches and pains, decreased appetite, increased triglyceride levels, insomnia, abdominal pain, back pain, dizziness, low blood pressure, anaphylaxis, infections, cancer, serum sickness, autoimmune thyroiditis, arterial and venous blood clots, congestive heart failure, bleeding, interstitial lung disease, hepatitis, gastrointestinal perforation, enterocolitis, mucositis, stomatitis, anemia, reduced white blood cell count, and/or hypothyroidism.
• use of a recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation described herein for localized delivery of an antibody to a subject increases antibody tissue accessibility and/or infiltration at a site of interest (e.g., in the skin, in a joint, in the eye, in the airway or lungs, etc.), as compared to antibody tissue accessibility and/or infiltration at the site of interest after systemic (e.g., intravenous or subcutaneous) administration of the purified antibody.
• a site of interest e.g., in the skin, in a joint, in the eye, in the airway or lungs, etc.
• systemic e.g., intravenous or subcutaneous
• use of a recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation described herein for localized delivery of an antibody to a subject increases antibody concentration at the site of interest (e.g., in the skin, in a joint, in the eye, in the airway or lungs, etc.), as compared to antibody concentration at the site of interest after systemic (e.g., intravenous or subcutaneous) administration of the purified antibody.
• site of interest e.g., in the skin, in a joint, in the eye, in the airway or lungs, etc.
• systemic e.g., intravenous or subcutaneous
• use of a recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation described herein for localized delivery of an antibody increases antibody concentration at a site of interest (e.g., in the skin, in a joint, in the eye, in the airway or lungs, etc.) by at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more, as compared to antibody concentration at the site of interest after systemic (e.g., intravenous or subcutaneous) administration of the purified antibody.
• a site of interest e.g., in the skin, in a joint, in the eye, in the airway or lungs, etc.
• use of a recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation described herein for localized delivery of an antibody increases antibody concentration at a site of interest (e.g., in the skin, in a joint, in the eye, in the airway or lungs, etc.) by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about lO-fold, at least about l5-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about lOO-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold, as compared to antibody concentration at the site of interest after systemic (e.g., intravenous or subcutaneous) administration of the purified
• prophylactic, palliative, and/or therapeutic relief of one or more signs or symptoms of a disease in a subject comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, medicaments, and/or pharmaceutical compositions or formulations described herein.
• the subject is a human.
• the disease may be any disease known in the art that may benefit from treatment with a therapeutic antibody, including, for example, psoriasis (e.g., chronic plaque psoriasis), atopic dermatitis, pyoderma gangrenosum, a blistering disease, pemphigus, pemphigus vulgaris, pemphigus foliaceus, an autoimmune bullous skin disorder, bullous pemphigoid, Behcet’s disease, cancer (e.g., skin cancer, breast cancer, lymphoma, colorectal cancer, head and neck cancer, etc.), hidradenitis suppurativa, arthritis, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, juvenile idiopathic arthritis, ankylosing spondylitis, axial spondylarthritis, reactive arthritis, enteropathic arthritis, autoimmune disease (e.g., multiple sclerosis, systemic lupus erythematosus
• the disease not cancer.
• the recombinant nucleic acids, viruses, medicaments, and/or pharmaceutical compositions or formulations described herein may be administered by any suitable method or route known in the art, including, without limitation, by oral administration, sublingual administration, buccal administration, intranasal administration, intratracheal administration, topical administration, rectal administration, via inhalation, transdermal administration, subcutaneous injection, intradermal injection, intravenous (IV) injection, intra-arterial injection, intramuscular injection, intracardiac injection, intraosseous injection,
• intraperitoneal injection transmucosal administration, vaginal administration, intravitreal administration, intraorbital administration, subconjunctival administration (e.g., the use of subconjunctival depots), suprachoroidal administration, subretinal administration, intra- articular administration, peri-articular administration, local administration, epicutaneous administration, or any combinations thereof.
• the present disclosure thus encompasses methods of delivering any of the recombinant nucleic acids, viruses, medicaments, and/or pharmaceutical compositions or formulations described herein to an individual (or a specific site or tissue thereof).
• the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation used in the methods of the present disclosure is administered cutaneously, topically, transdermally, subcutaneously, intradermally, transmucosally, sublingually, nasally, buccally, intranasally, intratracheally, intravitreally, subconjunctivally, suprachoroidally, subretinally, intraarticularly, or via inhalation to the subject.
• the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered topically to the subject.
• the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered intradermally to the subject.
• the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered orally, sublingually, buccally, nasally, intranasally, intratracheally, or via inhalation to the subject.
• the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered intraarticularly to the subject.
• the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered intraorbitally, intravitreally, subconjunctivally, suprachoroidally, subretinally, or topically (to the eye) of the subject.
• the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered once to the subject. In some embodiments, the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered at least twice (e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, etc.) to the subject.
• At least about 1 hour e.g., at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 15 days, at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, at least about 60 days, at least about 70 days, at least about 80 days, at least about 90 days, at least about 100 days, at least about 120 days, etc.) pass between administrations (e.g., between the first and second administrations, between the second and third administrations, etc.).
• administrations e.g., between the first and second administrations, between the second and third administrations, etc.
• the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered one, two, three, four, five or more times per day to the subject. In some embodiments, the recombinant nucleic acid, virus, medicament and/or pharmaceutical composition or formulation is administered to one or more affected and/or unaffected areas of the subject.
• Other aspects of the present disclosure relate to a method of administering an antibody to the epidermis and/or dermis of a subject comprising topically, transdermally, and/or intradermally administering an effective amount of any of the recombinant nucleic acids, viruses, medicaments, and/or pharmaceutical compositions or formulations described herein to the subject.
• the subject is not exposed to the antibody systemically (e.g., it is not detectable in the serum).
• the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered topically.
• the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered
• the subject is a human.
• the subject suffers from a disease or disorder of the skin.
• the subject suffers from one or more of psoriasis, atopic dermatitis, pyoderma gangrenosum, a blistering disease, pemphigus, pemphigus vulgaris, pemphigus foliaceus, an autoimmune bullous skin disorder, bullous pemphigoid, Befu j et’s disease, skin cancer, and/or hidradenitis suppurativa.
• the disease or disorder is not cancer (e.g., is not skin cancer).
• one or more portions of the skin of the subject is abraded or made more permeable prior to treatment with a recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation described herein.
• Any suitable method of abrading the skin or increasing skin permeability known in the art may be used, including, for example, use of a dermal roller, repeated use of adhesive strips to remove layers of skin cells (tape stripping), scraping with a scalpel or blade, use of sandpaper, use of chemical permeation enhancers (e.g., cell-penetrating polypeptides) or electrical energy, use of sonic or ultrasonic energy, use of light (e.g., laser) energy, use of micron-sized needles or blades with a length suitable to pierce but not completely pass through the epidermis, etc.
• any suitable method of abrading the skin or increasing skin permeability known in the art may be used, including, for example, use of a dermal roller, repeated use of adhesive strips
• the subject is not exposed to the antibody systemically (e.g., it is not detectable in the serum).
• the recombinant nucleic acid, virus, medicament, and/or composition is administered sublingually.
• the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered topically.
• the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered buccally.
• the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered intranasally. In some embodiments, the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered via inhalation. In some embodiments, the subject is a human.
• aspects of the present disclosure relate to a method of administering an antibody to the airway or lungs of a subject comprising orally, sublingually, nasally, intranasally, intratracheally, via inhalation, or buccally administering an effective amount of any of the recombinant nucleic acids, viruses, medicaments, and/or pharmaceutical compositions or formulations described herein to the subject.
• the subject is not exposed to the antibody systemically (e.g., it is not detectable in the serum).
• the recombinant nucleic acid, virus, medicament, and/or composition is administered sublingually.
• the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered buccally. In some embodiments, the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered intranasally. In some embodiments, the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation is administered via inhalation. In some embodiments, the subject is a human. In some embodiments, the subject suffers from a disease or disorder of the airway or lungs (e.g., a respiratory disease such as asthma, lung cancer, respiratory infections, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, etc.).
• a respiratory disease such as asthma, lung cancer, respiratory infections, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, etc.
• Other aspects of the present disclosure relate to a method of administering an antibody to one or more joints of a subject comprising intraarticularly administering an effective amount of any of the recombinant nucleic acids, viruses, medicaments, and/or pharmaceutical compositions or formulations described herein to the subject.
• the subject is not exposed to the antibody systemically (e.g., it is not detectable in the serum).
• the subject suffers from a disease of the joints.
• the subject suffers from one or more of arthritis, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, juvenile idiopathic arthritis, ankylosing spondylitis, axial
• the subject is a human.
• aspects of the present disclosure relate to a method of administering an antibody to one or both eyes of a subject comprising topically, intravitreally, intraorbitally, subconjunctivally, subretinally, or suprachoroidally administering an effective amount of any of the recombinant nucleic acids, viruses, medicaments, and/or pharmaceutical compositions or formulations described herein to the subject.
• the subject is not exposed to the antibody systemically (e.g., it is not detectable in the serum).
• the subject suffers from a disease of the eye.
• the subject suffers from an autoimmune disease that effects the eyes.
• the subject suffers from uveal melanoma or thyroid eye disease.
• the subject is a human.
• prokaryotic cells comprising any of the recombinant nucleic acids described herein.
• Any suitable host cell known in the art may be used, including, for example: prokaryotic cells including eubacteria, such as Gram-negative or Gram-positive organisms, for example Enterobacteriaceae such as Escherichia (e.g., E. coli ), Enterobacter, Erminia, Klebsiella, Proteus, Salmonella (e.g., S. typhimurium), Serratia (e.g., S. marcescans), and Shigella, as well as Bacilli such as B. subtilis and B.
• eubacteria such as Gram-negative or Gram-positive organisms
• Enterobacteriaceae such as Escherichia (e.g., E. coli )
• Enterobacter Erminia
• Klebsiella Proteus
• Salmonella e.g., S. typhimurium
• Serratia
• licheniformis fungal cells (e.g., S. cerevisiae ); insect cells (e.g., S2 cells, etc.); and mammalian cells, including monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture), baby hamster kidney cells (BHK, ATCC CCL 10), mouse Sertoli cells (TM4), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, LS4 cells, human hepatom
• the host cell is a human or non-human primate cell.
• the host cells are cells from a cell line. Examples of suitable host cells or cell lines may include, but are not limited to, 293, HeLa, SH-Sy5y, Hep G2, CACO-2, A549, L929, 3T3, K562, CHO-K1, MDCK, HUVEC, Vero, N20, COS-7, PSN1, VCaP, CHO cells, and the like.
• the recombinant nucleic acid is a herpes simplex viral vector. In some embodiments, the recombinant nucleic acid is a herpes simplex virus amplicon. In some embodiments, the recombinant nucleic acid is an HSV-l amplicon or HSV-l hybrid amplicon. In some embodiments, a host cell comprising a helper virus is contacted with an HSV-l amplicon or HSV-l hybrid amplicon described herein, resulting in the production of a virus comprising one or more recombinant nucleic acids described herein. In some embodiments, the virus is collected from the supernatant of the contacted host cell. Methods of generating virus by contacting host cells comprising a helper virus with an HSV- 1 amplicon or HSV-l hybrid amplicon are known in the art
• the host cell is a complementing host cell.
• the complementing host cell expresses one or more genes that are inactivated in any of the viral vectors described herein.
• the complementing host cell is contacted with a recombinant herpes viral genome (e.g., a recombinant herpes simplex viral genome) described herein.
• contacting a complementing host cell with a recombinant herpes virus genome results in the production of a herpes virus comprising one or more recombinant nucleic acids described herein.
• the virus is collected from the supernatant of the contacted host cell. Methods of generating virus by contacting complementing host cells with a recombinant herpes simplex virus are generally described in W02015/009952 and/or WO2017/176336.
• Certain aspects of the present disclosure relate to an article of manufacture or a kit comprising any of the recombinant nucleic acids, viruses, medicaments, and/or
• the article of manufacture or kit comprises a package insert comprising instructions for administering the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation (e.g., to provide a method of locally delivering an antibody to one or more tissues of the subject (such as the epidermis and/or dermis of a subject) in need thereof by administering the recombinant nucleic acid, virus, medicament, and/or pharmaceutical composition or formulation).
• Suitable containers for the recombinant nucleic acids, viruses, medicaments and/or pharmaceutical compositions or formulations may include, for example, bottles, vials, bags, tubes, and syringes.
• the container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy).
• the container comprises a label on, or associated with the container, wherein the label indicates directions for use.
• the article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, package inserts, and the like.
• Embodiment 1 a recombinant herpes simplex virus (HSV) genome comprising one or more polynucleotides encoding an antibody.
• HSV herpes simplex virus
• Embodiment 2 the recombinant genome of embodiment 1, wherein the antibody is an antibody fragment.
• Embodiment 3 the recombinant genome of embodiment 2, wherein the antibody fragment is a Fab, Fab’, Fab’-SF, F(ab’)2, Fv, scFv, or scFv-Fc fragment.
• Embodiment 4 the recombinant genome of embodiment 1, wherein the antibody is a full-length antibody.
• Embodiment 5 the recombinant genome of any one of embodiments 1-4, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, a monoclonal antibody, or a multispecific antibody.
• Embodiment 6 the recombinant genome of any one of embodiments 1-5, wherein the antibody is an IgA, IgD, IgE, IgG, or IgM antibody.
• Embodiment 7 the recombinant genome of any one of embodiments 1-6, wherein the antibody is an IgG antibody.
• Embodiment 8 the recombinant genome of embodiment 7, wherein the IgG antibody is an IgGl, IgG2, IgG3, or IgG4 antibody.
• Embodiment 9 the recombinant genome of any one of embodiments 1-8, wherein the antibody is an agonist antibody.
• Embodiment 10 the recombinant genome of any one of embodiments 1-8, wherein the antibody is an antagonist antibody.
• Embodiment 11 the recombinant genome of any one of embodiments 1-10, wherein the antibody comprises a heavy chain variable region comprising an HVR-H1, an HVR-H2, and an HVR-H3, wherein the HVR-H1 comprises a sequence selected from the group consisting of SEQ ID NOS: 1-59, the HVR-H2 comprises a sequence selected from the group consisting of SEQ ID NOS: 60-122, and/or the HVR-H3 comprises a sequence selected from the group consisting of SEQ ID NOS: 123-185.
• the antibody comprises a heavy chain variable region comprising an HVR-H1, an HVR-H2, and an HVR-H3, wherein the HVR-H1 comprises a sequence selected from the group consisting of SEQ ID NOS: 1-59, the HVR-H2 comprises a sequence selected from the group consisting of SEQ ID NOS: 60-122, and/or the HVR-H3 comprises a sequence selected from the group consisting of SEQ ID NOS: 123
• Embodiment 12 the recombinant genome of embodiment 11, wherein the heavy chain variable region comprises a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 355-419.
• Embodiment 13 the recombinant genome of any one of embodiments 1-12, wherein the antibody comprises a light chain variable region comprising an HVR-L1, an HVR-L2, and an HVR-L3, wherein the HVR-L1 comprises a sequence selected from the group consisting of SEQ ID NOS: 186-242, the HVR-L2 comprises a sequence selected from the group consisting of SEQ ID NOS: 243-294, and/or the HVR-L3 comprises a sequence selected from the group consisting of SEQ ID NOS: 295-354.
• Embodiment 14 the recombinant genome of embodiment 13, wherein the light chain variable region comprises a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NOS: 420-482.
• Embodiment 15 the recombinant genome of any one of embodiments 1-14, wherein the recombinant genome is a recombinant HSV-l genome, a recombinant HSV-2 genome, or any derivatives thereof.
• Embodiment 16 the recombinant genome of any one of embodiments 1-15, wherein the recombinant genome comprises an inactivating mutation in a herpes simplex virus gene.
• Embodiment 17 the recombinant genome of embodiment 16, wherein the herpes simplex virus gene is selected from the group consisting of Infected Cell Protein (ICP) 0, ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), Long Unique Region (UL) 41, and UL55.
• ICP Infected Cell Protein
• ICP4 ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), Long Unique Region (UL) 41, and UL55.
• Embodiment 18 the recombinant genome of embodiment 17, wherein the recombinant genome comprises an inactivating mutation in one or both copies of the ICP4 gene.
• Embodiment 19 the recombinant genome of embodiment 17 or 18, wherein the recombinant genome comprises an inactivating mutation in the ICP22 gene.
• Embodiment 20 the recombinant genome of any one of embodiments 17-19, wherein the recombinant genome comprises an inactivating mutation in the UL41 gene.
• Embodiment 21 the recombinant genome of any one of embodiments 17-20, wherein the recombinant genome comprises an inactivating mutation in the ICP0 gene.
• Embodiment 22 the recombinant genome of any one of embodiments 17-21, wherein the recombinant genome comprises an inactivating mutation in the ICP27 gene.
• Embodiment 23 the recombinant genome of any one of embodiments 16-22, wherein the inactivating mutation is a deletion of the coding sequence of the gene(s).
• Embodiment 24 the recombinant genome of any one of embodiments 1-23, wherein the recombinant genome has reduced cytotoxicity when introduced into a target cell as compared to a wild-type herpes simplex virus genome.
• Embodiment 25 the recombinant genome of embodiment 24, wherein the target cell is a human cell.
• Embodiment 26 the recombinant genome of embodiment 24 or 25, wherein the target cell is a keratinocyte or fibroblast.
• Embodiment 27 the recombinant genome of any one of embodiments 1-2, wherein the recombinant genome comprises the one or more polynucleotides within one or more viral gene loci.
• Embodiment 28 the recombinant genome of embodiment 27, wherein the recombinant genome comprises one or more polynucleotides within one or both of the ICP4 viral gene loci.
• Embodiment 29 the recombinant genome of embodiment 27 or 28, wherein the recombinant genome comprises the one or more polynucleotides within the ICP22 viral gene locus.
• Embodiment 30 the recombinant genome of any one of embodiments 27-29, wherein the recombinant genome comprises the one or more polynucleotides within the UL41 viral gene locus.
• Embodiment 31 a herpes simplex virus (HSV) comprising the recombinant genome of any one of embodiments 1-30.
• HSV herpes simplex virus
• Embodiment 32 the virus of embodiment 31, wherein the HSV is replication competent.
• Embodiment 33 the virus of embodiment 31, wherein the HSV is replication defective.
• Embodiment 34 the virus of any one of embodiments 31-33, wherein the HSV has reduced cytotoxicity as compared to a wild-type herpes simplex virus.
• Embodiment 35 the virus of any one of embodiments 31-34, wherein the HSV is a herpes simplex type 1 virus, a herpes simplex type 2 virus, or any derivatives thereof.
• Embodiment 36 a pharmaceutical composition comprising the recombinant genome of any one of embodiments 1-30 or the virus of any one of embodiments 31-35 and a pharmaceutically acceptable excipient.
• Embodiment 37 the pharmaceutical composition of embodiment 36, wherein the pharmaceutically acceptable excipient is suitable for topical, transdermal, subcutaneous, intradermal, transmucosal, sublingual, nasal, buccal, intraorbital, intravitreal,
• Embodiment 38 the pharmaceutical composition of embodiment 36 or 37, wherein the pharmaceutically acceptable excipient is suitable for topical administration.
• Embodiment 39 the pharmaceutical composition of any one of embodiments 36-
• the pharmaceutically acceptable excipient comprises a hydroxypropyl methylcellulose gel.
• Embodiment 40 the pharmaceutical composition of any one of embodiments 36-
• the pharmaceutically acceptable excipient comprises a phosphate buffer.
• Embodiment 41 the pharmaceutical composition of any one of embodiments 36-
• the pharmaceutically acceptable excipient comprises glycerol
• Embodiment 42 the pharmaceutical composition of any one of embodiments 36-
• the pharmaceutically acceptable excipient comprises a lipid carrier.
• Embodiment 43 the pharmaceutical composition of any one of embodiments 36-
• the pharmaceutically acceptable excipient comprises a nanoparticle carrier.
• Embodiment 44 a method of administering an antibody to a subject, the method comprises administering to the subject an effective amount of the virus of any one of embodiments 31-35 or the pharmaceutical composition of any one of embodiments 36-43.
• Embodiment 45 a method of providing prophylactic, palliative, and/or therapeutic relief of one or more signs or symptoms of a disease in a subject, the method comprising administering to the subject an effective amount of the virus of any one of embodiments 31- 35 or the pharmaceutical composition of any one of embodiments 36-43.
• Embodiment 46 the method of embodiment 44 or 45, wherein the virus or composition is administered topically, transdermally, subcutaneously, intradermally, transmucosally, sublingually, nasally, buccally, intravitreally, subconjunctivally, suprachoroidally, intraarticularly, or via inhalation.
• Embodiment 47 the method of embodiment45 or 46, wherein the disease is selected from the group consisting of psoriasis, atopic dermatitis, pyoderma gangrenosum, a blistering disease, pemphigus, pemphigus vulgaris, pemphigus foliaceus, an autoimmune bullous skin disorder, bullous pemphigoid, Behi j et’s disease, cancer, hidradenitis suppurativa, arthritis, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, juvenile idiopathic arthritis, ankylosing spondylitis, axial spondylarthritis, reactive arthritis, enteropathic arthritis, autoimmune disease, melanoma, uveal melanoma, and thyroid eye disease.
• the disease is selected from the group consisting of psoriasis, atopic dermatitis, pyoderma gangrenosum, a blistering
• Embodiment 48 a method of administering an antibody to the epidermis and/or dermis of a subject, the method comprising topically transdermally or intradermally administering to the subject an effective amount of the virus of any one of embodiments 31- 35 or the pharmaceutical composition of any one of embodiments 36-43.
• Embodiment 49 the method of embodiment 48, wherein the skin of the subject is abraded prior to administration.
• Embodiment 50 a method of administering an antibody to the mucosa of a subject, the method comprising topically, transmucosally, sublingually, nasally, or buccally administering to the subject an effective amount of the virus of any one of embodiments 31- 35 or the pharmaceutical composition of any one of embodiments 36-43.
• Embodiment 51 a method of administering an antibody to one or more joints of a subject, the method comprising intraarticularly administering to the subject an effective amount of the virus of any one of embodiments 31-35 or the pharmaceutical composition of any one of embodiments 36-43.
• Embodiment 52 a method of administering an antibody to one or both eyes of a subject, the method comprising topically, intraorbitally, intravitreally, subconjunctivally, or suprachoroidally administering to the subject an effective amount of the virus of any one of embodiments 31-35 or the pharmaceutical composition of any one of embodiments 36-43.
• Embodiment 53 the method of any one of embodiments 44-52, wherein the subject is a human.
• Embodiment 54 the method of any one of embodiments 44-53, wherein the subject is not exposed to the antibody systemically.
• Example 1 modified herpes simplex virus vectors encoding antibodies
• a herpes simplex virus genome (FIG. 1A) is first modified to inactivate one or more herpes simplex virus genes. Such modifications may decrease the toxicity of the genome in mammalian cells.
• variants of these modified/attenuated recombinant viral constructs are generated such that they carry one or more polynucleotides encoding the desired antibody.
• variants include: 1) a recombinant AICP4/AICP22-modified HSV-l genome comprising expression cassettes containing the coding sequence of an antibody fragment (e.g., an scFv-Fc) under the control of a heterologous promoter integrated at each ICP4 locus (FIG. IB); 2) a recombinant AICP4-modified HSV-l genome comprising expression cassettes containing the coding sequence of an antibody fragment (e.g., an scFv-Fc) under the control of a heterologous promoter integrated at each ICP4 locus (FIG.
• AICP4/AICP22-modified HSV-l genome comprising expression cassettes containing the coding sequence of an antibody heavy chain under the control of a first heterologous promoter and the coding sequence of an antibody light chain under the control of a second heterologous promoter on opposite strands of DNA integrated at each ICP4 locus (FIG. IF); 6) a recombinant AICP4-modified HSV-l genome comprising expression cassettes containing the coding sequence of an antibody heavy chain under the control of a first heterologous promoter and the coding sequence of an antibody light chain under the control of a second heterologous promoter on opposite strands of DNA integrated at each ICP4 locus (FIG. IF); 6) a recombinant AICP4-modified HSV-l genome comprising expression cassettes containing the coding sequence of an antibody heavy chain under the control of a first heterologous promoter and the coding sequence of an antibody light chain under the control of a second heterologous promoter on opposite strands
• a recombinant AICP4/AICP22-modified HSV-l genome comprising expression cassettes encoding a polycistronic mRNA under the control of a heterologous promoter integrated at each of the ICP4 loci, where the polycistronic mRNA contains the coding sequence of an antibody heavy and light chain separated by an internal ribosomal entry site (IRES) (FIG.
• ICP4 internal ribosomal entry site
• a recombinant AICP4-modified HSV-l genome comprising expression cassettes encoding a polycistronic mRNA under the control of a heterologous promoter integrated at each of the ICP4 loci, where the polycistronic mRNA contains the coding sequence of an antibody heavy and light chain separated by an internal ribosomal entry site (IRES) (FIG.
• IRS internal ribosomal entry site
• a recombinant AICP4/AICP22/AUL41 -modified HSV-l genome comprising expression cassettes containing the coding sequence of an antibody heavy chain under the control of a heterologous promoter integrated at each of the ICP4 loci, and an expression cassette containing the coding sequence of an antibody light chain under the control of a heterologous promoter integrated at the UL41 and ICP22 loci (FIG.
• a recombinant AICP4/AICP22/AUL41 -modified HSV-l genome comprising expression cassettes containing the coding sequence of an antibody light chain under the control of a heterologous promoter integrated at each of the ICP4 loci, and an expression cassette containing the coding sequence of an antibody heavy chain under the control of a heterologous promoter integrated at the UL41 and ICP22 loci (FIG.
• IK 11
• a recombinant AICP4/AUL41 -modified HSV-l genome comprising expression cassettes containing the coding sequence of an antibody heavy chain under the control of a heterologous promoter integrated at each of the ICP4 loci, and an expression cassette containing the coding sequence of a polycistronic mRNA under the control of a heterologous promoter integrated at the UL41 locus, where the polycistronic mRNA contains two copies of the coding sequence of an antibody light chain separated by an internal ribosomal entry site (IRES) (FIG.
• IRS internal ribosomal entry site
• a recombinant AICP4/AICP22-modified HSV-l genome comprising expression cassettes containing the coding sequence of an antibody heavy chain under the control of a heterologous promoter integrated at each of the ICP4 loci, and an expression cassette containing the coding sequence of a polycistronic mRNA under the control of a heterologous promoter integrated at the ICP22 locus, where the polycistronic mRNA contains two copies of the coding sequence of an antibody light chain separated by an internal ribosomal entry site (IRES) (FIG.
• IRS internal ribosomal entry site
• a recombinant AICP4/AICP22/AUL41 -modified HSV-l genome comprising expression cassettes containing the coding sequence of an antibody light chain under the control of a heterologous promoter integrated at the UL41 locus, and an expression cassette containing the coding sequence of an antibody heavy chain under the control of a heterologous promoter integrated at the ICP22 locus (FIG. IN).
• the modified herpes simplex virus genome may be engineered as described above, except that the coding sequence for the full-length antibody heavy chain is replaced with the coding sequence of an antibody heavy chain variable region and constant region 1 (CHi), such that the recombinant herpes simplex virus produces a Fab fragment.
• modified herpes simplex virus genome vectors are transfected into engineered Vero cells that are modified to express one or more herpes virus genes.
• engineered Vero cells secrete in the supernatant of the cell culture a replication defective herpes simplex virus with the modified genomes packaged therein. The supernatant is then collected, concentrated, and sterile filtered through a 5 pm filter.
• Example 2 construction and validation of recombinant herpes viruses encoding antibodies
• a recombinant, attenuated HSV vector was designed to express a full-length antibody.
• a polynucleotide construct was generated which encoded, from 5’ to 3’, a first ORF encoding the antibody light chain (with a leader sequence), a synthetic IRES, and second ORF encoding the antibody heavy chain (with a leader sequence) (e.g., as depicted in FIGS. 1H and II).
• the IRES-based construct (encoded in an expression cassette which further contained a heterologous promoter and a polyA sequence) was then inserted into both copies of the HSV1 ICP4 gene locus.
• This first viral construct (HSV-flAb2) was engineered to express a chimeric anti-human CD20 IgGl antibody containing a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 356 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 421.
• HSV 1 vectors were constructed to express single-chain antibodies from a single ORF (see e.g., FIGS IB and 1C).
• single-chain antibodies were constructed to contain a leader sequence, a heavy chain variable region sequence, a light chain variable region sequence, a linker polypeptide linking the heavy and light chain variable regions, and an Fc region (i.e., an scFv-Fc antibody).
• each single-chain antibody Two different variants of each single-chain antibody were designed to contain both possible relative orientations of the light and heavy chain variable region sequences: the“Fcl” variant of each antibody contained, from n-terminus to c- terminus, a leader sequence-a heavy chain variable region-a linker sequence-a light chain variable region-and an Fc region; the“Fc2” variant of each antibody contained, from n- terminus to c-terminus, a leader sequence-a light chain variable region-a linker sequence-a heavy chain variable region-and an Fc region.
• These single-chain antibody constructs (encoded in an expression cassette which further contained a heterologous promoter and a polyA sequence) were then inserted into both copies of the HSV1 ICP4 gene locus.
• Table 4 A summary of the sequences employed in each scFv-Fc antibody tested is provided in Table 4 below.
• Virus plaques were identified and picked from lawns of engineered cells, and the isolates were individually screened in Vero cells to identify vectors that produced the encoded antibodies, as described above.
• Cell supernatants were harvested from the infected Vero cells and tested for the presence of secreted antibody by ELISA, according to the manufacturer’s instructions (Abeam, cat. no. ab 195215 for single-chain antibodies containing human Fc sequences; Abeam, cat. no. abl51276 for single-chain antibodies containing mouse Fc sequences).
• Table 5 below provides the average calculated concentration of each antibody secreted into the supernatant from infected cells.
• HaCaTs Immortalized human keratinocytes
• MOI 0 mock infected
• MOIs multiplicities of infection
• the antibody encoding vectors used in this experiment were: HSV-AblFcl (a single-chain human anti- human TNFa antibody), HSV-AblFc2 (a single-chain human anti-human TNFa antibody), FlSV-Ab2Fc2 (a single-chain chimeric anti-human CD20 antibody), FlSV-Ab66Fcl (a single chain mouse anti-mouse IL-4Ra antibody), and FlSV-Ab66Fc2 (a single-chain mouse anti mouse IL-4Ra antibody).
• HSV-AblFcl a single-chain human anti-human TNFa antagonist antibody, to inhibit ELISA-based detection of TNFa was examined.
• HaCaTs HSV-AblFcl
• HSV-AblFcl HSV-AblFcl
• MOI MOI of 0.3, 1, and 3 for 48 hours.
• cell supernatants were harvested and cleared by centrifugation at 11,000 x 6 for 5 minutes.
• Recombinant human TNFa 1000 pg/mL was added into the clarified cell supernatants and incubated for 2 hours at 37°C.
• An anti-TNFa ELISA was then performed (Abeam, cat. no. abl81421) to determine whether the single chain AblFcl produced by the engineered virus could mediate suppression of TNFa detection.
• the detectable level of TNFa was significantly reduced in cell supernatants harvested from infected human cells, as compared to mock-infected cells (FIG. 3),
• engineered herpes viral vectors present a novel method of administering multiple types of antibodies to humans.
• Example 3 herpes virus-encoded antibodies in a mouse atopic dermatitis (AD) model
• Atopic dermatitis is a chronic, relapsing, and often intensely pruritic inflammatory disorder of the skin.
• the main cause of the disease appears to be a defect of the epidermal barrier resulting from a combination of a genetic predisposition, functioning of the immune system, and environmental factors.
• atopic dermatitis such as antibodies targeting the IL-4 (e.g., dupilumab), IL-l2/23p40 (e.g., ustekinumab), IL-13 (e.g., tralokinumab, lebrikizumab), IL-17 (e.g., secukinumab), and IL-31 (e.g., nemolizumab) pathways) have been explored in the clinical setting.
• IL-4 e.g., dupilumab
• IL-l2/23p40 e.g., ustekinumab
• IL-13 e.g., tralokinumab, lebrikizumab
• IL-17 e.g., secukinumab
• IL-31 e.g., nemolizumab
• vitamin D3 The topical application of vitamin D3, or its synthetic analogs, induces AD-like inflammation in mouse skin.
• the skin inflammation model induced by the vitamin D analog calcipotriol (MC903) has recently gained increased attention, as topical application of MC903 induces high levels of TSLP and the infiltration of group 2 (IL-5 + and IL-l3 + ) innate lymphoid cells to the skin, thereby resembling some immune perturbations observed in skin lesions of humans with AD.
• Topical MC903 administration has also been shown to induce increased IL-4 signaling in treated animals (Martel et al, Yale J Biol Med (2017)).
• DNA and RNA were isolated using an AllPrep DNA/RNA Mini kit (Qiagen) according to the manufacturer’s protocol.
• Qiagen DNA/RNA Mini kit
• 50ng of DNA or RNA were used per reaction in a total reaction volume of 25pL.
• DNA quantification was determined by qPCR analysis using a Taqman ® Fast Advanced Master Mix (Applied Biosystems); RNA quantification was determined by qRT-PCR analysis using Quantabio 1-Step RT-qPCR ToughMix. All samples were run in duplicate or triplicate.
• A-11029) was applied at a 1:200 dilution in antibody diluent buffer for 30 minutes at room temperature in a humidified chamber. Slides were once again washed three times with TBST, and the stained sections were mounted with mounting media (ProLongTM Gold Antifade Mountant with DAPI, ThermoFisher, cat. no. P36931) and covered with a coverslip. The sections were imaged after dehydration (approximately 24 hours) using an ECHO Fluorescence Microscope.
• H&E staining 5 pm sections were taken from cryopreserved tissue, mounted on slides, and air dried for up to 1 hour. The dried slides were rehydrated by soaking in double-distilled water for 2 minutes at room temperature. Sections were then incubated in Hematoxylin Gill 2x (VWR) for 2 minutes at room temperature, followed by being dipped 2 to 3 times in acid alcohol, dipped 3 to 4 times in Blue in Ammonia water, and incubated in eosin (Eosin Y Solution 1%, VWR) for 2 minutes.
• VWR Hematoxylin Gill 2x
• MC903 induced atopic dermatitis- like symptoms in treated animals.
• 14 C57BL/6J mice were used (two mice per group).
• MC903 was prepared in ethanol (EtOH) at a concentration of IOOmM.
• Mice were anaesthetized with isoflurane, their backs were shaved and treated with a chemical hair removal agent, and 25pL of EtOH or MC903/EtOH was applied to the left and right ears (both sides) and to 4 marked dorsal sites ( ⁇ 2 cm 2 each) on Day 1.
• the mice were then retreated with EtOH or MC903/EtOH at the same sites on Days 2, 3, 4, and 5.
• HSV-AblFcl or vehicle control
• HSV-AblFcl or vehicle control
• a gel carrier to the left and right ears and 2 dorsal sites of MC903 treatment
• HSV-AblFcl or vehicle control
• mice were then euthanized on Days 7 and 9, respectively.
• the HSV-AbFcl vector (fully human anti-TNFa single-chain antibody) was used in this experiment as a proof-of-concept to assess antibody expression in AD-like lesions since this virus was appropriately purified and characterized at the time of study initiation.
• Table 6 provides a summary of the experimental design.
• Ear and dorsal skin tissue sections were prepared for immunofluorescent analysis from animals exposed to MC903 on Days 1-5 and treated topically with FlSV-AblFcl (compounded in a methylcellulose gel carrier) or vehicle control (compounded in a methylcellulose gel carrier) on Day 5 or Day 7.
• FIG. 6A shows representative immunofluorescence images of ear and dorsal skin harvested on Day 7 after topical treatment with FlSV-AblFcl or vehicle control on Day 5.
• FIG. 6B shows representative immunofluorescence images of ear and dorsal skin harvested on Day 9 after topical treatment with FlSV-AblFcl or vehicle control on Day 7.
• dupilumab an anti-human IL-4Ra antibody
• dupilumab is the only FDA-approved antibody for treating atopic dermatitis at present, which is administered systemically by bi weekly subcutaneous injections.
• Flere a recombinant herpes virus (FlSV-Ab66Fcl) was engineered to express a dupilumab surrogate antibody that targeted mouse IL-4Ra (Ab66Fcl, a mouse anti-mouse IL-4Ra single-chain antibody), and was tested for its ability to reduce MC903-mediated ear thickening.
• a dupilumab surrogate antibody that targeted mouse IL-4Ra (Ab66Fcl, a mouse anti-mouse IL-4Ra single-chain antibody)
• mice 8 C57BL/6J mice were used (two mice per group).
• MC903 was prepared in ethanol (EtOFl) at a concentration of IOOmM. The backs of the mice were shaved with electric clippers and treated with a chemical hair removal compound on Day -2.
• mice were anaesthetized with isoflurane and 25pL of EtOFl or MC903/EtOFl was applied to the left and right ears (both sides) and to 4 marked dorsal sites ( ⁇ 2 cm 2 each).
• the mice were retreated with EtOH or MC903/EtOH at the same sites on Days 2, 3, 4, and 5 of the study.
• select cohorts of animals received a total of five topical treatments with HSV- Ab66Fcl or vehicle control formulated in a gel carrier to the left and right ears and 2 dorsal sites of EtOH or MC903/EtOH treatment, and received five total intradermal injections of HSV-Ab66Fcl or vehicle control to the remaining 2 dorsal sites of EtOH or EtOH/MC903 treatment, per the study schedule.
• Table 6 provides a summary of the experimental design.
• ear thickness was measured for both ears on each mouse daily from Days 1-10 using a digital caliper (FIG. 8A).
• MC903 exposure induced significant ear thickening in treated mice, as compared to ethanol exposure.
• the average ear thickness at Day 10 for ethanol/vehicle treated animals was 0.19 mm, while the average thickness at this same time point for MC903/vehicle treated animals was 0.6125 mm.
• a significant reduction in ear thickening was observed by Day 5 in the MC903/Ab66Fcl treated ears as compared to the MC903/vehicle treated ears, which carried through to the end of the experiment.
• the antibody caused no appreciable change in ear thickness of ethanol exposed ears (compare EtOH/veh to EtOH/Ab66 in FIG. 8A), confirming specificity of the antibody for reducing this atopic dermatitis-like phenotype.
• W02017060289 were similar, the differences in the therapeutic approaches were stark.
• the study described in W02017060289 required systemic administration of the therapeutic antibody via high dose intraperitoneal injection, and included an antibody pre-treatment step where the first dose of antibody was administered three days before MC903 treatment was initiated.
• the present example used local (as opposed to systemic) administration of the drug product, and did not employ any antibody pre-treatment (the first HSV-Ab66Fcl application occurred after the first MC903 application).

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